Chaitan Khosla
Wells H. Rauser and Harold M. Petiprin Professor and Professor of Chemistry and, by courtesy, of Biochemistry
Chemical Engineering
Bio
Research in this laboratory focuses on problems where deep insights into enzymology and metabolism can be harnessed to improve human health.
For the past two decades, we have studied and engineered enzymatic assembly lines called polyketide synthases that catalyze the biosynthesis of structurally complex and medicinally fascinating antibiotics in bacteria. An example of such an assembly line is found in the erythromycin biosynthetic pathway. Our current focus is on understanding the structure and mechanism of this polyketide synthase. At the same time, we are developing methods to decode the vast and growing number of orphan polyketide assembly lines in the sequence databases.
For more than a decade, we have also investigated the pathogenesis of celiac disease, an autoimmune disorder of the small intestine, with the goal of discovering therapies and related management tools for this widespread but overlooked disease. Ongoing efforts focus on understanding the pivotal role of transglutaminase 2 in triggering the inflammatory response to dietary gluten in the celiac intestine.
Academic Appointments
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Professor, Chemical Engineering
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Professor, Chemistry
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Professor (By courtesy), Biochemistry
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Member, Bio-X
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Institute Scholar, Sarafan ChEM-H
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Director, Innovative Medicines Accelerator (IMA)
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Member, Stanford Cancer Institute
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Member, Wu Tsai Neurosciences Institute
Administrative Appointments
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Baker Family Director, Stanford ChEM-H (2012 - 2020)
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Director, Innovative Medicines Accelerator (2020 - Present)
Honors & Awards
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Member, National Academy of Sciences (2020)
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Arthur C. Cope Scholar Award, American Chemical Society (2009)
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Member, National Academy of Engineering (2009)
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Professional Progress Award, American Institute of Chemical Engineers (2008)
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Member, American Academy of Arts and Sciences (2007)
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Fellow, American Association for the Advancement of Science (2006)
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Pure Chemistry Award, American Chemical Society (2000)
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Alan T. Waterman Award, National Science Foundation (1999)
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Eli Lilly Award in Biological Chemistry, American Chemical Society (1999)
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Allan P. Colburn Award, American Institute of Chemical Engineers (1997)
Boards, Advisory Committees, Professional Organizations
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Scientific Policy Committee Member, SLAC National Accelerator Laboratory (2014 - 2020)
Professional Education
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Postdoc, John Innes Centre, U.K., Genetics (1992)
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PhD, California Institute of Technology, Chemical Engineering (1990)
Current Research and Scholarly Interests
Research in this laboratory focuses on problems where deep insights into enzymology and metabolism can be harnessed to improve human health.
For the past two decades, we have studied and engineered enzymatic assembly lines called polyketide synthases that catalyze the biosynthesis of structurally complex and medicinally fascinating antibiotics in bacteria. An example of such an assembly line is found in the erythromycin biosynthetic pathway. Our current focus is on understanding the structure and mechanism of this polyketide synthase. At the same time, we are developing methods to decode the vast and growing number of orphan polyketide assembly lines in the sequence databases.
For more than a decade, we have also investigated the pathogenesis of celiac disease, an autoimmune disorder of the small intestine, with the goal of discovering therapies and related management tools for this widespread but overlooked disease. Ongoing efforts focus on understanding the pivotal role of transglutaminase 2 in triggering the inflammatory response to dietary gluten in the celiac intestine.
Clinical Trials
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COVID-19 Outpatient Pragmatic Platform Study (COPPS) - Camostat Sub-Protocol
Not Recruiting
The overall objective of this study is to efficiently evaluate the clinical efficacy and safety of different investigational therapeutics among adults who have COVID-19 but are not yet sick enough to require hospitalization. The overall hypothesis is that through an adaptive trial design, potential effective therapies (single and combination) may be identified for this group of patients. COVID-19 Outpatient Pragmatic Platform Study (COPPS) is a pragmatic platform protocol designed to evaluate COVID-19 treatments by assessing their ability to reduce viral shedding (Viral Domain) or improve clinical outcomes (Clinical Domain). To be included into the platform, every investigational product will collect data for both Domain primary endpoints. Individual treatments to be evaluated in the platform will be described in separate sub-protocols.
Stanford is currently not accepting patients for this trial. For more information, please contact Study Team, 650-721-9316.
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COVID-19 Outpatient Pragmatic Platform Study (COPPS) - Master Protocol
Not Recruiting
The overall objective of this study is to efficiently evaluate the clinical efficacy and safety of different investigational therapeutics among adults who have COVID-19 but are not yet sick enough to require hospitalization. The overall hypothesis is that through an adaptive trial design, potential effective therapies (single and combination) may be identified for this group of patients. COVID-19 Outpatient Pragmatic Platform Study (COPPS) is a pragmatic platform protocol designed to evaluate COVID-19 treatments by assessing their ability to reduce viral shedding (Viral Domain) or improve clinical outcomes (Clinical Domain). To be included into the platform, every investigational product will collect data for both Domain primary endpoints. Individual treatments to be evaluated in the platform will be described in separate sub-protocols.
Stanford is currently not accepting patients for this trial. For more information, please contact Study Team, 650-721-9316.
2024-25 Courses
- Biochemistry II
CHEM 183, CHEMENG 183, CHEMENG 283 (Win) -
Independent Studies (8)
- Advanced Undergraduate Research
CHEM 190 (Aut, Win, Spr, Sum) - Directed Instruction/Reading
CHEM 90 (Aut, Win, Spr, Sum) - Graduate Research in Chemical Engineering
CHEMENG 600 (Aut, Win, Spr, Sum) - Out-of-Department Advanced Research Laboratory in Bioengineering
BIOE 191X (Aut, Win, Spr, Sum) - Research and Special Advanced Work
CHEM 200 (Aut, Win, Spr, Sum) - Research in Chemistry
CHEM 301 (Aut, Win, Spr, Sum) - Undergraduate Honors Research in Chemical Engineering
CHEMENG 190H (Aut, Win, Spr, Sum) - Undergraduate Research in Chemical Engineering
CHEMENG 190 (Aut, Win, Spr, Sum)
- Advanced Undergraduate Research
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Prior Year Courses
2023-24 Courses
- Biochemistry II
CHEM 183, CHEMENG 183, CHEMENG 283 (Win) - Foundational Biology for Engineers
CHEMENG 55, ENGR 55 (Aut)
2022-23 Courses
- Special Topics in Biocatalysis
CHEMENG 503 (Aut)
2021-22 Courses
- Special Topics in Biocatalysis
CHEMENG 503 (Aut)
- Biochemistry II
Stanford Advisees
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Doctoral Dissertation Reader (AC)
Lexie Adams, Xujun Cao, Sriya Chitti, Chiu-Chun Chou, L Handy, Alby Joseph, Christina Lee, Michelle Lee, Jack Liu, Kwamina Nyame, Micah Olivas, Elizabeth Park, Prima Dewi Sinawang, Jonathan Yang -
Postdoctoral Faculty Sponsor
Heewon Cho, Antonio Del Rio Flores, Ricardo Hernandez Arriaza, Lin Liu, Fu Chen Yang, Jinping Yang -
Doctoral Dissertation Advisor (AC)
Harrison Besser, Krystal Brodsky, Nina Fatuzzo, Elizabeth Karas, Shreya Kishore, Seokyoung Lee, Dylan Reil, Agnele Sewa, Alex Soohoo -
Doctoral Dissertation Co-Advisor (AC)
Katie Antilla -
Postdoctoral Research Mentor
Xiaowen Ding
All Publications
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Characterization of the Flavin-Dependent Monooxygenase Involved in the Biosynthesis of the Nocardiosis-Associated Polyketide†.
Biochemistry
2024
Abstract
Some species of the Nocardia genus harbor a highly conserved biosynthetic gene cluster designated as the NOCardiosis-Associated Polyketide (NOCAP) synthase that produces a unique glycolipid natural product. The NOCAP glycolipid is composed of a fully substituted benzaldehyde headgroup linked to a polyfunctional alkyl tail and an O-linked disaccharide composed of 3-α-epimycarose and 2-O-methyl-α-rhamnose. Incorporation of the disaccharide unit is preceded by a critical step involving hydroxylation by NocapM, a flavin monooxygenase. In this study, we employed biochemical, spectroscopic, and kinetic analyses to explore the substrate scope of NocapM. Our findings indicate that NocapM catalyzes hydroxylation of diverse aromatic substrates, although the observed coupling between NADPH oxidation and substrate hydroxylation varies widely from substrate to substrate. Our in-depth biochemical characterization of NocapM provides a solid foundation for future mechanistic studies of this enzyme as well as its utilization as a practical biocatalyst.
View details for DOI 10.1021/acs.biochem.4c00480
View details for PubMedID 39433512
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Structural basis for intermodular communication in assembly-line polyketide biosynthesis.
Nature chemical biology
2024
Abstract
Assembly-line polyketide synthases (PKSs) are modular multi-enzyme systems with considerable potential for genetic reprogramming. Understanding how they selectively transport biosynthetic intermediates along a defined sequence of active sites could be harnessed to rationally alter PKS product structures. To investigate functional interactions between PKS catalytic and substrate acyl carrier protein (ACP) domains, we employed a bifunctional reagent to crosslink transient domain-domain interfaces of a prototypical assembly line, the 6-deoxyerythronolide B synthase, and resolved their structures by single-particle cryogenic electron microscopy (cryo-EM). Together with statistical per-particle image analysis of cryo-EM data, we uncovered interactions between ketosynthase (KS) and ACP domains that discriminate between intra-modular and inter-modular communication while reinforcing the relevance of conformational asymmetry during the catalytic cycle. Our findings provide a foundation for the structure-based design of hybrid PKSs comprising biosynthetic modules from different naturally occurring assembly lines.
View details for DOI 10.1038/s41589-024-01709-y
View details for PubMedID 39179672
View details for PubMedCentralID 6935866
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Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state.
Proceedings of the National Academy of Sciences of the United States of America
2024; 121 (28): e2407066121
Abstract
Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient gamma-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for gamma-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.
View details for DOI 10.1073/pnas.2407066121
View details for PubMedID 38959038
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Enterocyte-derived and catalytically active transglutaminase 2 in the gut lumen of mice: Implications for celiac disease.
Gastroenterology
2024
View details for DOI 10.1053/j.gastro.2024.05.029
View details for PubMedID 38825048
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Structure and Mechanisms of Assembly-Line Polyketide Synthases.
Annual review of biochemistry
2024
Abstract
Three decades of studies on the multifunctional 6-deoxyerythronolide B synthase have laid a foundation for understanding the chemistry and evolution of polyketide antibiotic biosynthesis by a large family of versatile enzymatic assembly lines. Recent progress in applying chemical and structural biology tools to this prototypical assembly-line polyketide synthase (PKS) and related systems has highlighted several features of their catalytic cycles and associated protein dynamics. There is compelling evidence that multiple mechanisms have evolved in this enzyme family to channel growing polyketide chains along uniquely defined sequences of 10-100 active sites, each of which is used only once in the overall catalytic cycle of an assembly-line PKS. Looking forward, one anticipates major advances in our understanding of the mechanisms by which the free energy of a repetitive Claisen-like reaction is harnessed to guide the growing polyketide chain along the assembly line in a manner that is kinetically robust yet evolutionarily adaptable.
View details for DOI 10.1146/annurev-biochem-080923-043654
View details for PubMedID 38663033
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Past, present and future of non-invasive tests to assess gluten exposure, celiac disease activity, and end-organ damage.
Gastroenterology
2024
Abstract
Although many biomarkers have been proposed and several are in widespread clinical use, there is no single readout or combination of readouts that correlates tightly with gluten exposure, disease activity or end-organ damage in treated celiac disease patients. Challenges to developing and evaluating better biomarkers include significant interindividual variability related to immune amplification of gluten exposure and how effects of immune activation are manifest. Furthermore, the current "gold standard" for assessment of end-organ damage, small intestinal biopsy, is itself highly imperfect, such that a marker that is actually a better reflection of the "ground truth" may indeed appear to perform poorly. The goal of this review is to review past and present efforts to establish robust non-invasive tools for monitoring treated celiac disease patients and to highlight emerging tools that may prove to be useful in clinical practice.
View details for DOI 10.1053/j.gastro.2024.01.053
View details for PubMedID 38670279
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Discovery and Characterization of the Fully Decorated Nocardiosis-Associated Polyketide Natural Product.
Journal of the American Chemical Society
2024
Abstract
The genomes of 40 strains of Nocardia, most of which were associated with life-threatening human infections, encode a highly conserved assembly line polyketide synthase designated as the NOCAP (NOCardiosis-Associated Polyketide) synthase, whose product structure has been previously described. Here we report the structure and inferred biosynthetic pathway of the fully decorated glycolipid natural product. Its structure reveals a fully substituted benzaldehyde headgroup harboring an unusual polyfunctional tail and an O-linked disaccharide comprising a 3-α-epimycarose and 2-O-methyl-α-rhamnose whose installation requires flavin monooxygenase-dependent hydroxylation of the polyketide product. Production of the fully decorated glycolipid was verified in cultures of two patient-derived Nocardia species. In both E. coli and Nocardia spp., the glycolipid was only detected in culture supernatants, consistent with data from genetic knockout experiments implicating roles for two dedicated proteins in installing the second sugar substituent only after the monoglycosyl intermediate is exported across the bacterial cell membrane. With the NOCAP product in hand, the stage is set for investigating the evolutionary benefit of this polyketide biosynthetic pathway for Nocardia strains capable of infecting human hosts.
View details for DOI 10.1021/jacs.3c13670
View details for PubMedID 38295028
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Celiac disease: mechanisms and emerging therapeutics.
Trends in pharmacological sciences
2023
Abstract
Celiac disease (CeD) is a widespread, gluten-induced, autoimmune disorder that lacks any medicinal therapy. Towards the goal of developing non-dietary treatments for CeD, research has focused on elucidating its molecular and cellular etiology. A model of pathogenesis has emerged centered on interactions between three molecular families: specific class II MHC proteins on antigen-presenting cells (APCs), deamidated gluten-derived peptides, and T cell receptors (TCRs) on inflammatory CD4+ T cells. Growing evidence suggests that this pathogenic axis can be pharmacologically targeted to protect patients from some of the adverse effects of dietary gluten. Further studies have revealed the existence of additional host and environmental contributors to disease initiation and tissue damage. This review summarizes our current understanding of CeD pathogenesis and how it is being harnessed for therapeutic design and development.
View details for DOI 10.1016/j.tips.2023.09.006
View details for PubMedID 37839914
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Targeted Lysosomal Degradation of Secreted and Cell Surface Proteins through the LRP-1 Pathway.
Journal of the American Chemical Society
2023
Abstract
Protein dysregulation has been characterized as the cause of pathogenesis in many different diseases. For proteins lacking easily druggable pockets or catalytically active sites, targeted protein degradation is an attractive therapeutic approach. While several methods for targeted protein degradation have been developed, there remains a demand for lower molecular weight molecules that promote efficient degradation of their targets. In this work, we describe the synthesis and validation of a series of heterobifunctional molecules that bind a protein of interest through a small molecule ligand while targeting them to the lysosome using a short gluten peptide that leverages the TG2/LRP-1 pathway. We demonstrate that this approach can be used to effectively endocytose and degrade representative secreted, cell surface, and transmembrane proteins, notably streptavidin, the vitamin B12 receptor, cubilin, and integrin αvβ5. Optimization of these prototypical molecules could generate pharmacologically relevant LYTAC agents.
View details for DOI 10.1021/jacs.3c05109
View details for PubMedID 37590164
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Genomic mining and diversity of assembly line polyketide synthases.
Open biology
2023; 13 (8): 230096
Abstract
Assembly line polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for synthesizing many medicinally relevant natural products with remarkable structural variety and biological activity. The decrease in cost of genomic sequencing paired with development of computational tools like antiSMASH presents an opportunity to survey the vast diversity of assembly line PKS. Mining the genomic data in the National Center for Biotechnology Information database, our updated catalogue (https://orphanpkscatalog2022.stanford.edu/catalog) presented in this article revealed 8799 non-redundant assembly line polyketide synthase clusters across 4083 species, representing a threefold increase over the past 4 years. Additionally, 95% of the clusters are 'orphan clusters' for which natural products are neither chemically nor biologically characterized. Our analysis indicates that the diversity of assembly line PKSs remains vastly under-explored and also highlights the promise of a genomics-driven approach to natural product discovery.
View details for DOI 10.1098/rsob.230096
View details for PubMedID 37528731
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Discovery and Characterization of Antibody Probes of Module 2 of the 6-Deoxyerythronolide B Synthase.
Biochemistry
2023
Abstract
Fragment antigen-binding domains of antibodies (Fabs) are powerful probes of structure-function relationships of assembly line polyketide synthases (PKSs). We report the discovery and characterization of Fabs interrogating the structure and function of the ketosynthase-acyltransferase (KS-AT) core of Module 2 of the 6-deoxyerythronolide B synthase (DEBS). Two Fabs (AC2 and BB1) were identified to potently inhibit the catalytic activity of Module 2. Both AC2 and BB1 were found to modulate ACP-mediated reactions catalyzed by this module, albeit by distinct mechanisms. AC2 primarily affects the rate (kcat), whereas BB1 increases the KM of an ACP-mediated reaction. A third Fab, AA5, binds to the KS-AT fragment of DEBS Module 2 without altering either parameter; it is phenotypically reminiscent of a previously characterized Fab, 1B2, shown to principally recognize the N-terminal helical docking domain of DEBS Module 3. Crystal structures of AA5 and 1B2 bound to the KS-AT fragment of Module 2 were solved to 2.70 and 2.65 A resolution, respectively, and revealed entirely distinct recognition features of the two antibodies. The new tools and insights reported here pave the way toward advancing our understanding of the structure-function relationships of DEBS Module 2, arguably the most well-studied module of an assembly line PKS.
View details for DOI 10.1021/acs.biochem.3c00156
View details for PubMedID 37184546
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Evaluation of acebilustat, a selective inhibitor of leukotriene B4 biosynthesis, for treatment of outpatients with mild-moderate COVID-19 disease: A randomized, double-blind, placebo- controlled Phase 2 trial.
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
2023
Abstract
The vast majority of COVID-19 disease occurs in outpatients where treatment is limited to anti-virals for high-risk subgroups. Acebilustat, a leukotriene B4 (LTB4) inhibitor, has potential to reduce inflammation and symptom duration.In a single-center trial spanning Delta and Omicron variants, outpatients were randomized to 100 mg of oral acebilustat or placebo for 28 days. Patients reported daily symptoms via electronic query through Day 28 with phone follow-up on Day 120 and collected nasal swabs on Days 1-10. The primary outcome was sustained symptom resolution to Day 28. Secondary 28-day outcomes included time to first symptom resolution, area under the curve (AUC) of longitudinal daily symptom scores; duration of viral shedding through Day 10; and symptoms on Day 120.Sixty participants were randomized to each study arm. At enrollment, median duration and number of symptoms were 4 (IQR 3-5) days and 9 (IQR 7-11) symptoms. Most patients (90%) were vaccinated with 73% having neutralizing antibodies. A minority (44%) of participants (35% in the acebilustat arm and 53% in placebo) had sustained symptom resolution at Day 28 (HR 0.6, 95% CI 0.34-1.04, p = 0.07 favoring placebo). There was no difference in mean AUC of symptom scores over 28 days (difference in mean of AUC 9.4, 95% CI -42.1-60.9, p=0.72). Acebilustat did not impact viral shedding or symptoms at Day 120.Sustained symptoms through Day 28 were common in this low-risk population. Despite this, LTB4 antagonism with acebilustat did not shorten symptom duration in outpatients with COVID-19.
View details for DOI 10.1093/cid/ciad187
View details for PubMedID 36996150
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Challenges in Harnessing Shared Within-Host Severe Acute Respiratory Syndrome Coronavirus 2 Variation for Transmission Inference.
Open forum infectious diseases
2023; 10 (2): ofad001
Abstract
The limited variation observed among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consensus sequences makes it difficult to reconstruct transmission linkages in outbreak settings. Previous studies have recovered variation within individual SARS-CoV-2 infections but have not yet measured the informativeness of within-host variation for transmission inference.We performed tiled amplicon sequencing on 307 SARS-CoV-2 samples, including 130 samples from 32 individuals in 14 households and 47 longitudinally sampled individuals, from 4 prospective studies with household membership data, a proxy for transmission linkage.Consensus sequences from households had limited diversity (mean pairwise distance, 3.06 single-nucleotide polymorphisms [SNPs]; range, 0-40). Most (83.1%, 255 of 307) samples harbored at least 1 intrahost single-nucleotide variant ([iSNV] median, 117; interquartile range [IQR], 17-208), above a minor allele frequency threshold of 0.2%. Pairs in the same household shared significantly more iSNVs (mean, 1.20 iSNVs; 95% confidence interval [CI], 1.02-1.39) than did pairs in different households infected with the same viral clade (mean, 0.31 iSNVs; 95% CI, .28-.34), a signal that decreases with increasingly stringent minor allele frequency thresholds. The number of shared iSNVs was significantly associated with an increased odds of household membership (adjusted odds ratio, 1.35; 95% CI, 1.23-1.49). However, the poor concordance of iSNVs detected across sequencing replicates (24.8% and 35.0% above a 0.2% and 1% threshold) confirms technical concerns that current sequencing and bioinformatic workflows do not consistently recover low-frequency within-host variants.Shared within-host variation may augment the information in consensus sequences for predicting transmission linkages. Improving sensitivity and specificity of within-host variant identification will improve the informativeness of within-host variation.
View details for DOI 10.1093/ofid/ofad001
View details for PubMedID 36751652
View details for PubMedCentralID PMC9898879
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LRP-1 links post-translational modifications to efficient presentation of celiac disease-specific Tcell antigens.
Cell chemical biology
2022
Abstract
Celiac disease (CeD) is an autoimmune disorder in which gluten-derived antigens trigger inflammation. Antigenic peptides must undergo site-specific deamidation to be presentable to CD4+ Tcells in an HLA-DQ2 or -DQ8 restricted manner. While the biochemical basis for this post-translational modification is understood, its localization in the patient's intestine remains unknown. Here, we describe a mechanism by which gluten peptides undergo deamidation and concentration in the lysosomes of antigen-presenting cells, explaining how the concentration of gluten peptides necessary to elicit an inflammatory response in CeD patients is achieved. A ternary complex forms between a gluten peptide, transglutaminase-2 (TG2), and ubiquitous plasma protein alpha2-macroglobulin, and is endocytosed by LRP-1. The covalent TG2-peptide adduct undergoes endolysosomal decoupling, yielding the expected deamidated epitope. Our findings invoke a pathogenic role for dendritic cells and/or macrophages in CeD and implicate TG2 in the lysosomal clearance of unwanted self and foreign extracellular proteins.
View details for DOI 10.1016/j.chembiol.2022.12.002
View details for PubMedID 36608691
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Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells.
The Journal of biological chemistry
2022: 102848
Abstract
In eukaryotes carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for β-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for post-translational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did β-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MG cells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic post-translational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase (CRAT) is believed to catalyze acetyl group transfer from LAC to Coenzyme A, CRAT-/- U87MG cells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase (CROT) as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.
View details for DOI 10.1016/j.jbc.2022.102848
View details for PubMedID 36587768
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Early immune markers of clinical, virological, and immunological outcomes in patients with COVID-19: a multi-omics study.
eLife
2022; 11
Abstract
The great majority of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, there is substantial heterogeneity in SARS-CoV-2-specific memory immune responses following infection. There remains a critical need to identify host immune biomarkers predictive of clinical and immunological outcomes in SARS-CoV-2-infected patients.Leveraging longitudinal samples and data from a clinical trial (N=108) in SARS-CoV-2-infected outpatients, we used host proteomics and transcriptomics to characterize the trajectory of the immune response in COVID-19 patients. We characterized the association between early immune markers and subsequent disease progression, control of viral shedding, and SARS-CoV-2-specific T cell and antibody responses measured up to 7 months after enrollment. We further compared associations between early immune markers and subsequent T cell and antibody responses following natural infection with those following mRNA vaccination. We developed machine-learning models to predict patient outcomes and validated the predictive model using data from 54 individuals enrolled in an independent clinical trial.We identify early immune signatures, including plasma RIG-I levels, early IFN signaling, and related cytokines (CXCL10, MCP1, MCP-2, and MCP-3) associated with subsequent disease progression, control of viral shedding, and the SARS-CoV-2-specific T cell and antibody response measured up to 7 months after enrollment. We found that several biomarkers for immunological outcomes are shared between individuals receiving BNT162b2 (Pfizer-BioNTech) vaccine and COVID-19 patients. Finally, we demonstrate that machine-learning models using 2-7 plasma protein markers measured early within the course of infection are able to accurately predict disease progression, T cell memory, and the antibody response post-infection in a second, independent dataset.Early immune signatures following infection can accurately predict clinical and immunological outcomes in outpatients with COVID-19 using validated machine-learning models.Support for the study was provided from National Institute of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID) (U01 AI150741-01S1 and T32-AI052073), the Stanford's Innovative Medicines Accelerator, National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA) DP1DA046089, and anonymous donors to Stanford University. Peginterferon lambda provided by Eiger BioPharmaceuticals.
View details for DOI 10.7554/eLife.77943
View details for PubMedID 36239699
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Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2).
Biochemistry
2022
Abstract
Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here, we use structure-based drug prototyping to identify two classes of promising leads that noncompetitively inhibit UCK2 activity. In the process, we have identified a hitherto unknown allosteric site at the intersubunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.
View details for DOI 10.1021/acs.biochem.2c00451
View details for PubMedID 36190114
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In vivo visualization and molecular targeting of the cardiac conduction system.
The Journal of clinical investigation
2022
Abstract
Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects to CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab, that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo providing a proof-of-principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging and arrhythmia management.
View details for DOI 10.1172/JCI156955
View details for PubMedID 35951416
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Latiglutenase Protects the Mucosa and Attenuates Symptom Severity in Patients with Celiac Disease Exposed to a Gluten Challenge.
Gastroenterology
2022
Abstract
BACKGROUND & AIMS: Gluten ingestion in celiac disease (CeD) patients can lead to gastrointestinal symptoms and small intestinal mucosal injury METHODS: This gluten-challenge (GC) Phase 2 trial was double-blind, placebo-controlled, and assessed the efficacy and safety of a 1,200 mg dose of IMGX003 in CeD patients exposed to 2 g of gluten per day for 6 weeks. The change in the ratio of villus height to crypt depth (Vh:Cd) was the primary endpoint. Secondary endpoints included densities of intraepithelial lymphocytes (IEL) and symptom severity. These endpoints were evaluated by ANCOVA. Additional endpoints included serology and gluten-immunogenic peptides (GIP) in urine RESULTS: Fifty (50) patients were randomized, and 43 patients completed (n=21 IMGX003, n=22 placebo). The mean DeltaVh:Cd (primary endpoint) for IMGX003 vs. placebo was -0.04 vs. -0.35 (p = .057). The mean DeltaIEL (secondary endpoint) for IMGX003 vs. placebo was 9.8 vs. 24.8 (p = .018). The mean change (worsening) in symptom severity (secondary endpoint) for IMGX003 vs. placebo was 0.22 vs. 1.63 (abdominal pain, p = .231), 0.96 vs. 3.29 (bloating, p = .204), and 0.02 vs. 3.20 (tiredness, p = .113). The 3 x 2-week trend-line significance for these symptoms, respectively, were p = .014, .030 and .002 CONCLUSION: IMGX003 reduced gluten-induced intestinal mucosal damage and symptom severity.CLINICALTRIALS: gov number NCT03585478.
View details for DOI 10.1053/j.gastro.2022.07.071
View details for PubMedID 35931103
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A Mouse Model of Celiac Disease.
Current protocols
2022; 2 (8): e515
Abstract
The design and use of mouse models that reproduce key features of human diseases are critical to advance our understanding of the pathogenesis of autoimmune diseases and to test new therapeutic strategies. Celiac disease is a unique organ-specific autoimmune-like disorder occurring in genetically susceptible individuals carrying HLA-DQ2 or HLA-DQ8 molecules who consume gluten. The key histological characteristic of the disease in humans is the destruction of the lining of the small intestine, a feature that has been difficult to reproduce in immunocompetent animal models. This unit describes the DQ8-Dd -villin-IL-15 transgenic mouse model of CeD, which was engineered based on the knowledge acquired from studying CeD patients' intestinal samples, and which represents the first animal model that develops villous atrophy in an HLA- and gluten-dependent manner without administration of any adjuvant. We provide detailed protocols for inducing and monitoring intestinal tissue damage, evaluating the cytotoxic properties of intraepithelial lymphocytes that mediate enterocyte lysis, and assessing the activation of the enzyme transglutaminase 2, which contributes to the generation of highly immunogenic gluten peptides. Detailed protocols to prepare pepsin-trypsin digested gliadin (PT-gliadin) or chymotrypsin-digested gliadin (CT-gliadin), which allow antibody detection against native or deamidated gluten peptides, are also provided in this unit. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of celiac-like disease in DQ8-Dd -villin-IL-15tg mice Basic Protocol 2: Histological assessment of villous atrophy Support Protocol 1: Morphometric assessment of villous/crypt ratio Support Protocol 2: Evaluation of epithelial cells renewal Support Protocol 3: Evaluation of the density of intraepithelial lymphocytes Basic Protocol 3: Analysis of cytotoxic intraepithelial lymphocytes Basic Protocol 4: Transglutaminase 2 activation and measurement of antibodies against native and deamidated gluten peptides Support Protocol 4: Preparation of CT-gliadin Support Protocol 5: Preparation of PT-gliadin.
View details for DOI 10.1002/cpz1.515
View details for PubMedID 35994521
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Engineering site-selective incorporation of fluorine into polyketides.
Nature chemical biology
2022
Abstract
Although natural products and synthetic small molecules both serve important medicinal functions, their structures and chemical properties are relatively distinct. To expand the molecular diversity available for drug discovery, one strategy is to blend the effective attributes of synthetic and natural molecules. A key feature found in synthetic compounds that is rare in nature is the use of fluorine to tune drug behavior. We now report a method to site-selectively incorporate fluorine into complex structures to produce regioselectively fluorinated full-length polyketides. We engineered a fluorine-selective trans-acyltransferase to produce site-selectively fluorinated erythromycin precursors in vitro. We further demonstrated that these analogs could be produced in vivo in Escherichia coli on engineering of the fluorinated extender unit pool. By using engineered microbes, elaborate fluorinated compounds can be produced by fermentation, offering the potential for expanding the identification and development of bioactive fluorinated small molecules.
View details for DOI 10.1038/s41589-022-01070-y
View details for PubMedID 35817967
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Favipiravir for treatment of outpatients with asymptomatic or uncomplicated COVID-19: a double-blind randomized, placebo-controlled, phase 2 trial.
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
2022
Abstract
Favipiravir is an oral, RNA-dependent RNA polymerase inhibitor with in vitro activity against SARS-CoV2. Despite limited data, favipiravir is administered to patients with COVID-19 in several countries.We conducted a phase 2 double-blind randomized controlled outpatient trial of favipiravir in asymptomatic or mildly symptomatic adults with a positive SARS-CoV2 RT-PCR within 72 hours of enrollment. Participants were randomized 1: 1 to receive placebo or favipiravir (1800mg BID Day 1, 800 mg BID Days 2-10). The primary outcome was SARS-CoV-2 shedding cessation in a modified intention-to-treat (mITT) cohort of participants with positive enrollment RT-PCRs. Using SARS-CoV-2 amplicon-based sequencing, we assessed favipiravir's impact on mutagenesis.From July 8, 2020 - March 23, 2021, we randomized 149 participants with 116 included in the mITT cohort. The participants' mean age was 43 years (SD 12.5) and 57 (49%) were women. We found no difference in time to shedding cessation by treatment arm overall (HR 0.76 favoring placebo, 95% confidence interval [CI] 0.48-1.20) or in sub-group analyses (age, sex, high-risk comorbidities, seropositivity or symptom duration at enrollment). We observed no difference in time to symptom resolution (initial: HR 0.84, 95% CI 0.54-1.29; sustained: HR 0.87, 95% CI 0.52-1.45). We detected no difference in accumulation of transition mutations in the viral genome during treatment.Our data do not support favipiravir use at commonly used doses in outpatients with uncomplicated COVID-19. Further research is needed to ascertain if higher doses of favipiravir are effective and safe for patients with COVID-19.
View details for DOI 10.1093/cid/ciac312
View details for PubMedID 35446944
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KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19.
Science (New York, N.Y.)
2022: eabi9591
Abstract
Here we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from celiac disease patients' leukocytes in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, which correlated with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity post infection. Our results indicate that in both species, these regulatory CD8+ T cells act uniquely to suppress pathogenic T cells in autoimmune and infectious diseases.
View details for DOI 10.1126/science.abi9591
View details for PubMedID 35258337
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Fragment antigen binding domains (Fabs) as tools to study assembly-line polyketide synthases.
Synthetic and systems biotechnology
1800; 7 (1): 506-512
Abstract
The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions. Therefore, discovering tools that aid crystallization is crucial. In this review, the versatility of fragment-antigen binding domains (Fabs) as protein crystallization chaperones is discussed. Fabs have aided the crystallization of membrane-bound and soluble proteins as well as RNA. The ability to bind three Fabs onto a single protein target has demonstrated their potential for crystallization of challenging proteins. We describe a high-throughput workflow for identifying Fabs to aid the crystallization of a protein of interest (POI) by leveraging phage display technologies and differential scanning fluorimetry (DSF). This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases (PKSs), which harbor flexible domains and assume transient conformations. PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds. Despite years of research studying these megasynthases, their overall topology has remained elusive. One Fab in particular, 1B2, has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic (cryoEM) analyses of multiple modules from distinct assembly-line PKSs. Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM, thereby enabling the visualization of intact PKS modules at near-atomic (3-5A) resolution. The identification of PKS-binding Fabs can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.
View details for DOI 10.1016/j.synbio.2021.12.003
View details for PubMedID 34977395
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An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease.
Nature communications
2022; 13 (1): 888
Abstract
Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, impeding our knowledge of potentially diverse peptide species that encounter the immune system. Here, we develop a liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. We detect over 600 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and ~5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differ from controls. Our results provide a new foundation for understanding gluten immunogenicity, improving CeD management, and characterizing the dietary and urinary peptidomes.
View details for DOI 10.1038/s41467-022-28353-1
View details for PubMedID 35173144
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Early non-neutralizing, afucosylated antibody responses are associated with COVID-19 severity.
Science translational medicine
1800: eabm7853
Abstract
A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated IgG antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were instead highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc gamma receptor (FcgammaR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from COVID-19 patients induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine-elicited IgG did not promote an inflammatory lung response. Together, these results show that IgG-FcgammaR interactions are able to regulate inflammation in the lung and may define distinct lung activities associated with the IgG that are associated with severe COVID-19 and protection against infection with SARS-CoV-2.
View details for DOI 10.1126/scitranslmed.abm7853
View details for PubMedID 35040666
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Solution Structure and Conformational Flexibility of a Polyketide Synthase Module.
JACS Au
1800; 1 (12): 2162-2171
Abstract
Polyketide synthases (PKSs) are versatile C-C bond-forming enzymes that are broadly distributed in bacteria and fungi. The polyketide compound family includes many clinically useful drugs such as the antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering lovastatin. Harnessing PKSs for custom compound synthesis remains an open challenge, largely because of the lack of knowledge about key structural properties. Particularly, the domains-well characterized on their own-are poorly understood in their arrangement, conformational dynamics, and interplay in the intricate quaternary structure of modular PKSs. Here, we characterize module 2 from the 6-deoxyerythronolide B synthase by small-angle X-ray scattering and cross-linking mass spectrometry with coarse-grained structural modeling. The results of this hybrid approach shed light on the solution structure of a cis-AT type PKS module as well as its inherent conformational dynamics. Supported by a directed evolution approach, we also find that acyl carrier protein (ACP)-mediated substrate shuttling appears to be steered by a nonspecific electrostatic interaction network.
View details for DOI 10.1021/jacsau.1c00043
View details for PubMedID 34977887
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Prospects for Antibacterial Discovery and Development
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2021; 143 (50): 21127-21142
Abstract
The rising prevalence of multidrug-resistant bacteria is an urgent health crisis that can only be countered through renewed investment in the discovery and development of antibiotics. There is no panacea for the antibacterial resistance crisis; instead, a multifaceted approach is called for. In this Perspective we make the case that, in the face of evolving clinical needs and enabling technologies, numerous validated antibacterial targets and associated lead molecules deserve a second look. At the same time, many worthy targets lack good leads despite harboring druggable active sites. Creative and inspired techniques buoy discovery efforts; while soil screening efforts frequently lead to antibiotic rediscovery, researchers have found success searching for new antibiotic leads by studying underexplored ecological niches or by leveraging the abundance of available data from genome mining efforts. The judicious use of "polypharmacology" (i.e., the ability of a drug to alter the activities of multiple targets) can also provide new opportunities, as can the continued search for inhibitors of resistance enzymes with the capacity to breathe new life into old antibiotics. We conclude by highlighting available pharmacoeconomic models for antibacterial discovery and development while making the case for new ones.
View details for DOI 10.1021/jacs.1c10200
View details for Web of Science ID 000750819800018
View details for PubMedID 34860516
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Properties of a "Split-and-Stuttering" Module of an Assembly Line Polyketide Synthase
JOURNAL OF ORGANIC CHEMISTRY
2021; 86 (16): 11100-11106
Abstract
Notwithstanding the "one-module-one-elongation-cycle" paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as "stuttering", have been derived through in vivo and in vitro analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a trans-AT subfamily PKS that is also naturally split across two polypeptides. Previous work has shown that Module 5 of the NOCAP (nocardiosis associated polyketide) synthase iterates precisely three times in the biosynthesis of its polyketide product, resulting in an all-trans-configured triene moiety in the polyketide product. Here, we describe the intrinsic catalytic properties of this NOCAP synthase module. Through complementary experiments in vitro and in E. coli, the "split-and-stuttering" module was shown to catalyze up to five elongation cycles, although its dehydratase domain ceased to function after three cycles. Unexpectedly, the central olefinic group of this truncated product had a cis configuration. Our findings set the stage for further in-depth analysis of a structurally and functionally unusual PKS module with contextual biosynthetic plasticity.
View details for DOI 10.1021/acs.joc.1c00120
View details for Web of Science ID 000687851300016
View details for PubMedID 33755455
View details for PubMedCentralID PMC8380650
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An Unusual "OR" Gate for Allosteric Regulation of Mammalian Transglutaminase 2 in the Extracellular Matrix
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2021; 143 (28): 10537-10540
Abstract
Transglutaminase 2 (TG2) is a highly expressed mammalian enzyme whose biological function is unclear, although its catalytic activity in the small intestine appears necessary for celiac disease (CeD) pathogenesis. While TG2 activity is reversibly regulated by multiple allosteric mechanisms, their roles under fluctuating physiological conditions are not well understood. Here, we demonstrate that extracellular TG2 activity is competitively controlled by the mutually exclusive binding of a high-affinity Ca2+ ion or the formation of a strained disulfide bond. Binding of Ca2+ at the high-affinity site does not activate TG2 per se, but it protects against oxidative enzyme deactivation while preserving the ability of Ca2+ ions to occupy weaker binding sites capable of allosteric TG2 activation. In contrast, disulfide bond formation competitively occludes the high-affinity Ca2+ site while resulting in complete TG2 inactivation. Because both outcomes are comparably favorable under typical extracellular conditions, subtle changes in the availability of redox catalysts or promoters in the extracellular matrix can dramatically alter steady-state TG2 activity. Thus, TG2 harbors a molecular "OR" gate that determines its catalytic fate upon export from cells.
View details for DOI 10.1021/jacs.1c04616
View details for Web of Science ID 000677544800008
View details for PubMedID 34232639
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The COVID-19 Outpatient Pragmatic Platform Study (COPPS): Study design of a multi-center pragmatic platform trial.
Contemporary clinical trials
2021: 106509
Abstract
More than 3000 clinical trials related to COVID-19 have been registered through clinicaltrials.gov. With so many trials, there is a risk that many will be inconclusive due to being underpowered or due to an inability to recruit patients. At academic medical centers, multiple trials are competing for the same resources; the success of one may come at the expense of another. The COVID-19 Outpatient Pragmatic Protocol Study (COPPS) is a flexible phase 2, multi-site, randomized, blinded trial based at Stanford University designed to overcome these issues by simultaneously evaluating multiple COVID-19 treatments in the outpatient setting in one common platform with shared controls. This approach reduces the overall number of patients required for statistical power, while improving the likelihood that any enrolled patient receives active treatment. The platform study has two main domains designed to evaluate COVID-19 treatments by assessing their ability to reduce viral shedding (Viral Domain), measured with self-collected nasal swabs, or improve clinical outcomes (Clinical Domain), measured through self-reported symptomology data. Data are collected on both domains for all participants enrolled. Participants are followed over a 28-day period. COPPS has the advantage of pragmatism created around its workflow that is also appealing to potential participants because of a lower probability of inactive treatment. At the conclusion of this clinical trial we expect to have identified potentially effective therapeutic strategy/ies for treating COVID-19 in the outpatient setting, which will have a transformative impact on medicine and public health.
View details for DOI 10.1016/j.cct.2021.106509
View details for PubMedID 34274494
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GRINS: Genetic elements that recode assembly-line polyketide synthases and accelerate their diversification
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2021; 118 (26)
View details for DOI 10.1073/pnas.2100751118|1of8
View details for Web of Science ID 000671767800007
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GRINS: Genetic elements that recode assembly-line polyketide synthases and accelerate their diversification.
Proceedings of the National Academy of Sciences of the United States of America
2021; 118 (26)
Abstract
Assembly-line polyketide synthases (PKSs) are large and complex enzymatic machineries with a multimodular architecture, typically encoded in bacterial genomes by biosynthetic gene clusters. Their modularity has led to an astounding diversity of biosynthesized molecules, many with medical relevance. Thus, understanding the mechanisms that drive PKS evolution is fundamental for both functional prediction of natural PKSs as well as for the engineering of novel PKSs. Here, we describe a repetitive genetic element in assembly-line PKS genes which appears to play a role in accelerating the diversification of closely related biosynthetic clusters. We named this element GRINS: genetic repeats of intense nucleotide skews. GRINS appear to recode PKS protein regions with a biased nucleotide composition and to promote gene conversion. GRINS are present in a large number of assembly-line PKS gene clusters and are particularly widespread in the actinobacterial genus Streptomyces While the molecular mechanisms associated with GRINS appearance, dissemination, and maintenance are unknown, the presence of GRINS in a broad range of bacterial phyla and gene families indicates that these genetic elements could play a fundamental role in protein evolution.
View details for DOI 10.1073/pnas.2100751118
View details for PubMedID 34162709
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50 Years Ago in TheJournalofPediatrics: Association of Type 1 Diabetes Mellitus and Celiac Disease: Then and Now.
The Journal of pediatrics
2021; 230: 70
View details for DOI 10.1016/j.jpeds.2020.10.050
View details for PubMedID 33632400
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Association of Type 1 Diabetes Mellitus and Celiac Disease: Then and Now
JOURNAL OF PEDIATRICS
2021; 230: 70
View details for Web of Science ID 000621364700015
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Peginterferon Lambda-1a for treatment of outpatients with uncomplicated COVID-19: a randomized placebo-controlled trial.
Nature communications
2021; 12 (1): 1967
Abstract
Type III interferons have been touted as promising therapeutics in outpatients with coronavirus disease 2019 (COVID-19). We conducted a randomized, single-blind, placebo-controlled trial (NCT04331899) in 120 outpatients with mild to moderate COVID-19 to determine whether a single, 180 mcg subcutaneous dose of Peginterferon Lambda-1a (Lambda) within 72 hours of diagnosis could shorten the duration of viral shedding (primary endpoint) or symptoms (secondary endpoint). In both the 60 patients receiving Lambda and 60 receiving placebo, the median time to cessation of viral shedding was 7 days (hazard ratio [HR] = 0.81; 95% confidence interval [CI] 0.56 to 1.19). Symptoms resolved in 8 and 9 days in Lambda and placebo, respectively, and symptom duration did not differ significantly between groups (HR 0.94; 95% CI 0.64 to 1.39). Both Lambda and placebo were well-tolerated, though liver transaminase elevations were more common in the Lambda vs. placebo arm (15/60 vs 5/60; p = 0.027). In this study, a single dose of subcutaneous Peginterferon Lambda-1a neither shortened the duration of SARS-CoV-2 viral shedding nor improved symptoms in outpatients with uncomplicated COVID-19.
View details for DOI 10.1038/s41467-021-22177-1
View details for PubMedID 33785743
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Mapping the catalytic conformations of an assembly-line polyketide synthase module.
Science (New York, N.Y.)
2021; 374 (6568): 729-734
Abstract
[Figure: see text].
View details for DOI 10.1126/science.abi8358
View details for PubMedID 34735239
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SARS-CoV-2 subgenomic RNA kinetics in longitudinal clinical samples
Open Forum Infectious Diseases
2021
View details for DOI 10.1093/ofid/ofab310
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Structure and Mechanism of the Ketosynthase-Chain Length Factor Didomain from a Prototypical Polyunsaturated Fatty Acid Synthase.
Biochemistry
2020
Abstract
Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential ingredients of the human diet. They are synthesized by LC-PUFA synthases (PFASs) expressed in marine bacteria and other organisms. PFASs are large enzyme complexes that are homologous to mammalian fatty acid synthases and microbial polyketide synthases. One subunit of each PFAS harbors consecutive ketosynthase (KSc) and chain length factor (CLF) domains that collectively catalyze the elongation of a nascent fatty acyl chain via iterative carbon-carbon bond formation. We report the X-ray crystal structure of the KS-CLF didomain from a well-studied PFAS in Moritella marina. Our structure, in combination with biochemical analysis, provides a foundation for understanding the mechanism of substrate recognition and chain length control by the KS-CLF didomain as well as its interaction with a cognate acyl carrier protein partner.
View details for DOI 10.1021/acs.biochem.0c00785
View details for PubMedID 33283513
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Antibody Probes of Module 1 of the 6-Deoxyerythronolide B Synthase Reveal an Extended Conformation During Ketoreduction.
Journal of the American Chemical Society
2020
Abstract
The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase (PKS) that synthesizes the macrocyclic core of the antibiotic erythromycin. Each of its six multidomain modules presumably sample distinct conformations, as biosynthetic intermediates tethered to their acyl carrier proteins interact with multiple active sites during the courses of their catalytic cycles. The spatiotemporal details underlying these protein dynamics remain elusive. Here, we investigate one aspect of this conformational flexibility using two domain-specific monoclonal antibody fragments (Fabs) isolated from a very large naive human antibody library. Both Fabs, designated 1D10 and 2G10, were bound specifically and with high affinity to the ketoreductase domain of DEBS module 1 (KR1). Comparative kinetic analysis of stand-alone KR1 as well as a truncated bimodular derivative of DEBS revealed that 1D10 inhibited KR1 activity whereas 2G10 did not. Co-crystal structures of each KR1-Fab complex provided a mechanistic rationale for this difference. A hybrid PKS module harboring KR1 was engineered, whose individual catalytic domains have been crystallographically characterized at high resolution. Size exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS) of this hybrid module bound to 1D10 provided further support for the catalytic relevance of the "extended" model of a PKS module. Our findings reinforce the power of monoclonal antibodies as tools to interrogate structure-function relationships of assembly line PKSs.
View details for DOI 10.1021/jacs.0c05133
View details for PubMedID 32786753
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Challenges and opportunities for engineering assembly-line polyketide biosynthesis in Escherichia coli.
Metabolic engineering communications
2020; 10: e00106
Abstract
Assembly-line polyketide synthases generate natural products that have led to many live-saving drugs. The use of E.coli as a heterologous host for reconstituting these enormous and complex enzymatic machines has and will continue to be a critical strategy for understanding them. Here, we concisely summarize successful examples in exploiting E.coli for assembly-line polyketide biosynthesis as well as offer examples of new challenges in which this approach is primed to tackle.
View details for DOI 10.1016/j.mec.2019.e00106
View details for PubMedID 32547924
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When the Quest for a Cure Is Personal
CELL
2020; 181 (1): 19
Abstract
We asked three researchers how their personal connection to disease has affected them and what lessons it has taught them along the way.
View details for Web of Science ID 000523319000007
View details for PubMedID 32243789
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Complete Reconstitution and Deorphanization of the 3 MDa Nocardiosis-Associated Polyketide Synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2020; 142 (13): 5952–57
Abstract
Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line polyketide synthase (PKS) that presumably synthesizes an unknown polyketide. Here, we report the discovery and structure elucidation of the NOCAP (nocardiosis-associated polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly line PKS in vitro and of using these approaches to "deorphanize" a complete assembly line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.
View details for DOI 10.1021/jacs.0c00904
View details for Web of Science ID 000526393700009
View details for PubMedID 32182063
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IL-15, gluten and HLA-DQ8 drive tissue destruction in coeliac disease.
Nature
2020
Abstract
Coeliac disease is a complex, polygenic inflammatory enteropathy caused by exposure to dietary gluten that occurs in a subset of genetically susceptible individuals whoexpress either the HLA-DQ8 or HLA-DQ2 haplotypes1,2. The need to develop non-dietary treatments is now widely recognized3, but no pathophysiologically relevant gluten- and HLA-dependent preclinical model exists. Furthermore, although studies in humans have led to major advances in our understanding of the pathogenesis of coeliac disease4, the respective roles of disease-predisposing HLA molecules, and of adaptive and innate immunity in the development of tissue damage, have not been directly demonstrated. Here we describe a mouse model that reproduces the overexpression ofinterleukin-15 (IL-15) in the gut epithelium and lamina propria that is characteristic of active coeliac disease, expresses the predisposing HLA-DQ8 molecule, and develops villous atrophy after ingestion of gluten. Overexpression of IL-15 in both the epithelium and the lamina propria is required for the development of villous atrophy, which demonstrates the location-dependent central role of IL-15 in the pathogenesis of coeliac disease. In addition, CD4+ T cells and HLA-DQ8 have a crucial role in the licensing of cytotoxic T cells to mediate intestinal epithelial cell lysis. We also demonstrate a role for the cytokine interferon-gamma (IFNgamma) and theenzyme transglutaminase 2 (TG2) in tissue destruction. By reflecting the complex interaction between gluten, genetics and IL-15-driven tissue inflammation, this mouse model provides the opportunity to both increase our understanding of coeliac disease, and develop new therapeutic strategies.
View details for DOI 10.1038/s41586-020-2003-8
View details for PubMedID 32051586
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Genome-wide analysis of targets of macrolide antibiotics in mammalian cells.
The Journal of biological chemistry
2020
Abstract
Macrolide antibiotics, such as erythromycin and josamycin, are polyketide natural products harboring 14-16-membered macrocyclic lactone rings to which various sugars are attached. These antibiotics are extensively used in the clinic because of their ability to inhibit bacterial protein synthesis. More recently, some macrolides have been shown to also possess anti-inflammatory and other therapeutic activities in mammalian cells. To better understand the targets and effects of this drug class in mammalian cells, we used a genome-wide shRNA screen in K562 cancer cells to identify genes that modulate cellular sensitivity to josamycin. Among the most sensitizing hits were proteins involved in mitochondrial translation and the mitochondrial unfolded protein response, glycolysis and the mitogen-activated protein kinase signaling cascade. Further analysis revealed that cells treated with josamycin or other antibacterials exhibited impaired oxidative phosphorylation and metabolic shifts to glycolysis. Interestingly, we observed that knockdown of the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) gene, which contributes to p38 MAPK signaling, sensitized cells to only josamycin but not to other antibacterials. There is a growing interest in better characterizing the therapeutic effects and toxicities of antibiotics in mammalian cells to guide new applications in both cellular and clinical studies. To our knowledge, this is the first report of an unbiased genome-wide screen to investigate the effects of a clinically used antibiotic on human cells.
View details for DOI 10.1074/jbc.RA119.010770
View details for PubMedID 31915244
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Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism.
Cell chemical biology
2020
Abstract
Genome-wide analysis of the mode of action of GSK983, a potent antiviral agent, led to the identification of dihydroorotate dehydrogenase as its target along with the discovery that genetic knockdown of pyrimidine salvage sensitized cells to GSK983. Because GSK983 is an ineffective antiviral in the presence of physiological uridine concentrations, we explored combining GSK983 with pyrimidine salvage inhibitors. We synthesized and evaluated analogs of cyclopentenyl uracil (CPU), an inhibitor of uridine salvage. We found that CPU was converted into its triphosphate in cells. When combined with GSK983, CPU resulted in large drops in cellular UTP and CTP pools. Consequently, CPU-GSK983 suppressed dengue virus replication in the presence of physiological concentrations of uridine. In addition, the CPU-GSK983 combination markedly enhanced the effect of RNA-dependent RNA polymerase (RdRp) inhibition on viral infection. Our findings highlight a new host-targeting strategy for potentiating the antiviral activity of RdRp inhibitors.
View details for DOI 10.1016/j.chembiol.2020.05.002
View details for PubMedID 32442424
View details for PubMedCentralID PMC7241336
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Evolution and Diversity of Assembly-Line Polyketide Synthases
CHEMICAL REVIEWS
2019; 119 (24): 12524–47
Abstract
Assembly-line polyketide synthases (PKSs) are among the most complex protein machineries known in nature, responsible for the biosynthesis of numerous compounds used in the clinic. Their present-day diversity is the result of an evolutionary path that has involved the emergence of a multimodular architecture and further diversification of assembly-line PKSs. In this review, we provide an overview of previous studies that investigated PKS evolution and propose a model that challenges the currently prevailing view that gene duplication has played a major role in the emergence of multimodularity. We also analyze the ensemble of orphan PKS clusters sequenced so far to evaluate how large the entire diversity of assembly-line PKS clusters and their chemical products could be. Finally, we examine the existing techniques to access the natural PKS diversity in natural and heterologous hosts and describe approaches to further expand this diversity through engineering.
View details for DOI 10.1021/acs.chemrev.9b00525
View details for Web of Science ID 000505627700009
View details for PubMedID 31838842
View details for PubMedCentralID PMC6935866
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Latiglutenase Treatment for Celiac Disease: Symptom and Quality of Life Improvement for Seropositive Patients on a Gluten-Free Diet.
GastroHep
2019; 1 (6): 293–301
Abstract
Background: Celiac disease (CD) is a widespread autoimmune disease triggered by dietary gluten that can lead to severe gastrointestinal symptoms. Because there is no available treatment other than a lifelong gluten-free diet, many patients continue to experience chronic symptoms.Aim: In this analysis we report on the efficacy of latiglutenase, an orally administered enzyme treatment, for improving multiple gluten-induced symptoms and consequent quality of life (QOL) due to inadvertent gluten consumption.Methods: This analysis is based on data from the CeliAction study of symptomatic patients (ALV003-1221; NCT01917630). Patients were treated with latiglutenase or placebo for 12 weeks and instructed to respond to a symptom diary daily and to multiple QOL questionnaires at weeks 0, 6, and 12 of the treatment periods as secondary endpoints. The results were stratified by serostatus.Results: 398 patients completed the 12-week CDSD study. In seropositive, but not seronegative, CD patients a statistically significant and dose-dependent improvement was seen in the severity and frequency of abdominal pain, bloating, tiredness, and constipation. In subjects receiving 900 mg latiglutenase, improvements (p-values) in the severity of these symptoms for week 12 were 58% (0.038), 44% (0.023), 21% (0.164), and 104% (0.049) respectively, relative to placebo-dosed subjects. The reduction in symptoms trended higher for more symptomatic patients. Similar results were observed for the QOL outcome measures.Conclusions: Although this study was not powered to definitively establish the benefit of latiglutenase in seropositive CD patients, such patients appear to show symptomatic and QOL benefit from using latiglutenase with meals.
View details for DOI 10.1002/ygh2.371
View details for PubMedID 32313451
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Discovery of small molecule inhibitors of human uridine-cytidine kinase 2 by high-throughput screening.
Bioorganic & medicinal chemistry letters
2019
Abstract
Clinically relevant inhibitors of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in mammalian de novo pyrimidine synthesis, have strong antiviral and anticancer activity in vitro. However, they are ineffective in vivo due to efficient uridine salvage by infected or rapidly dividing cells. The pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2), a 29 kDa protein that forms a tetramer in its active state, is necessary for uridine salvage. Notwithstanding the pharmacological potential of this target, no medicinally tractable inhibitors of the human enzyme have been reported to date. We therefore established and miniaturized an in vitro assay for UCK2 activity and undertook a high-throughput screen against a 40,000-compound library to generate drug-like leads. The structures, activities, and modes of inhibition of the most promising hits are described. Notably, our screen yielded non-competitive UCK2 inhibitors which were able to suppress nucleoside salvage in cells both in the presence and absence of DHODH inhibitors.
View details for DOI 10.1016/j.bmcl.2019.08.010
View details for PubMedID 31420268
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Tunable Enzymatic Synthesis of the Immunomodulator Lipid IVA To Enable Structure-Activity Analysis
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2019; 141 (24): 9474–78
Abstract
The Lipid A family of glycolipids, found in the outer membranes of all Gram-negative bacteria, exhibits considerable structural diversity in both lipid and glycan moieties. The lack of facile methods to prepare analogues of these natural products represents a major roadblock in understanding the relationship between their structure and immunomodulatory activities. Here we present a modular, cell-free multienzymatic platform to access these structure-activity relationships. By individually purifying 19 Escherichia coli proteins and reconstituting them in vitro in the presence of acetyl-CoA, UDP- N-acetylglucosamine, NADPH, and ATP, we have developed a system capable of synthesizing Lipid IVA, the first bioactive intermediate in the Lipid A pathway. Our reconstituted multienzyme system revealed considerable promiscuity for orthologs with distinct substrate specificity, as illustrated by swapping enzymes from distantly related cyanobacterial and Pseudomonas species. Analysis of the agonistic and antagonistic activities of the resulting products against the THP-1 human monocytic cell line revealed hitherto unrecognized trends, while opening the door to harnessing the potent biological activities of these complex glycolipid natural products.
View details for DOI 10.1021/jacs.9b03066
View details for Web of Science ID 000471835600008
View details for PubMedID 31184877
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Engineering of Chimeric Polyketide Synthases Using SYNZIP Docking Domains
ACS CHEMICAL BIOLOGY
2019; 14 (3): 426–33
View details for DOI 10.1021/acschembio.8b01060
View details for Web of Science ID 000461844100015
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Engineering of Chimeric Polyketide Synthases Using SYNZIP Docking Domains.
ACS chemical biology
2019
Abstract
Engineering of assembly line polyketide synthases (PKSs) to produce novel bioactive compounds has been a goal for over 20 years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain-domain interactions are evolutionarily optimized and, if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a noncovalent docking domain, or SYNZIP domain, mediated interface. These chimeric systems exhibited non-native domain-domain interactions during intermodular polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in which a noncovalent interface was introduced between the condensing and processing parts of a module, resulting in non-native domain interactions during the extender unit acylation and polyketide chain elongation steps of their catalytic cycles. We show that the natural PKS docking domains can be efficiently substituted with SYNZIP domains and that the newly introduced noncovalent interface between the condensing and processing parts of a module can be harnessed for PKS engineering. Additionally, we established SYNZIP domains as a new tool for engineering PKSs by efficiently bridging non-native interfaces without perturbing PKS activity.
View details for PubMedID 30682239
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From Active Sites to Machines: A Challenge for Enzyme Chemists.
Israel journal of chemistry
2019; 59 (1-2): 37-40
Abstract
As researchers who study enzyme chemistry embrace increasingly complex systems, especially biological machines, our attention is also shifting from steps involving covalent bond formation or cleavage to those that exclusively involve changes in non-covalent bonding. Assembly line polyketide synthases are an example of this growing challenge. By now, the chemical reactions underpinning polyketide biosynthesis can be unequivocally mapped to well-defined active sites and are, for the most part, readily explicable in the language of physical organic chemistry. Yet, all of these insights merely serve as a backdrop to the real problem of explaining how the catalytic functions of dozens of active sites are synchronized in order to allow these remarkable machines to turn over with remarkable specificity. Notwithstanding the fact that the time-honored language of physical organic chemistry can teach us a lot, it is often insufficient to describe many of these events, and must therefore evolve.
View details for DOI 10.1002/ijch.201800098
View details for PubMedID 31762490
View details for PubMedCentralID PMC6874407
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From Active Sites to Machines: A Challenge for Enzyme Chemists
ISRAEL JOURNAL OF CHEMISTRY
2019; 59 (1-2): 37–40
View details for DOI 10.1002/ijch.201800098
View details for Web of Science ID 000462773200006
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Substrates, inhibitors, and probes of mammalian transglutaminase 2.
Analytical biochemistry
2019: 113560
Abstract
Transglutaminase 2 (TG2) is a ubiquitous but enigmatic mammalian protein to which a number of biological functions have been ascribed but not definitively proven. As a member of the transglutaminase family, TG2 can catalyze deamidation or alternatively transamidation of selected Gln residues in proteins and peptides. It is also known to harbor other enzymatic properties, including protein disulfide isomerase, GTP-dependent signal transduction, and ATP dependent protein kinase activity. Given its multifunctional chemistry, it is unsurprising that a long list of proteins from the mammalian proteome have been identified as substrates and/or binding partners; however, the biological relevance of none of these protein-protein interactions has been clarified as yet. Remarkably, the most definitive insights into the biology of TG2 stem from its pathophysiological role in gluten peptide deamidation in celiac disease. Meanwhile our understanding of TG2 chemistry has been leveraged to engineer a spectrum of inhibitors and other molecular probes of TG2 biology in vivo. This review summarizes our current knowledge of the enzymology and regulation of human TG2 with a focus on its physiological substrates as well as tool molecules whose engineering was inspired by their identities.
View details for DOI 10.1016/j.ab.2019.113560
View details for PubMedID 31874171
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In Vivo Measurement of Redox-Regulated TG2 Activity
FUNCTIONAL DISULPHIDE BONDS: METHODS AND PROTOCOLS
2019; 1967: 263–74
View details for DOI 10.1007/978-1-4939-9187-7_16
View details for Web of Science ID 000486462500017
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In Vivo Measurement of Redox-Regulated TG2 Activity.
Methods in molecular biology (Clifton, N.J.)
2019; 1967: 263–74
Abstract
Transglutaminase 2 (TG2) is a ubiquitous mammalian enzyme that is implicated in a variety of physiological processes and human diseases. Normally, extracellular TG2 is catalytically dormant due to formation of an allosteric disulphide bond between Cys370 and 371 of the enzyme. In this protocol, we describe a method to reduce this disulphide bond in living mice and to monitor the resulting in vivo TG2 activity. Briefly, exogenous thioredoxin-1 protein (TRX) is prepared and administered as a specific, physiologically relevant reductant of the Cys370-371 disulphide along with the small molecule 5-biotinamidopentylamine (5-BP) as a TG2 activity probe. Tissue cryosections are then analyzed by immunohistochemistry to ascertain the extent of 5-BP incorporation, which serves as a record of the redox state of TG2 in vivo. This protocol focuses on the modulation and measurement of TG2 in the small intestine, but we encourage investigators to evaluate it in their organ(s) of interest.
View details for PubMedID 31069776
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A tribute to Professor Jay Bailey: A pioneer in biochemical engineering
AICHE JOURNAL
2018; 64 (12): 4179–81
View details for DOI 10.1002/aic.16441
View details for Web of Science ID 000449983800002
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A Tribute to James E. Bailey
AICHE JOURNAL
2018; 64 (12): 4178
View details for DOI 10.1002/aic.16455
View details for Web of Science ID 000449983800001
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Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase
BIOCHEMISTRY
2018; 57 (43): 6201–8
View details for DOI 10.1021/acs.biochem.8b00886
View details for Web of Science ID 000449123400008
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Discovery and Characterization of a Thioesterase-Specific Monoclonal Antibody That Recognizes the 6-Deoxyerythronolide B Synthase.
Biochemistry
2018
Abstract
Assembly line polyketide synthases (PKSs) are large multimodular enzymes responsible for the biosynthesis of diverse antibiotics in bacteria. Structural and mechanistic analysis of these megasynthases can benefit from the discovery of reagents that recognize individual domains or linkers in a site-specific manner. Monoclonal antibodies not only have proven themselves as premier tools in analogous applications but also have the added benefit of constraining the conformational flexibility of their targets in unpredictable but often useful ways. Here we have exploited a library based on the naive human antibody repertoire to discover a Fab (3A6) that recognizes the terminal thioesterase (TE) domain of the 6-deoxyerythronolide B synthase with high specificity. Biochemical assays were used to verify that 3A6 binding does not inhibit enzyme turnover. The co-crystal structure of the TE-3A6 complex was determined at 2.45 A resolution, resulting in atomic characterization of this protein-protein recognition mechanism. Fab binding had minimal effects on the structural integrity of the TE. In turn, these insights were used to interrogate via small-angle X-ray scattering the solution-phase conformation of 3A6 complexed to a catalytically competent PKS module and bimodule. Altogether, we have developed a high-affinity monoclonal antibody tool that recognizes the TE domain of the 6-deoxyerythronolide B synthase while maintaining its native function.
View details for PubMedID 30289692
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Interleukin 4 is inactivated via selective disulfide-bond reduction by extracellular thioredoxin
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2018; 115 (35): 8781-8786
View details for DOI 10.1073/pnas.1805288115
View details for Web of Science ID 000442861600057
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Interleukin 4 is inactivated via selective disulfide-bond reduction by extracellular thioredoxin.
Proceedings of the National Academy of Sciences of the United States of America
2018
Abstract
Thioredoxin 1 (TRX), an essential intracellular redox regulator, is also secreted by mammalian cells. Recently, we showed that TRX activates extracellular transglutaminase 2 via reduction of an allosteric disulfide bond. In an effort to identify other extracellular substrates of TRX, macrophages derived from THP-1 cells were treated with NP161, a small-molecule inhibitor of secreted TRX. NP161 enhanced cytokine outputs of alternatively activated macrophages, suggesting that extracellular TRX regulated the activity of interleukin 4 (IL-4) and/or interleukin 13 (IL-13). To test this hypothesis, the C35S mutant of human TRX was shown to form a mixed disulfide bond with recombinant IL-4 but not IL-13. Kinetic analysis revealed a kcat/KM value of 8.1 muM-1min-1 for TRX-mediated recognition of IL-4, which established this cytokine as the most selective partner of extracellular TRX to date. Mass spectrometry identified the C46-C99 bond of IL-4 as the target of TRX, consistent with the essential role of this disulfide bond in IL-4 activity. To demonstrate the physiological relevance of our biochemical findings, recombinant TRX was shown to attenuate IL-4-dependent proliferation of cultured TF-1 erythroleukemia cells and also to inhibit the progression of chronic pancreatitis in an IL-4-driven mouse model of this disease. By establishing that IL-4 is posttranslationally regulated by TRX-promoted reduction of a disulfide bond, our findings highlight a novel regulatory mechanism of the type 2 immune response that is specific to IL-4 over IL-13.
View details for PubMedID 30104382
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Cystamine and Disulfiram Inhibit Human Transglutaminase 2 via an Oxidative Mechanism
BIOCHEMISTRY
2018; 57 (24): 3359–63
Abstract
The catalytic activity of transglutaminase 2 (TG2), a ubiquitously expressed mammalian enzyme, is regulated by multiple post-translational mechanisms. Because elevated activity of TG2 in the extracellular matrix is associated with organ-specific diseases such as celiac disease and renal fibrosis, there is growing therapeutic interest in inhibitors of this enzyme. Cystamine, a symmetric disulfide compound, is one of the earliest reported TG2 inhibitors. Despite its widespread use as a tool compound to block TG2 activity in vitro and in vivo, its mechanism of action has remained unclear. Here, we demonstrate that cystamine irreversibly inhibits human TG2 ( kinh/ Ki = 1.2 mM-1 min-1) via a mechanism fundamentally distinct from those proposed previously. Through mass spectrometric disulfide mapping and site-directed mutagenesis, we show that cystamine promotes the formation of a physiologically relevant disulfide bond between Cys370 and Cys371 that allosterically abrogates the catalytic activity of human TG2. This discovery led us to evaluate clinically useful thiol → disulfide oxidants for TG2 inhibitory activity. It is demonstrated that disulfiram, a relatively safe oral thiuram disulfide, is a fairly potent TG2 inhibitor ( kinh/ Ki = 8.3 mM-1 min-1) and may therefore provide a practical tool for clinically validating this emerging therapeutic target in intestinal disorders such as celiac disease.
View details for DOI 10.1021/acs.biochem.8b00204
View details for Web of Science ID 000436026000006
View details for PubMedID 29570977
View details for PubMedCentralID PMC6008213
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Structure-Function Analysis of the Extended Conformation of a Polyketide Synthase Module
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2018; 140 (21): 6518–21
Abstract
Catalytic modules of assembly-line polyketide synthases (PKSs) have previously been observed in two very different conformations-an "extended" architecture and an "arch-shaped" architecture-although the catalytic relevance of neither has been directly established. By the use of a fully human naïve antigen-binding fragment (Fab) library, a high-affinity antibody was identified that bound to the extended conformation of a PKS module, as verified by X-ray crystallography and tandem size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS). Kinetic analysis proved that this antibody-stabilized module conformation was fully competent for catalysis of intermodular polyketide chain translocation as well as intramodular polyketide chain elongation and functional group modification of a growing polyketide chain. Thus, the extended conformation of a PKS module is fully competent for all of its essential catalytic functions.
View details for PubMedID 29762030
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HEx: A heterologous expression platform for the discovery of fungal natural products
SCIENCE ADVANCES
2018; 4 (4): eaar5459
Abstract
For decades, fungi have been a source of U.S. Food and Drug Administration-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.
View details for PubMedID 29651464
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Endoplasmic reticulum-resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2
JOURNAL OF BIOLOGICAL CHEMISTRY
2018; 293 (8): 2640–49
Abstract
Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX).
View details for PubMedID 29305423
View details for PubMedCentralID PMC5827427
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Transglutaminase 2 in pulmonary and cardiac tissue remodeling in experimental pulmonary hypertension
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY
2017; 313 (5): L752–L762
Abstract
Tissue matrix remodeling and fibrosis leading to loss of pulmonary arterial and right ventricular compliance are important features of both experimental and clinical pulmonary hypertension (PH). We have previously reported that transglutaminase 2 (TG2) is involved in PH development while others have shown it to be a cross-linking enzyme that participates in remodeling of extracellular matrix in fibrotic diseases in general. In the present studies, we used a mouse model of experimental PH (Sugen 5416 and hypoxia; SuHypoxia) and cultured primary human cardiac and pulmonary artery adventitial fibroblasts to evaluate the relationship of TG2 to the processes of fibrosis, protein cross-linking, extracellular matrix collagen accumulation, and fibroblast-to-myofibroblast transformation. We report here that TG2 expression and activity as measured by serotonylated fibronectin and protein cross-linking activity along with fibrogenic markers are significantly elevated in lungs and right ventricles of SuHypoxic mice with PH. Similarly, TG2 expression and activity, protein cross-linking activity, and fibrogenic markers are significantly increased in cultured cardiac and pulmonary artery adventitial fibroblasts in response to hypoxia exposure. Pharmacological inhibition of TG2 activity with ERW1041E significantly reduced hypoxia-induced cross-linking activity and synthesis of collagen 1 and α-smooth muscle actin in both the in vivo and in vitro studies. TG2 short interfering RNA had a similar effect in vitro. Our results suggest that TG2 plays an important role in hypoxia-induced pulmonary and right ventricular tissue matrix remodeling in the development of PH.
View details for PubMedID 28775095
View details for PubMedCentralID PMC5792178
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Biosynthesis and structure-activity relationships of the lipid a family of glycolipids
CURRENT OPINION IN CHEMICAL BIOLOGY
2017; 40: 127–37
Abstract
Lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram-negative bacteria, is a potent elicitor of innate immune responses in mammals. A typical LPS molecule is composed of three different structural domains: a polysaccharide called the O-antigen, a core oligosaccharide, and Lipid A. Lipid A is the amphipathic glycolipid moiety of LPS. It stimulates the immune system by tightly binding to Toll-like receptor 4. More recently, Lipid A has also been shown to activate intracellular caspase-4 and caspase-5. An impressive diversity is observed in Lipid A structures from different Gram-negative bacteria, and it is well established that subtle changes in chemical structure can result in dramatically different immune activities. For example, Lipid A from Escherichia coli is highly toxic to humans, whereas a biosynthetic precursor called Lipid IVA blocks this toxic activity, and monophosphoryl Lipid A from Salmonella minnesota is a vaccine adjuvant. Thus, an understanding of structure-activity relationships in this glycolipid family could be used to design useful immunomodulatory agents. Here we review the biosynthesis, modification, and structure-activity relationships of Lipid A.
View details for PubMedID 28942130
View details for PubMedCentralID PMC5696077
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The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography
BIOCHEMISTRY
2017; 56 (36): 4751–56
Abstract
The crystal structure of the trans-acyltransferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for the direct delivery of the sample into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes for polyketide synthase reaction dynamics are discussed.
View details for DOI 10.1021/acs.biochem.7b00711
View details for Web of Science ID 000410867600005
View details for PubMedID 28832129
View details for PubMedCentralID PMC5721673
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Latiglutenase Improves Symptoms in Seropositive Celiac Disease Patients While on a Gluten-Free Diet
DIGESTIVE DISEASES AND SCIENCES
2017; 62 (9): 2428–32
Abstract
Celiac disease (CD) is a widespread condition triggered by dietary gluten and treated with a lifelong gluten-free diet (GFD); however, inadvertent exposure to gluten can result in episodic symptoms. A previous trial of latiglutenase (clinicaltrials.gov; NCT01917630), an orally administered mixture of two recombinant gluten-specific proteases, was undertaken in symptomatic subjects with persistent injury. The primary endpoint for histologic improvement was not met, presumably due to a trial effect. In this post hoc analysis, we investigated the efficacy of latiglutenase for reducing symptoms in subgroups of the study participants based on their seropositivity.The study involved symptomatic CD patients following a GFD for at least one year prior to randomization. Patients were treated for 12 weeks with latiglutenase or placebo. Of 398 completed patients, 173 (43%) were seropositive at baseline. Symptoms were recorded daily, and weekly symptom scores were compiled. p values were calculated by analysis of covariance.A statistically significant, dose-dependent reduction was detected in the severity and frequency of symptoms in seropositive but not seronegative patients. The severity of abdominal pain and bloating was reduced by 58 and 44%, respectively, in the cohort receiving the highest latiglutenase dose (900 mg, n = 14) relative to placebo (n = 54). Symptom improvement increased from week 6 to week 12. There was also a trend toward greater symptom improvement with greater baseline symptom severity.Seropositive CD patients show symptomatic improvement from latiglutenase taken with meals and would benefit from the availability of this treatment.
View details for PubMedID 28755266
View details for PubMedCentralID PMC5709215
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A B-Cell Gene Signature Correlates With the Extent of Gluten-Induced Intestinal Injury in Celiac Disease.
Cellular and molecular gastroenterology and hepatology
2017; 4 (1): 1-17
Abstract
Celiac disease (CeD) provides an opportunity to study autoimmunity and the transition in immune cells as dietary gluten induces small intestinal lesions.Seventy-three celiac disease patients on a long-term, gluten-free diet ingested a known amount of gluten daily for 6 weeks. A peripheral blood sample and intestinal biopsy specimens were taken before and 6 weeks after initiating the gluten challenge. Biopsy results were reported on a continuous numeric scale that measured the villus-height-to-crypt-depth ratio to quantify gluten-induced intestinal injury. Pooled B and T cells were isolated from whole blood, and RNA was analyzed by DNA microarray looking for changes in peripheral B- and T-cell gene expression that correlated with changes in villus height to crypt depth, as patients maintained a relatively healthy intestinal mucosa or deteriorated in the face of a gluten challenge.Gluten-dependent intestinal damage from baseline to 6 weeks varied widely across all patients, ranging from no change to extensive damage. Genes differentially expressed in B cells correlated strongly with the extent of intestinal damage. A relative increase in B-cell gene expression correlated with a lack of sensitivity to gluten whereas their relative decrease correlated with gluten-induced mucosal injury. A core B-cell gene module, representing a subset of B-cell genes analyzed, accounted for the correlation with intestinal injury.Genes comprising the core B-cell module showed a net increase in expression from baseline to 6 weeks in patients with little to no intestinal damage, suggesting that these individuals may have mounted a B-cell immune response to maintain mucosal homeostasis and circumvent inflammation. DNA microarray data were deposited at the GEO repository (accession number: GSE87629; available: https://www.ncbi.nlm.nih.gov/geo/).
View details for DOI 10.1016/j.jcmgh.2017.01.011
View details for PubMedID 28508029
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C-Thiourea.
ACS chemical biology
2017
Abstract
Reactive oxygen species (ROS) are essential cellular metabolites widely implicated in many diseases including cancer, inflammation, and cardiovascular and neurodegenerative disorders. Yet, ROS signaling remains poorly understood, and their measurements are a challenge due to high reactivity and instability. Here, we report the development of (13)C-thiourea as a probe to detect and measure H2O2 dynamics with high sensitivity and spatiotemporal resolution using hyperpolarized (13)C magnetic resonance spectroscopic imaging. In particular, we show (13)C-thiourea to be highly polarizable and to possess a long spin-lattice relaxation time (T1), which enables real-time monitoring of ROS-mediated transformation. We also demonstrate that (13)C-thiourea reacts readily with H2O2 to give chemically distinguishable products in vitro and validate their detection in vivo in a mouse liver. This study suggests that (13)C-thiourea is a promising agent for noninvasive detection of H2O2 in vivo. More broadly, our findings outline a viable clinical application for H2O2 detection in patients with a range of diseases.
View details for DOI 10.1021/acschembio.7b00130
View details for PubMedID 28452454
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Elucidation of the Stereospecificity of C-Methyltransferases from trans-AT Polyketide Synthases
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2017; 139 (17): 6102-6105
Abstract
S-Adenosyl methionine (SAM)-dependent C-methyltransferases are responsible for the C2-methylation of 3-ketoacyl-acyl carrier protein (ACP) intermediates to give the corresponding 2-methy-3-ketoacyl-ACP products during bacterial polyketide biosynthesis mediated by trans-AT polyketide synthases that lack integrated acyl transferase (AT) domains. A coupled ketoreductase (KR) assay was used to assign the stereochemistry of the C-methyltransferase-catalyzed reaction. Samples of chemoenzymatically generated 3-ketopentanoyl-ACP (9) were incubated with SAM and BonMT2 from module 2 of the bongkrekic acid polyketide synthase. The resulting 2-methyl-3-ketopentanoyl-ACP (10) was incubated separately with five (2R)- or (2S)-methyl specific KR domains. Analysis of the derived 2-methyl-3-hydroxypentanoate methyl esters (8) by chiral GC-MS established that the BonMT2-catalyzed methylation generated exclusively (2R)-2-methyl-3-ketopentanoyl-ACP ((2R)-10). Identical results were also obtained with three additional C-methyltransferases-BaeMT9, DifMT1, and MupMT1-from the bacillaene, difficidin, and mupirocin trans-AT polyketide synthases.
View details for DOI 10.1021/jacs.7b02911
View details for Web of Science ID 000400802300021
View details for PubMedID 28430424
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Human pyrimidine nucleotide biosynthesis as a target for antiviral chemotherapy.
Current opinion in biotechnology
2017; 48: 127-134
Abstract
The development of broad-spectrum, host-acting antiviral therapies remains an important but elusive goal in anti-infective drug discovery. To replicate efficiently, viruses not only depend on their hosts for an adequate supply of pyrimidine nucleotides, but also up-regulate pyrimidine nucleotide biosynthesis in infected cells. In this review, we outline our understanding of mammalian de novo and salvage metabolic pathways for pyrimidine nucleotide biosynthesis. The available spectrum of experimental and FDA-approved drugs that modulate individual steps in these metabolic pathways is also summarized. The logic of a host-acting combination antiviral therapy comprised of inhibitors of dihydroorotate dehydrogenase and uridine/cytidine kinase is discussed.
View details for DOI 10.1016/j.copbio.2017.03.010
View details for PubMedID 28458037
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Heterologous expression of diverse propionyl-CoA carboxylases affects polyketide production in Escherichia coli.
journal of antibiotics
2017
View details for DOI 10.1038/ja.2017.38
View details for PubMedID 28400575
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Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease
SCIENCE
2017; 356 (6333): 44-?
Abstract
Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.
View details for DOI 10.1126/science.aah5298
View details for PubMedID 28386004
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Exploring vectorial chain translocation in assembly line polyketide synthases
AMER CHEMICAL SOC. 2017
View details for Web of Science ID 000430568501116
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Combinatorial enzymatic synthesis of lipid A analogs
AMER CHEMICAL SOC. 2017
View details for Web of Science ID 000430568501390
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Cholestyramine as a promising, strong anion exchange resin for direct capture of genetic biomarkers from raw pancreatic fluids
BIOTECHNOLOGY AND BIOENGINEERING
2017; 114 (4): 934-938
Abstract
The ability to capture cell-free DNA from the gastrointestinal tract, in a minimally invasive manner, could enhance our ability to diagnose gastrointestinal disease, or gain a better understanding of the spatial mapping of the intestinal microbiota. We, therefore, sought to identify a class of capture agents that could directly and efficiently sequester genetic material from intestinal fluids. As a particular case study, we examined the ability to capture DNA from pancreatic secretions, for potential application in enabling the sequestration of early, genetic biomarkers of pancreatic disease. We hypothesized that the cholestyramine series of strong cation exchange resins, which are FDA approved for the treatment of high cholesterol, may be capable of capturing DNA from pancreatic secretions. We identified a particular cholestyramine resin, DOWEX 1 × 2 100-200 mesh, which is able to efficiently capture and purify DNA from pancreatic fluid. Using only 200 μL of pancreatic secretions, we are able to recover 247 ± 182 ng of amplifiable human DNA, giving an estimated pancreatic fluid DNA content of 1.23 ± 0.91 ng/μL. To our knowledge, this is the first demonstration of a material that can effectively capture and purify DNA directly from untreated pancreatic fluids. Thus, our approach could hold high utility for the in vivo capture of DNA and disease biomarkers if incorporated into an appropriate sampling device. Biotechnol. Bioeng. 2016;9999: 1-5. © 2016 Wiley Periodicals, Inc.
View details for DOI 10.1002/bit.26207
View details for Web of Science ID 000395650600022
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Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis.
Journal of the American Chemical Society
2017; 139 (8): 3283-3292
Abstract
The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR(0)) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR(0) domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP(+) in the presence of redox-inactive, recombinant NanKR1(0) or NanKR5(0), from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR7(0) from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR(0)-catalyzed isotope exchange of the reversibly generated, transiently formed oxidation product [2-(2)H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR(0) domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR(0) domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.
View details for DOI 10.1021/jacs.7b00278
View details for PubMedID 28157306
View details for PubMedCentralID PMC5332327
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Thioredoxin-1 Selectively Activates Transglutaminase 2 in the Extracellular Matrix of the Small Intestine: IMPLICATIONS FOR CELIAC DISEASE.
journal of biological chemistry
2017; 292 (5): 2000-2008
Abstract
Transglutaminase 2 (TG2) catalyzes transamidation or deamidation of its substrates and is ordinarily maintained in a catalytically inactive state in the intestine and other organs. Aberrant TG2 activity is thought to play a role in celiac disease, suggesting that a better understanding of TG2 regulation could help to elucidate the mechanistic basis of this malady. Structural and biochemical analysis has led to the hypothesis that extracellular TG2 activation involves reduction of an allosteric disulfide bond by thioredoxin-1 (TRX), but cellular and in vivo evidence for this proposal is lacking. To test the physiological relevance of this hypothesis, we first showed that macrophages exposed to pro-inflammatory stimuli released TRX in sufficient quantities to activate their extracellular pools of TG2. By using the C35S mutant of TRX, which formed a metastable mixed disulfide bond with TG2, we demonstrated that these proteins specifically recognized each other in the extracellular matrix of fibroblasts. When injected into mice and visualized with antibodies, we observed the C35S TRX mutant bound to endogenous TG2 as its principal protein partner in the small intestine. Control experiments showed no labeling of TG2 knock-out mice. Intravenous administration of recombinant TRX in wild-type mice, but not TG2 knock-out mice, led to a rapid rise in intestinal transglutaminase activity in a manner that could be inhibited by small molecules targeting TG2 or TRX. Our findings support the potential pathophysiological relevance of TRX in celiac disease and establish the Cys(370)-Cys(371) disulfide bond of TG2 as one of clearest examples of an allosteric disulfide bond in mammals.
View details for DOI 10.1074/jbc.M116.767988
View details for PubMedID 28003361
View details for PubMedCentralID PMC5290969
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Elucidation of the Cryptic Methyl Group Epimerase Activity of Dehydratase Domains from Modular Polyketide Synthases Using a Tandem Modules Epimerase Assay.
Journal of the American Chemical Society
2017; 139 (28): 9507–10
Abstract
Dehydratase (DH) domains of cryptic function are often found in polyketide synthase (PKS) modules that produce epimerized (2S)-2-methyl-3-ketoacyl-ACP (acyl carrier protein) intermediates. A combination of tandem equilibrium isotope exchange (EIX) and a newly developed Tandem Modules Epimerase assay revealed the intrinsic epimerase activity of NanDH1 and NanDH5, from modules 1 and 5, respectively, of the nanchangmycin (1) PKS as well of NigDH1, from module 1 of the nigericin (3) PKS. Unexpectedly, all three epimerase-active DH domains were also found to possess intrinsic dehydratase activity, whereas the conventional DH domains, EryDH4, from module 4 of the erythromycin synthase, and NanDH2 from module 2 of the nanchangmycin synthase, were shown to have cryptic epimerase activity.
View details for PubMedID 28682630
View details for PubMedCentralID PMC5546871
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Intracellular TG2 Activity Increases Microtubule Stability but is not Sufficient to Prompt Neurite Growth.
Neuroscience bulletin
2017; 33 (1): 103–6
View details for PubMedID 27815680
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Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars.
Cell
2017; 171 (2): 427–39.e21
Abstract
Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.
View details for PubMedID 28985565
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Celiac Disease: Lessons for and from Chemical Biology.
ACS chemical biology
2017; 12 (6): 1455–59
Abstract
Celiac disease is a lifelong immune disorder of the small intestine where inflammation is triggered by dietary gluten. There is an urgent need for the development of nondietary therapies for this widespread but overlooked disease. More fundamentally, a molecular understanding of gluten-induced pathogenesis in celiac disease has the potential to provide new insights into mucosal immunology. Over the past two decades, three pathogenically critical molecules-gluten, TG2, and HLA-DQ2-have served as focal points for collaborative efforts between biologists, chemists, engineers, and clinicians with an interest in celiac disease. This perspective summarizes a few examples of such multidisciplinary research directions with an emphasis on groundbreaking clinical studies that have profoundly informed the trajectory of subsequent molecular investigations. Examples of future challenges in fundamental and translational celiac disease research are also discussed.
View details for PubMedID 28165712
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Cholestyramine as a promising, strong anion exchange resin for direct capture of genetic biomarkers from raw pancreatic fluids.
Biotechnology and bioengineering
2016
Abstract
The ability to capture cell-free DNA from the gastrointestinal tract, in a minimally invasive manner, could enhance our ability to diagnose gastrointestinal disease, or gain a better understanding of the spatial mapping of the intestinal microbiota. We, therefore, sought to identify a class of capture agents that could directly and efficiently sequester genetic material from intestinal fluids. As a particular case study, we examined the ability to capture DNA from pancreatic secretions, for potential application in enabling the sequestration of early, genetic biomarkers of pancreatic disease. We hypothesized that the cholestyramine series of strong cation exchange resins, which are FDA approved for the treatment of high cholesterol, may be capable of capturing DNA from pancreatic secretions. We identified a particular cholestyramine resin, DOWEX 1 × 2 100-200 mesh, which is able to efficiently capture and purify DNA from pancreatic fluid. Using only 200 μL of pancreatic secretions, we are able to recover 247 ± 182 ng of amplifiable human DNA, giving an estimated pancreatic fluid DNA content of 1.23 ± 0.91 ng/μL. To our knowledge, this is the first demonstration of a material that can effectively capture and purify DNA directly from untreated pancreatic fluids. Thus, our approach could hold high utility for the in vivo capture of DNA and disease biomarkers if incorporated into an appropriate sampling device. Biotechnol. Bioeng. 2016;9999: 1-5. © 2016 Wiley Periodicals, Inc.
View details for DOI 10.1002/bit.26207
View details for PubMedID 27800600
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Partial In Vitro Reconstitution of an Orphan Polyketide Synthase Associated with Clinical Cases of Nocardiosis.
ACS chemical biology
2016; 11 (9): 2636-2641
Abstract
Although a few well-characterized polyketide synthases (PKSs) have been functionally reconstituted in vitro from purified protein components, the use of this strategy to decode "orphan" assembly line PKSs has not been described. To begin investigating a PKS found only in Nocardia strains associated with clinical cases of nocardiosis, we reconstituted in vitro its five terminal catalytic modules. In the presence of octanoyl-CoA, malonyl-CoA, NADPH, and S-adenosyl methionine, this pentamodular PKS system yielded unprecedented octaketide and heptaketide products whose structures were partially elucidated using mass spectrometry and NMR spectroscopy. The PKS has several notable features, including a "split, stuttering" module and a terminal reductive release mechanism. Our findings pave the way for further analysis of this unusual biosynthetic gene cluster whose natural product may enhance the infectivity of its producer strains in human hosts.
View details for DOI 10.1021/acschembio.6b00489
View details for PubMedID 27384917
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Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase.
Biochemistry
2016; 55 (32): 4476-4484
Abstract
Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C-C bond formation. S315 played a role in coupling decarboxylation to C-C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs.
View details for DOI 10.1021/acs.biochem.6b00639
View details for PubMedID 27441852
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Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases.
journal of biological chemistry
2016; 291 (31): 16404-16415
Abstract
The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.
View details for DOI 10.1074/jbc.M116.730531
View details for PubMedID 27246853
View details for PubMedCentralID PMC4965586
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Recognition of acyl carrier proteins by ketoreductases in assembly line polyketide synthases.
journal of antibiotics
2016; 69 (7): 507-510
Abstract
Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed an ~10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed an ~10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules.
View details for DOI 10.1038/ja.2016.41
View details for PubMedID 27118242
View details for PubMedCentralID PMC4963262
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Structure and mechanism of assembly line polyketide synthases.
Current opinion in structural biology
2016; 41: 10-18
Abstract
Assembly line polyketide synthases (PKSs) are remarkable biosynthetic machines with considerable potential for structure-based engineering. Several types of protein-protein interactions, both within and between PKS modules, play important roles in the catalytic cycle of a multimodular PKS. Additionally, vectorial biosynthesis is enabled by the energetic coupling of polyketide chain elongation to the channeling of intermediates between successive modules. A combination of high-resolution analysis of smaller PKS components and lower resolution characterization of intact modules and bimodules has yielded insights into the structure and organization of a prototypical assembly line PKS. This review discusses our understanding of key structure-function relationships in this family of megasynthases, along with a recap of key unanswered questions in the field.
View details for DOI 10.1016/j.sbi.2016.05.009
View details for PubMedID 27266330
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Editorial overview: Next-generation therapeutics: Breaking new ground and making a difference for patients
CURRENT OPINION IN CHEMICAL BIOLOGY
2016; 32: 58-59
View details for DOI 10.1016/j.cbpa.2016.04.017
View details for Web of Science ID 000379105200007
View details for PubMedID 27206139
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Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification
NATURE CHEMICAL BIOLOGY
2016; 12 (5): 361-?
Abstract
Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.
View details for DOI 10.1038/NCHEMBIO.2050
View details for PubMedID 27018887
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Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues.
Biochemistry
2016; 55 (8): 1179-1186
Abstract
The role of the conserved active site tyrosine and serine residues in epimerization catalyzed by polyketide synthase ketoreductase (PKS KR) domains has been investigated. Both mutant and wild-type forms of epimerase-active KR domains, including the intrinsically redox-inactive EryKR3° and PicKR3° as well as redox-inactive mutants of EryKR1, were incubated with [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-2) and 0.05 equiv of NADP(+) in the presence of the redox-active, epimerase-inactive EryKR6 domain. The residual epimerase activity of each mutant was determined by tandem equilibrium isotope exchange, in which the first-order, time-dependent washout of isotope from 2 was monitored by liquid chromatography-tandem mass spectrometry with quantitation of the deuterium content of the diagnostic pantetheinate ejection fragment (4). Replacement of the active site Tyr or Ser residues, alone or together, significantly reduced the observed epimerase activity of each KR domain with minimal effect on substrate binding. Our results demonstrate that the epimerase and reductase activities of PKS KR domains share a common active site, with both reactions utilizing the same pair of Tyr and Ser residues.
View details for DOI 10.1021/acs.biochem.6b00024
View details for PubMedID 26863427
View details for PubMedCentralID PMC4775309
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A Turnstile Mechanism for the Controlled Growth of Biosynthetic Intermediates on Assembly Line Polyketide Synthases.
ACS central science
2016; 2 (1): 14-20
Abstract
Vectorial polyketide biosynthesis on an assembly line polyketide synthase is the most distinctive property of this family of biological machines, while providing the key conceptual tool for the bioinformatic decoding of new antibiotic pathways. We now show that the action of the entire assembly line is synchronized by a previously unrecognized turnstile mechanism that prevents the ketosynthase domain of each module from being acylated by a new polyketide chain until the product of the prior catalytic cycle has been passed to the downstream module from the corresponding acyl carrier protein domain. The turnstile is closed by virtue of tight coupling to the signature decarboxylative condensation reaction catalyzed by the ketosynthase domain of each polyketide synthase module. Reopening of the turnstile is coupled to the eventual chain translocation step that vacates the module. At the maximal rate of substrate turnover, one would expect the chain release step to initiate a cascade of chain translocation events that sequentially migrate back upstream, thereby repriming each module and setting up the assembly line for the next round of polyketide chain elongation.
View details for PubMedID 26878060
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Thiol-Disulfide Exchange Reactions in the Mammalian Extracellular Environment
ANNUAL REVIEW OF CHEMICAL AND BIOMOLECULAR ENGINEERING, VOL 7
2016; 7: 197-222
Abstract
Disulfide bonds represent versatile posttranslational modifications whose roles encompass the structure, catalysis, and regulation of protein function. Due to the oxidizing nature of the extracellular environment, disulfide bonds found in secreted proteins were once believed to be inert. This notion has been challenged by the discovery of redox-sensitive disulfides that, once cleaved, can lead to changes in protein activity. These functional disulfides are twisted into unique configurations, leading to high strain and potential energy. In some cases, cleavage of these disulfides can lead to a gain of function in protein activity. Thus, these motifs can be referred to as switches. We describe the couples that control redox in the extracellular environment, examine several examples of proteins with switchable disulfides, and discuss the potential applications of disulfides in molecular biology.
View details for DOI 10.1146/annurev-chembioeng-080615-033553
View details for Web of Science ID 000379322600009
View details for PubMedID 27023663
View details for PubMedCentralID PMC4899241
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Gluten Introduction, Breastfeeding, and Celiac Disease: Back to the Drawing Board.
American journal of gastroenterology
2016; 111 (1): 12-14
Abstract
This commentary by the leadership of the North American Society for the Study of Celiac Disease (NASSCD) concerns recent research findings regarding infant feeding practices. Celiac disease has increased markedly in recent decades, and seroprevalence studies indicate that this is a true rise, rather than one due to increased awareness and testing. Prior studies have suggested that infant feeding practices and timing of initial gluten exposure are central to the development of celiac disease. Two recent multicenter randomized trials tested strategies of early or delayed gluten introduction in infants, and neither strategy appeared to influence celiac disease risk. These studies also found that breastfeeding did not protect against the development of celiac disease. While disappointing, these results should spur the study of wider environmental risk factors beyond infant feeding, such as intrauterine and perinatal exposures as well as environmental influences later in life, including drug exposure, microbial infections, and the microbiome. Given that celiac disease can develop at any age, it is imperative to study these proposed triggers so as to elucidate the loss of tolerance to gluten and to develop future intervention strategies.
View details for DOI 10.1038/ajg.2015.219
View details for PubMedID 26259710
View details for PubMedCentralID PMC4720595
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An unprecedented dual antagonist and agonist of human Transglutaminase 2.
Bioorganic & medicinal chemistry letters
2015; 25 (21): 4922-4926
Abstract
Transglutaminase 2 (TG2) is a ubiquitously expressed, Ca(2+)-activated extracellular enzyme in mammals that is maintained in a catalytically dormant state by multiple mechanisms. Although its precise physiological role in the extracellular matrix remains unclear, aberrantly up-regulated TG2 activity is a hallmark of several maladies, including celiac disease. Previously, we reported the discovery of a class of acylideneoxoindoles as potent, reversible inhibitors of human TG2. Detailed analysis of one of those inhibitors (CK-IV-55) led to an unprecedented and striking observation. Whereas this compound was a non-competitive inhibitor (3.3±0.9 μM) of human TG2 at saturating Ca(2+) concentrations, it activated TG2 in the presence of sub-saturating but physiologically relevant Ca(2+) concentrations (0.5-0.7 mM). This finding was validated in a cellular model of TG2 activation and inhibition. Mutant TG2 analysis suggested that CK-IV-55 and its analogs bound to a low-affinity Ca(2+) binding site on the catalytic core of TG2. A mechanistic model for the dual agonistic/antagonistic action of CK-IV-55 on TG2 is presented, and the pathophysiological implications of basal activation of intestinal TG2 by small molecules are discussed.
View details for DOI 10.1016/j.bmcl.2015.05.006
View details for PubMedID 26004580
View details for PubMedCentralID PMC4607565
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In Vitro Reconstitution of Metabolic Pathways: Insights into Nature's Chemical Logic.
Synlett : accounts and rapid communications in synthetic organic chemistry
2015; 26 (8): 1008-1025
Abstract
In vitro analysis of metabolic pathways is becoming a powerful method to gain a deeper understanding of Nature's core biochemical transformations. With astounding advancements in biotechnology, purification of a metabolic pathway's constitutive enzymatic components is becoming a tractable problem, and such in vitro studies allow scientists to capture the finer details of enzymatic reaction mechanisms, kinetics, and the identity of organic product molecules. In this review, we present eleven metabolic pathways that have been the subject of in vitro reconstitution studies in the literature in recent years. In addition, we have selected and analyzed subset of four case studies within these eleven examples that exemplify remarkable organic chemistry occurring within biology. These examples serves as tangible reminders that Nature's biochemical routes obey the fundamental principles of organic chemistry, and the chemical mechanisms are reminiscent of those featured in traditional synthetic organic routes. The illustrations of biosynthetic chemistry depicted in this review may inspire the development of biomimetic chemistries via abiotic chemical techniques.
View details for DOI 10.1055/s-0034-1380264
View details for PubMedID 26207083
View details for PubMedCentralID PMC4507746
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Quo vadis, enzymology?
NATURE CHEMICAL BIOLOGY
2015; 11 (7): 438-441
View details for Web of Science ID 000356334600001
View details for PubMedID 26083060
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Therapeutic approaches for celiac disease
BEST PRACTICE & RESEARCH IN CLINICAL GASTROENTEROLOGY
2015; 29 (3): 503-521
Abstract
Celiac disease is a common, lifelong autoimmune disorder for which dietary control is the only accepted form of therapy. A strict gluten-free diet is burdensome to patients and can be limited in efficacy, indicating there is an unmet need for novel therapeutic approaches to supplement or supplant dietary therapy. Many molecular events required for disease pathogenesis have been recently characterized and inspire most current and emerging drug-discovery efforts. Genome-wide association studies (GWAS) confirm the importance of human leukocyte antigen genes in our pathogenic model and identify a number of new risk loci in this complex disease. Here, we review the status of both emerging and potential therapeutic strategies in the context of disease pathophysiology. We conclude with a discussion of how genes identified during GWAS and follow-up studies that enhance susceptibility may offer insight into developing novel therapies.
View details for DOI 10.1016/j.bpg.2015.04.005
View details for Web of Science ID 000356748500013
View details for PubMedCentralID PMC4465084
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Therapeutic approaches for celiac disease.
Best practice & research. Clinical gastroenterology
2015; 29 (3): 503-521
Abstract
Celiac disease is a common, lifelong autoimmune disorder for which dietary control is the only accepted form of therapy. A strict gluten-free diet is burdensome to patients and can be limited in efficacy, indicating there is an unmet need for novel therapeutic approaches to supplement or supplant dietary therapy. Many molecular events required for disease pathogenesis have been recently characterized and inspire most current and emerging drug-discovery efforts. Genome-wide association studies (GWAS) confirm the importance of human leukocyte antigen genes in our pathogenic model and identify a number of new risk loci in this complex disease. Here, we review the status of both emerging and potential therapeutic strategies in the context of disease pathophysiology. We conclude with a discussion of how genes identified during GWAS and follow-up studies that enhance susceptibility may offer insight into developing novel therapies.
View details for DOI 10.1016/j.bpg.2015.04.005
View details for PubMedID 26060114
View details for PubMedCentralID PMC4465084
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In Vitro Reconstitution of Metabolic Pathways: Insights into Nature's Chemical Logic
SYNLETT
2015; 26 (8): 1008-1025
Abstract
In vitro analysis of metabolic pathways is becoming a powerful method to gain a deeper understanding of Nature's core biochemical transformations. With astounding advancements in biotechnology, purification of a metabolic pathway's constitutive enzymatic components is becoming a tractable problem, and such in vitro studies allow scientists to capture the finer details of enzymatic reaction mechanisms, kinetics, and the identity of organic product molecules. In this review, we present eleven metabolic pathways that have been the subject of in vitro reconstitution studies in the literature in recent years. In addition, we have selected and analyzed subset of four case studies within these eleven examples that exemplify remarkable organic chemistry occurring within biology. These examples serves as tangible reminders that Nature's biochemical routes obey the fundamental principles of organic chemistry, and the chemical mechanisms are reminiscent of those featured in traditional synthetic organic routes. The illustrations of biosynthetic chemistry depicted in this review may inspire the development of biomimetic chemistries via abiotic chemical techniques.
View details for DOI 10.1055/s-0034-1380264
View details for Web of Science ID 000353918600003
View details for PubMedCentralID PMC4507746
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Discovery of Potent and Specific Dihydroisoxazole Inhibitors of Human Transglutaminase 2
JOURNAL OF MEDICINAL CHEMISTRY
2014; 57 (21): 9042-9064
Abstract
Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that catalyzes the posttranslational modification of glutamine residues on protein or peptide substrates. A growing body of literature has implicated aberrantly regulated activity of TG2 in the pathogenesis of various human inflammatory, fibrotic, and other diseases. Taken together with the fact that TG2 knockout mice are developmentally and reproductively normal, there is growing interest in the potential use of TG2 inhibitors in the treatment of these conditions. Targeted-covalent inhibitors based on the weakly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) scaffold have been widely used to study TG2 biology and are well tolerated in vivo, but these compounds have only modest potency, and their selectivity toward other transglutaminase homologues is largely unknown. In the present work, we first profiled the selectivity of existing inhibitors against the most pertinent TG isoforms (TG1, TG3, and FXIIIa). Significant cross-reactivity of these small molecules with TG1 was observed. Structure-activity and -selectivity analyses led to the identification of modifications that improved potency and isoform selectivity. Preliminary pharmacokinetic analysis of the most promising analogues was also undertaken. Our new data provides a clear basis for the rational selection of dihydroisoxazole inhibitors as tools for in vivo biological investigation.
View details for DOI 10.1021/jm501145a
View details for Web of Science ID 000344977400026
View details for PubMedCentralID PMC4234452
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Discovery of potent and specific dihydroisoxazole inhibitors of human transglutaminase 2.
Journal of medicinal chemistry
2014; 57 (21): 9042-9064
Abstract
Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that catalyzes the posttranslational modification of glutamine residues on protein or peptide substrates. A growing body of literature has implicated aberrantly regulated activity of TG2 in the pathogenesis of various human inflammatory, fibrotic, and other diseases. Taken together with the fact that TG2 knockout mice are developmentally and reproductively normal, there is growing interest in the potential use of TG2 inhibitors in the treatment of these conditions. Targeted-covalent inhibitors based on the weakly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) scaffold have been widely used to study TG2 biology and are well tolerated in vivo, but these compounds have only modest potency, and their selectivity toward other transglutaminase homologues is largely unknown. In the present work, we first profiled the selectivity of existing inhibitors against the most pertinent TG isoforms (TG1, TG3, and FXIIIa). Significant cross-reactivity of these small molecules with TG1 was observed. Structure-activity and -selectivity analyses led to the identification of modifications that improved potency and isoform selectivity. Preliminary pharmacokinetic analysis of the most promising analogues was also undertaken. Our new data provides a clear basis for the rational selection of dihydroisoxazole inhibitors as tools for in vivo biological investigation.
View details for DOI 10.1021/jm501145a
View details for PubMedID 25333388
View details for PubMedCentralID PMC4234452
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Role of hypoxia-induced transglutaminase 2 in pulmonary artery smooth muscle cell proliferation
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY
2014; 307 (7): L576-L585
Abstract
We previously reported that transglutaminase 2 (TG2) activity is markedly elevated in lungs of hypoxia-exposed rodent models of pulmonary hypertension (PH). Since vascular remodeling of pulmonary artery smooth muscle cells (PASMCs) is important in PH, we undertook the present study to determine whether TG2 activity is altered in PASMCs with exposure to hypoxia and whether that alteration participates in their proliferative response to hypoxia. Cultured distal bovine (b) and proximal human (h) PASMCs were exposed to hypoxia (3% O2) or normoxia (21% O2). mRNA and protein expression were determined by PCR and Western blot analyses. TG2 activity and function were visualized and determined by fluorescent labeled 5-pentylamine biotin incorporation and immunoblotting of serotonylated fibronectin. Cell proliferation was assessed by [(3)H]thymidine incorporation assay. At 24 h, both TG2 expression and activity were stimulated by hypoxia in bPASMCs. Activation of TG2 by hypoxia was blocked by inhibition of the extracellular calcium-sensing receptor or the transient receptor potential channel V4. In contrast, TG2 expression was blocked by inhibition of the transcription factor hypoxia-inducible factor-1α, supporting the presence of separate mechanisms for stimulation of activity and expression of TG2. Pulmonary arterial hypertension patient-derived hPASMCs were found to proliferate significantly more rapidly and respond to hypoxia more strongly than control-derived hPASMCs. Similar to bovine cells, hypoxia-induced proliferation of patient-derived cells was blocked by inhibition of TG2 activity. Our results suggest an important role for TG2, mediated by intracellular calcium fluxes and HIF-1α, in hypoxia-induced PASMC proliferation and possibly in vascular remodeling in PH.
View details for DOI 10.1152/ajplung.00162.2014
View details for Web of Science ID 000343245600008
View details for PubMedCentralID PMC4187037
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Role of hypoxia-induced transglutaminase 2 in pulmonary artery smooth muscle cell proliferation.
American journal of physiology. Lung cellular and molecular physiology
2014; 307 (7): L576-85
Abstract
We previously reported that transglutaminase 2 (TG2) activity is markedly elevated in lungs of hypoxia-exposed rodent models of pulmonary hypertension (PH). Since vascular remodeling of pulmonary artery smooth muscle cells (PASMCs) is important in PH, we undertook the present study to determine whether TG2 activity is altered in PASMCs with exposure to hypoxia and whether that alteration participates in their proliferative response to hypoxia. Cultured distal bovine (b) and proximal human (h) PASMCs were exposed to hypoxia (3% O2) or normoxia (21% O2). mRNA and protein expression were determined by PCR and Western blot analyses. TG2 activity and function were visualized and determined by fluorescent labeled 5-pentylamine biotin incorporation and immunoblotting of serotonylated fibronectin. Cell proliferation was assessed by [(3)H]thymidine incorporation assay. At 24 h, both TG2 expression and activity were stimulated by hypoxia in bPASMCs. Activation of TG2 by hypoxia was blocked by inhibition of the extracellular calcium-sensing receptor or the transient receptor potential channel V4. In contrast, TG2 expression was blocked by inhibition of the transcription factor hypoxia-inducible factor-1α, supporting the presence of separate mechanisms for stimulation of activity and expression of TG2. Pulmonary arterial hypertension patient-derived hPASMCs were found to proliferate significantly more rapidly and respond to hypoxia more strongly than control-derived hPASMCs. Similar to bovine cells, hypoxia-induced proliferation of patient-derived cells was blocked by inhibition of TG2 activity. Our results suggest an important role for TG2, mediated by intracellular calcium fluxes and HIF-1α, in hypoxia-induced PASMC proliferation and possibly in vascular remodeling in PH.
View details for DOI 10.1152/ajplung.00162.2014
View details for PubMedID 25128524
View details for PubMedCentralID PMC4187037
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The Convergence of Chemistry & Human Biology
DAEDALUS
2014; 143 (4): 43-48
View details for DOI 10.1162/DAED_a_00304
View details for Web of Science ID 000342858900005
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What can in vitro reconstitution of complex molecule metabolism teach the synthetic biologist?
AMER CHEMICAL SOC. 2014
View details for Web of Science ID 000349165102057
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Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
2014; 98 (15): 6689-6700
Abstract
Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.
View details for DOI 10.1007/s00253-014-5730-7
View details for Web of Science ID 000339880300015
View details for PubMedCentralID PMC4393947
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Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase.
Applied microbiology and biotechnology
2014; 98 (15): 6689-6700
Abstract
Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.
View details for DOI 10.1007/s00253-014-5730-7
View details for PubMedID 24752841
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Elucidation of the Cryptic Epimerase Activity of Redox-Inactive Ketoreductase Domains from Modular Polyketide Synthases by Tandem Equilibrium Isotope Exchange
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2014; 136 (29): 10190-10193
Abstract
Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such "KR(0)" domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-3b) with the EryKR3(0) domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP(+) resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR3(0)-catalyzed epimerization of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR3(0) from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR1(0) mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR(0) domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains.
View details for DOI 10.1021/ja5056998
View details for Web of Science ID 000339471900003
View details for PubMedID 25004372
View details for PubMedCentralID PMC4111212
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Comparative Analysis of the Substrate Specificity of trans- versus cis-Acyltransferases of Assembly Line Polyketide Synthases
BIOCHEMISTRY
2014; 53 (23): 3796-3806
Abstract
Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (kcat/KM) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT-null DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.
View details for DOI 10.1021/bi5004316
View details for Web of Science ID 000337645600010
View details for PubMedCentralID PMC4067149
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Comparative analysis of the substrate specificity of trans- versus cis-acyltransferases of assembly line polyketide synthases.
Biochemistry
2014; 53 (23): 3796-3806
Abstract
Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (kcat/KM) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT-null DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.
View details for DOI 10.1021/bi5004316
View details for PubMedID 24871074
View details for PubMedCentralID PMC4067149
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Use of transmission electron microscopy to identify nanocrystals of challenging protein targets
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (23): 8470-8475
Abstract
The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as "hits." We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.
View details for DOI 10.1073/pnas.1400240111
View details for Web of Science ID 000336976000051
View details for PubMedID 24872454
View details for PubMedCentralID PMC4060711
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Dihydroisoxazole inhibitors of Anopheles gambiae seminal transglutaminase AgTG3
MALARIA JOURNAL
2014; 13
View details for DOI 10.1186/1475-2875-13-210
View details for Web of Science ID 000339575400001
View details for PubMedID 24888439
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Architectures of Whole-Module and Bimodular Proteins from the 6-Deoxyerythronolide B Synthase
JOURNAL OF MOLECULAR BIOLOGY
2014; 426 (11): 2229-2245
Abstract
The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase produced by the actinomycete Saccharopolyspora erythraea that synthesizes the macrocyclic core of the antibiotic erythromycin 6-deoxyerythronolide B. The megasynthase is a 2-MDa trimeric complex composed of three unique homodimers assembled from the gene products DEBS1, DEBS2, and DEBS3, which are housed within the erythromycin biosynthetic gene cluster. Each homodimer contains two clusters of catalytically independent enzymatic domains, each referred to as a module, which catalyzes one round of polyketide chain extension and modification. Modules are named sequentially to indicate the order in which they are utilized during synthesis of 6-deoxyerythronolide B. We report small-angle X-ray scattering (SAXS) analyses of a whole module and a bimodule from DEBS, as well as a set of domains for which high-resolution structures are available. In all cases, the solution state was probed under previously established conditions ensuring that each protein is catalytically active. SAXS data are consistent with atomic-resolution structures of DEBS fragments. Therefore, we used the available high-resolution structures of DEBS domains to model the architectures of the larger protein assemblies using rigid-body refinement. Our data support a model in which the third module of DEBS forms a disc-shaped structure capable of caging the acyl carrier protein domain proximal to each active site. The molecular envelope of DEBS3 is a thin elongated ellipsoid, and the results of rigid-body modeling suggest that modules 5 and 6 stack collinearly along the 2-fold axis of symmetry.
View details for DOI 10.1016/j.jmb.2014.03.015
View details for Web of Science ID 000336699300007
View details for PubMedID 24704088
View details for PubMedCentralID PMC4284093
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Assembly line polyketide synthases: mechanistic insights and unsolved problems.
Biochemistry
2014; 53 (18): 2875-2883
Abstract
Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75-106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain-domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology.
View details for DOI 10.1021/bi500290t
View details for PubMedID 24779441
View details for PubMedCentralID PMC4020578
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Stanford Institute for Chemical Biology: At the crossroad between molecular engineering and human biology
AMER CHEMICAL SOC. 2014
View details for Web of Science ID 000348455200710
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The initiation ketosynthase (FabH) is the sole rate-limiting enzyme of the fatty acid synthase of Synechococcus sp. PCC 7002.
Metabolic engineering
2014; 22: 53-59
Abstract
Cyanobacteria are Gram-negative bacteria that are desirable hosts for biodiesel production, because they are photosynthetic, relatively fast growing, and can secrete products. We have reconstituted the fatty acid synthase (FAS) of the cyanobacterium Synechococcus sp. PCC 7002 and subjected it to in vitro kinetic analysis. Our data revealed that the overall rate of this metabolic pathway is exclusively limited by the FabH ketosynthase, which initiates product synthesis by condensing malonyl-ACP with acetyl-CoA to form acetoacetyl-ACP. This finding sharply contrasts with our previous findings that the Escherichia coli FAS is predominantly limited by its dehydratase (FabZ) and enoyl reductase (FabI) activities and that FabH activity is not limiting. We therefore reconstituted and analyzed a set of "hybrid" FASs. When the Synechococcus FabH was used to replace its counterpart in the reconstituted E. coli FAS, the resulting synthase was strongly limited by FabH activity. Conversely, replacement of the E. coli FabZ with its Synechococcus homolog dramatically alleviated the dependence of E. coli FAS activity on FabZ. In agreement with this finding, introduction of the E. coli FabH in the Synechococcus FAS virtually eliminated its dependence on this subunit, whereas substitution of the Synechococcus FabZ with its E. coli homolog shifted a substantial fraction of the overall flux control in the Synechococcus FAS to FabZ. Our findings demonstrate that the rate-limiting steps can differ dramatically between closely related bacterial fatty acid synthases, and that such regulatory behavior is fundamentally the property of the controlling enzyme(s).
View details for DOI 10.1016/j.ymben.2013.12.008
View details for PubMedID 24395007
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Rationale for Using Social Media to Collect Patient-Reported Outcomes in Patients with Celiac Disease.
Journal of gastrointestinal & digestive system
2014; 4 (1)
Abstract
Patients with celiac disease (CD) are increasingly interconnected through social media, exchanging patient experiences and health-tracking information between individuals through various web-based platforms. Social media represents potentially unique communication interface between gastroenterologists and active social media users - especially young adults and adolescents with celiac disease-regarding adherence to the strict gluten-free diet, gastrointestinal symptoms, and meaningful discussion about disease management. Yet, various social media platforms may be underutilized for research purposes to collect patient-reported outcomes data. In this commentary, we summarize the scientific rationale and potential for future growth of social media in patient-reported outcomes research, focusing on college freshmen with celiac disease as a case study and provide overview of the methodological approach. Finally, we discuss how social media may impact patient care in the future through increasing mobile technology use.
View details for PubMedID 25392743
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Architecture of Whole-Module and Bimodular Proteins from the 6-Deoxyerythronolide B Synthase
CELL PRESS. 2014: 466A
View details for DOI 10.1016/j.bpj.2013.11.2637
View details for Web of Science ID 000337000402590
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Computational identification and analysis of orphan assembly-line polyketide synthases
JOURNAL OF ANTIBIOTICS
2014; 67 (1): 89-97
Abstract
The increasing availability of DNA sequence data offers an opportunity for identifying new assembly-line polyketide synthases (PKSs) that produce biologically active natural products. We developed an automated method to extract and consolidate all multimodular PKS sequences (including hybrid PKS/non-ribosomal peptide synthetases) in the National Center for Biotechnology Information (NCBI) database, generating a non-redundant catalog of 885 distinct assembly-line PKSs, the majority of which were orphans associated with no known polyketide product. Two in silico experiments highlight the value of this search method and resulting catalog. First, we identified an orphan that could be engineered to produce an analog of albocycline, an interesting antibiotic whose gene cluster has not yet been sequenced. Second, we identified and analyzed a hitherto overlooked family of metazoan multimodular PKSs, including one from Caenorhabditis elegans. We also developed a comparative analysis method that identified sequence relationships among known and orphan PKSs. As expected, PKS sequences clustered according to structural similarities between their polyketide products. The utility of this method was illustrated by highlighting an interesting orphan from the genus Burkholderia that has no close relatives. Our search method and catalog provide a community resource for the discovery of new families of assembly-line PKSs and their antibiotic products.
View details for DOI 10.1038/ja.2013.125
View details for Web of Science ID 000330222300013
View details for PubMedID 24301183
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Macrolides and Antifungals via Biotransformation
NATURAL PRODUCTS IN MEDICINAL CHEMISTRY
2014; 60: 367-401
View details for Web of Science ID 000354997600013
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Elevated Transglutaminase 2 Activity Is Associated with Hypoxia-Induced Experimental Pulmonary Hypertension in Mice
ACS CHEMICAL BIOLOGY
2014; 9 (1): 266-275
Abstract
Previous studies in human patients and animal models have suggested that transglutaminase 2 (TG2) is upregulated in pulmonary hypertension (PH), a phenomenon that appears to be associated with the effects of serotonin (5-hydroxytryptamine; 5-HT) in this disease. Using chemical tools to interrogate and inhibit TG2 activity in vivo, we have shown that pulmonary TG2 undergoes marked post-translational activation in a mouse model of hypoxia-induced PH. We have also identified irreversible fluorinated TG2 inhibitors that may find use as non-invasive positron emission tomography probes for diagnosis and management of this debilitating, lifelong disorder. Pharmacological inhibition of TG2 attenuated the elevated right ventricular pressure but had no effect on hypertrophy of the right ventricle of the heart. A longitudinal study of pulmonary TG2 activity in PH patients is warranted.
View details for DOI 10.1021/cb4006408
View details for Web of Science ID 000330098800031
View details for PubMedID 24152195
View details for PubMedCentralID PMC3947056
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Dihydroisoxazole inhibitors of Anopheles gambiae seminal transglutaminase AgTG3.
Malaria journal
2014; 13: 210-?
Abstract
Current vector-based malaria control strategies are threatened by the rise of biochemical and behavioural resistance in mosquitoes. Researching mosquito traits of immunity and fertility is required to find potential targets for new vector control strategies. The seminal transglutaminase AgTG3 coagulates male Anopheles gambiae seminal fluids, forming a 'mating plug' that is required for male reproductive success. Inhibitors of AgTG3 can be useful both as chemical probes of A. gambiae reproductive biology and may further the development of new chemosterilants for mosquito population control.A targeted library of 3-bromo-4,5-dihydroxoisoxazole inhibitors were synthesized and screened for inhibition of AgTG3 in a fluorescent, plate-based assay. Positive hits were tested for in vitro activity using cross-linking and mass spectrometry, and in vivo efficacy in laboratory mating assays.A targeted chemical library was screened for inhibition of AgTG3 in a fluorescent plate-based assay using its native substrate, plugin. Several inhibitors were identified with IC50 < 10 μM. Preliminary structure-activity relationships within the library support the stereo-specificity and preference for aromatic substituents in the chemical scaffold. Both inhibition of plugin cross-linking and covalent modification of the active site cysteine of AgTG3 were verified. Administration of an AgTG3 inhibitor to A. gambiae males by intrathoracic injection led to a 15% reduction in mating plug transfer in laboratory mating assays.A targeted screen has identified chemical inhibitors of A. gambiae transglutaminase 3 (AgTG3). The most potent inhibitors are known inhibitors of human transglutaminase 2, suggesting a common binding pose may exist within the active site of both enzymes. Future efforts to develop additional inhibitors will provide chemical tools to address important biological questions regarding the role of the A. gambiae mating plug. A second use for transglutaminase inhibitors exists for the study of haemolymph coagulation and immune responses to wound healing in insects.
View details for DOI 10.1186/1475-2875-13-210
View details for PubMedID 24888439
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Metabolic Flux between Unsaturated and Saturated Fatty Acids Is Controlled by the FabA:FabB Ratio in the Fully Reconstituted Fatty Acid Biosynthetic Pathway of Escherichia coli
BIOCHEMISTRY
2013; 52 (46): 8304-8312
Abstract
The entire fatty acid biosynthetic pathway of Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from 14 purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H to the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multienzyme system. At steady state, a maximal turnover rate of 0.5 s(-1) was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. Via changes in these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximal turnover rate of the pathway. Our reconstituted system provides a powerful tool for understanding and engineering rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.
View details for DOI 10.1021/bi401116n
View details for Web of Science ID 000327044800014
View details for PubMedID 24147979
View details for PubMedCentralID PMC3858588
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In vitro reconstitution and analysis of the 6-deoxyerythronolide B synthase.
Journal of the American Chemical Society
2013; 135 (45): 16809-16812
Abstract
Notwithstanding an extensive literature on assembly line polyketide synthases such as the 6-deoxyerythronolide B synthase (DEBS), a complete naturally occurring synthase has never been reconstituted in vitro from purified protein components. Here, we describe the fully reconstituted DEBS and quantitatively characterize some of the properties of the assembled system that have never been explored previously. The maximum turnover rate of the complete hexamodular system is 1.1 min(-1), comparable to the turnover rate of a truncated trimodular derivative (2.5 min(-1)) but slower than that of a bimodular derivative (21 min(-1)). In the presence of similar concentrations of methylmalonyl- and ethylmalonyl-CoA substrates, DEBS synthesizes multiple regiospecifically modified analogues, one of which we have analyzed in detail. Our studies lay the foundation for biochemically interrogating and rationally engineering polyketide assembly lines in an unprecedented manner.
View details for DOI 10.1021/ja409048k
View details for PubMedID 24161212
View details for PubMedCentralID PMC3858836
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Coupled Methyl Group Epimerization and Reduction by Polyketide Synthase Ketoreductase Domains. Ketoreductase-Catalyzed Equilibrium Isotope Exchange
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2013; 135 (44): 16324-16327
Abstract
Incubation of [2-(2)H]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-1a) with the epimerizing ketoreductase domain EryKR1 in the presence of a catalytic amount NADP(+) (0.05 equiv) resulted in time- and cofactor-dependent washout of deuterium from 1a, as a result of equilibrium isotope exchange of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP. Incubations of [2-(2)H]-(2S,3S)-2-methyl-3-hydroxy-pentanoyl-SACP with RifKR7 and with NysKR1 also resulted in time-dependent loss of deuterium. By contrast, incubations of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP and [2-(2)H]-(2R,3R)-2-methyl-3-hydroxypentanoyl-SACP with the non-epimerizing ketoreductase domains EryKR6 and TylKR1, respectively, did not result in any significant washout of deuterium. The isotope exchange assay directly establishes that specific polyketide synthase ketoreductase domains also have an intrinsic epimerase activity, thus enabling mechanistic analysis of a key determinant of polyketide stereocomplexity.
View details for DOI 10.1021/ja408944s
View details for Web of Science ID 000326774300021
View details for PubMedID 24161343
View details for PubMedCentralID PMC3865775
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Working at the Triple Point
CHEMICAL ENGINEERING PROGRESS
2013; 109 (10): 27
View details for Web of Science ID 000326123800015
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The stanford institute for chemical biology.
ACS chemical biology
2013; 8 (9): 1860-1861
View details for DOI 10.1021/cb400641u
View details for PubMedID 24053754
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Assembly line antibiotic biosynthesis
AMER CHEMICAL SOC. 2013
View details for Web of Science ID 000329618405370
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Studies of the biosynthetic gene cluster of the guadinomines: Potent inhibitors of bacterial pathogenesis
AMER CHEMICAL SOC. 2013
View details for Web of Science ID 000329618401082
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Expanding the Fluorine Chemistry of Living Systems Using Engineered Polyketide Synthase Pathways
SCIENCE
2013; 341 (6150): 1089-1094
Abstract
Organofluorines represent a rapidly expanding proportion of molecules that are used in pharmaceuticals, diagnostics, agrochemicals, and materials. Despite the prevalence of fluorine in synthetic compounds, the known biological scope is limited to a single pathway that produces fluoroacetate. Here, we demonstrate that this pathway can be exploited as a source of fluorinated building blocks for introduction of fluorine into natural-product scaffolds. Specifically, we have constructed pathways involving two polyketide synthase systems, and we show that fluoroacetate can be used to incorporate fluorine into the polyketide backbone in vitro. We further show that fluorine can be inserted site-selectively and introduced into polyketide products in vivo. These results highlight the prospects for the production of complex fluorinated natural products using synthetic biology.
View details for DOI 10.1126/science.1242345
View details for Web of Science ID 000323933100038
View details for PubMedID 24009388
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Dietary gluten triggers concomitant activation of CD4(+) and CD8(+) alpha beta T cells and gamma delta T cells in celiac disease
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (32): 13073-13078
View details for DOI 10.1073/pnas.1311861110
View details for Web of Science ID 000322771100057
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Dietary gluten triggers concomitant activation of CD4+ and CD8+ aß T cells and ?d T cells in celiac disease.
Proceedings of the National Academy of Sciences of the United States of America
2013; 110 (32): 13073-13078
Abstract
Celiac disease is an intestinal autoimmune disease driven by dietary gluten and gluten-specific CD4(+) T-cell responses. In celiac patients on a gluten-free diet, exposure to gluten induces the appearance of gluten-specific CD4(+) T cells with gut-homing potential in the peripheral blood. Here we show that gluten exposure also induces the appearance of activated, gut-homing CD8(+) αβ and γδ T cells in the peripheral blood. Single-cell T-cell receptor sequence analysis indicates that both of these cell populations have highly focused T-cell receptor repertoires, indicating that their induction is antigen-driven. These results reveal a previously unappreciated role of antigen in the induction of CD8(+) αβ and γδ T cells in celiac disease and demonstrate a coordinated response by all three of the major types of T cells. More broadly, these responses may parallel adaptive immune responses to viral pathogens and other systemic autoimmune diseases.
View details for DOI 10.1073/pnas.1311861110
View details for PubMedID 23878218
View details for PubMedCentralID PMC3740842
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CYP3A4-Catalyzed Simvastatin Metabolism as a Non-Invasive Marker of Small Intestinal Health in Celiac Disease.
American journal of gastroenterology
2013; 108 (8): 1344-1351
Abstract
Histological examination of duodenal biopsies is the gold standard for assessing intestinal damage in celiac disease (CD). A noninvasive marker of disease status is necessary, because obtaining duodenal biopsies is invasive and not suitable for routine monitoring of CD patients. As the small intestine is a major site of cytochrome P450 3A4 (CYP3A4) activity and also the location of the celiac lesion, we investigated whether patients with active CD display abnormal pharmacokinetics of an orally administered CYP3A4 substrate, simvastatin (SV), which could potentially be used for noninvasive assessment of their small intestinal health.Preclinical experiments were performed in CYP3A4-humanized mice to examine the feasibility of the test. Subsequently, a clinical trial was undertaken with 11 healthy volunteers, 18 newly diagnosed patients with CD, and 25 celiac patients who had followed a gluten-free diet (GFD) for more than 1 year. The maximum concentration (Cmax) of orally administered SV plus its major non-CYP3A4-derived metabolite SV acid (SV equivalent (SVeq)) was measured, and compared with clinical, histological, and serological parameters.In CYP3A4-humanized mice, a marked decrease in SV metabolism was observed in response to enteropathy. In the clinical setting, untreated celiac patients displayed a significantly higher SVeq Cmax (46±24 nM) compared with treated patients (21±16 nM, P<0.001) or healthy subjects (19±11 nM, P<0.005). SVeq Cmax correctly predicted the diagnosis in 16/18 untreated celiac patients, and also the recovery status of all follow-up patients that exhibited normal or near-normal biopsies (Marsh 0-2). All patients with abnormal SVeq Cmax showed a reduction in the value after 1 year of following a GFD.SVeq Cmax is a promising noninvasive marker for assessment of small intestinal health. Further studies are warranted to establish its clinical utility for assessing gut status of patients with CD.
View details for DOI 10.1038/ajg.2013.151
View details for PubMedID 23732466
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Discovery and Mechanism of Type III Secretion System Inhibitors
ISRAEL JOURNAL OF CHEMISTRY
2013; 53 (8): 577-587
View details for DOI 10.1002/ijch.201300025
View details for Web of Science ID 000327739200009
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Nonproteinogenic Amino Acid Building Blocks for Nonribosomal Peptide and Hybrid Polyketide Scaffolds
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
2013; 52 (28): 7098-7124
Abstract
Freestanding nonproteinogenic amino acids have long been recognized for their antimetabolite properties and tendency to be uncovered to reactive functionalities by the catalytic action of target enzymes. By installing them regiospecifically into biogenic peptides and proteins, it may be possible to usher a new era at the interface between small molecule and large molecule medicinal chemistry. Site-selective protein functionalization offers uniquely attractive strategies for posttranslational modification of proteins. Last, but not least, many of the amino acids not selected by nature for protein incorporation offer rich architectural possibilities in the context of ribosomally derived polypeptides. This Review summarizes the biosynthetic routes to and metabolic logic for the major classes of the noncanonical amino acid building blocks that end up in both nonribosomal peptide frameworks and in hybrid nonribosomal peptide-polyketide scaffolds.
View details for DOI 10.1002/anie.201208344
View details for Web of Science ID 000321298800012
View details for PubMedCentralID PMC4634941
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Stereochemistry of Reductions Catalyzed by Methyl-Epimerizing Ketoreductase Domains of Polyketide Synthases
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2013; 135 (20): 7406-7409
Abstract
Ketoreductase (KR) domains from modular polyketide synthases (PKSs) catalyze the reduction of 2-methyl-3-ketoacyl acyl carrier protein (ACP) substrates and in certain cases epimerization of the 2-methyl group as well. The structural and mechanistic basis of epimerization is poorly understood, and only a small number of such KRs been studied. In this work, we studied three recombinant KR domains with putative epimerase activity: NysKR1 from module 1 of the nystatin PKS, whose stereospecificity can be predicted from both the protein sequence and the product structure; RifKR7 from module 7 of the rifamycin PKS, whose stereospecificity cannot be predicted from the protein sequence; and RifKR10 from module 10 of the rifamycin PKS, whose specificity is unclear from both the sequence and the structure. Each KR was individually incubated with NADPH and (2R)- or (2RS)-2-methyl-3-ketopentanoyl-ACP generated enzymatically in situ or via chemoenzymatic synthesis, respectively. Chiral GC-MS analysis revealed that each KR stereospecifically produced the corresponding (2S,3S)-2-methyl-3-hydroxypentanoyl-ACP in which the 2-methyl substituent had undergone KR-catalyzed epimerization. Thus, our results have led to the identification of a prototypical set of KR domains that generate (2S,3S)-2-methyl-3-hydroxyacyl products in the course of polyketide biosynthesis.
View details for DOI 10.1021/ja4014776
View details for Web of Science ID 000319551000005
View details for PubMedID 23659177
View details for PubMedCentralID PMC3699853
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Molecular insights into the biosynthesis of guadinomine, a type III secretion system inhibitor
AMER CHEMICAL SOC. 2013
View details for Web of Science ID 000324303602780
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Gluten-sensitive enteropathy coincides with decreased capability of intestinal T cells to secrete IL-17 and IL-22 in a macaque model for celiac disease
CLINICAL IMMUNOLOGY
2013; 147 (1): 40-49
Abstract
Celiac disease (CD) is an autoimmune disorder caused by intolerance to dietary gluten. The interleukin (IL)-17 and IL-22 function as innate regulators of mucosal integrity. Impaired but not well-understood kinetics of the IL-17/22 secretion was described in celiac patients. Here, the IL-17 and IL-22-producing intestinal cells were studied upon their in vitro stimulation with mitogens in class II major histocompatibility complex-defined, gluten-sensitive rhesus macaques. Pediatric biopsies were collected from distal duodenum during the stages of disease remission and relapse. Regardless of dietary gluten content, IL-17 and IL-22-producing cells consisted of CD4+ and CD8+ T lymphocytes as well as of lineage-negative (Lin-) cells. Upon introduction of dietary gluten, capability of intestinal T cells to secrete IL-17/22 started to decline (p<0.05), which was paralleled with gradual disruption of epithelial integrity. These data indicate that IL-17/22-producing cells play an important role in maintenance of intestinal mucosa in gluten-sensitive primates.
View details for DOI 10.1016/j.clim.2013.02.012
View details for Web of Science ID 000317810200007
View details for PubMedID 23518597
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Mechanism and Specificity of an Acyltransferase Domain from a Modular Polyketide Synthase
BIOCHEMISTRY
2013; 52 (11): 1839-1841
Abstract
Acyltransferase (AT) domains of modular polyketide synthases exercise tight control over the choice of α-carboxyacyl-CoA substrates, but the mechanistic basis for this specificity is unknown. We show that whereas the specificity for the electrophilic malonyl or methylmalonyl component is primarily expressed in the first half-reaction (formation of the acyl-enzyme intermediate), the second half-reaction shows comparable specificity for the acyl carrier protein that carries the nucleophilic pantetheine arm. We also show that currently used approaches for engineering AT domain specificity work mainly by degrading specificity for the natural substrate rather than by enhancing specificity for alternative substrates.
View details for DOI 10.1021/bi400185v
View details for Web of Science ID 000316520000001
View details for PubMedID 23452124
View details for PubMedCentralID PMC3612939
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Analysis and Refactoring of the A-74528 Biosynthetic Pathway
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2013; 135 (10): 3752-3755
Abstract
A-74528 is a C30 polyketide natural product that functions as an inhibitor of 2',5'-oligoadenylate phosphodiesterase (2'-PDE), a key regulatory enzyme of the interferon pathway. Modulation of 2'-PDE represents a unique therapeutic approach for regulating viral infections. The gene cluster responsible for biosynthesis of A-74528 yields minute amounts of this natural product together with considerably larger quantities of a structurally dissimilar C30 cytotoxic agent, fredericamycin. Through construction and analysis of a series of knockout mutants, we identified the genes necessary for A-74528 biosynthesis. Remarkably, the formation of six stereocenters and the regiospecific formation of six rings in A-74528 appear to be catalyzed by only two tailoring enzymes, a cyclase and an oxygenase, in addition to the core polyketide synthase. The inferred pathway was genetically refactored in a heterologous host, Streptomyces coelicolor CH999, to produce 3 mg/L A-74528 in the absence of fredericamycin.
View details for DOI 10.1021/ja311579s
View details for Web of Science ID 000316244200006
View details for PubMedID 23442197
View details for PubMedCentralID PMC3601660
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Selective Inhibition of Extracellular Thioredoxin by Asymmetric Disulfides
JOURNAL OF MEDICINAL CHEMISTRY
2013; 56 (3): 1301-1310
Abstract
Whereas the role of mammalian thioredoxin (Trx) as an intracellular protein cofactor is widely appreciated, its function in the extracellular environment is not well-understood. Only few extracellular targets of Trx-mediated thiol-disulfide exchange are known. For example, Trx activates extracellular transglutaminase 2 (TG2) via reduction of an intramolecular disulfide bond. Because hyperactive TG2 is thought to play a role in various diseases, understanding the biological role of extracellular Trx may provide critical insight into the pathogenesis of these disorders. Starting from a clinical-stage asymmetric disulfide lead, we have identified analogs with >100-fold specificity for Trx. Structure-activity relationship and computational docking model analyses have provided insights into the features important for enhancing potency and specificity. The most active compound identified had an IC(50) below 0.1 μM in cell culture and may be appropriate for in vivo use to interrogate the role of extracellular Trx in health and disease.
View details for DOI 10.1021/jm301775s
View details for Web of Science ID 000315182100055
View details for PubMedID 23327656
View details for PubMedCentralID PMC3574193
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Engineering the acyltransferase substrate specificity of assembly line polyketide synthases.
Journal of the Royal Society, Interface / the Royal Society
2013; 10 (85): 20130297-?
Abstract
Polyketide natural products act as a broad range of therapeutics, including antibiotics, immunosuppressants and anti-cancer agents. This therapeutic diversity stems from the structural diversity of these small molecules, many of which are produced in an assembly line manner by modular polyketide synthases. The acyltransferase (AT) domains of these megasynthases are responsible for selection and incorporation of simple monomeric building blocks, and are thus responsible for a large amount of the resulting polyketide structural diversity. The substrate specificity of these domains is often targeted for engineering in the generation of novel, therapeutically active natural products. This review outlines recent developments that can be used in the successful engineering of these domains, including AT sequence and structural data, mechanistic insights and the production of a diverse pool of extender units. It also provides an overview of previous AT domain engineering attempts, and concludes with proposed engineering approaches that take advantage of current knowledge. These approaches may lead to successful production of biologically active 'unnatural' natural products.
View details for DOI 10.1098/rsif.2013.0297
View details for PubMedID 23720536
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Regulation of the activities of the mammalian transglutaminase family of enzymes.
Protein science
2012; 21 (12): 1781-1791
Abstract
Mammalian transglutaminases catalyze post-translational modifications of glutamine residues on proteins and peptides through transamidation or deamidation reactions. Their catalytic mechanism resembles that of cysteine proteases. In virtually every case, their enzymatic activity is modulated by elaborate strategies including controlled gene expression, allostery, covalent modification, and proteolysis. In this review, we focus on our current knowledge of post-translational regulation of transglutaminase activity by physiological as well as synthetic allosteric agents. Our discussion will primarily focus on transglutaminase 2, but will also compare and contrast its regulation with Factor XIIIa as well as transglutaminases 1 and 3. Potential structure-function relationships of known mutations in human transglutaminases are analyzed.
View details for DOI 10.1002/pro.2162
View details for PubMedID 23011841
View details for PubMedCentralID PMC3575910
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Regulation of the activities of the mammalian transglutaminase family of enzymes
PROTEIN SCIENCE
2012; 21 (12): 1781-1791
Abstract
Mammalian transglutaminases catalyze post-translational modifications of glutamine residues on proteins and peptides through transamidation or deamidation reactions. Their catalytic mechanism resembles that of cysteine proteases. In virtually every case, their enzymatic activity is modulated by elaborate strategies including controlled gene expression, allostery, covalent modification, and proteolysis. In this review, we focus on our current knowledge of post-translational regulation of transglutaminase activity by physiological as well as synthetic allosteric agents. Our discussion will primarily focus on transglutaminase 2, but will also compare and contrast its regulation with Factor XIIIa as well as transglutaminases 1 and 3. Potential structure-function relationships of known mutations in human transglutaminases are analyzed.
View details for DOI 10.1002/pro.2162
View details for Web of Science ID 000311616200001
View details for PubMedCentralID PMC3575910
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Molecular Insights into the Biosynthesis of Guadinomine: A Type III Secretion System Inhibitor
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2012; 134 (42): 17797-17806
Abstract
Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509 with a novel mode of action. With an IC(50) of 14 nM, guadinomine B is the most potent known inhibitor of the type III secretion system (TTSS) of Gram-negative bacteria. TTSS activity is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli , Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. The guadinomine (gdn) biosynthetic gene cluster has been cloned and sequenced and includes 26 open reading frames spanning 51.2 kb. It encodes a chimeric multimodular polyketide synthase, a nonribosomal peptide synthetase, along with enzymes responsible for the biosynthesis of the unusual aminomalonyl-acyl carrier protein extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by targeted disruption of the gene cluster as well as by heterologous expression and analysis of key enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these scarce but potentially important natural products.
View details for DOI 10.1021/ja308622d
View details for Web of Science ID 000310103800078
View details for PubMedID 23030602
View details for PubMedCentralID PMC3483642
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Natural product inhibitors of glucose-6-phosphate translocase
MEDCHEMCOMM
2012; 3 (8): 926-931
View details for DOI 10.1039/c2md20008b
View details for Web of Science ID 000306759900009
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Precursor Directed Biosynthesis of an Orthogonally Functional Erythromycin Analogue: Selectivity in the Ribosome Macrolide Binding Pocket
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2012; 134 (29): 12259-12265
Abstract
The macrolide antibiotic erythromycin A and its semisynthetic analogues have been among the most useful antibacterial agents for the treatment of infectious diseases. Using a recently developed chemical genetic strategy for precursor-directed biosynthesis and colony bioassay of 6-deoxyerythromycin D analogues, we identified a new class of alkynyl- and alkenyl-substituted macrolides with activities comparable to that of the natural product. Further analysis revealed a marked and unexpected dependence of antibiotic activity on the size and degree of unsaturation of the precursor. Based on these leads, we also report the precursor-directed biosynthesis of 15-propargyl erythromycin A, a novel antibiotic that not only is as potent as erythromycin A with respect to its ability to inhibit bacterial growth and cell-free ribosomal protein biosynthesis but also harbors an orthogonal functional group that is capable of facile chemical modification.
View details for DOI 10.1021/ja304682q
View details for PubMedID 22741553
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Role of transglutaminase 2 in celiac disease pathogenesis
SEMINARS IN IMMUNOPATHOLOGY
2012; 34 (4): 513-522
Abstract
A number of lines of evidence suggest that transglutaminase 2 (TG2) may be one of the earliest disease-relevant proteins to encounter immunotoxic gluten in the celiac gut. These and other investigations also suggest that the reaction catalyzed by TG2 on dietary gluten peptides is essential for the pathogenesis of celiac disease. If so, several questions are of critical significance. How is TG2 activated in the celiac gut? What are the disease-specific and general consequences of activating TG2? Can local inhibition of TG2 in the celiac intestine suppress gluten induced pathogenesis in a dose-responsive manner? And what are the long-term consequences of suppressing TG2 activity in the small intestinal mucosa? Answers to these questions will depend upon the development of judicious models and chemical tools. They also have the potential of yielding powerful next-generation drug candidates for treating this widespread but overlooked chronic disease.
View details for DOI 10.1007/s00281-012-0305-0
View details for Web of Science ID 000307305600005
View details for PubMedID 22437759
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Role of a Conserved Arginine Residue in Linkers between the Ketosynthase and Acyltransferase Domains of Multimodular Polyketide Synthases
BIOCHEMISTRY
2012; 51 (18): 3708-3710
Abstract
The role of interdomain linkers in modular polyketide synthases is poorly understood. Analysis of the 6-deoxyerythronolide B synthase (DEBS) has yielded a model in which chain elongation is governed by interactions between the acyl carrier protein domain and the ketosynthase domain plus an adjacent linker. Alanine scanning mutagenesis of the conserved residues of this linker in DEBS module 3 led to the identification of the R513A mutant with a markedly reduced rate of chain elongation. Limited proteolysis supported a structural role for this Arg. Our findings highlight the importance of domain-linker interactions in assembly line polyketide biosynthesis.
View details for DOI 10.1021/bi300399u
View details for Web of Science ID 000303628200002
View details for PubMedID 22509729
View details for PubMedCentralID PMC3348408
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Resolving Multiple Protein-Peptide Binding Events: Implication for HLA-DQ2 Mediated Antigen Presentation in Celiac Disease
CHEMISTRY-AN ASIAN JOURNAL
2012; 7 (5): 992-999
Abstract
Techniques that can effectively separate protein-peptide complexes from free peptides have shown great value in major histocompatibility complex (MHC)-peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to an MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE-FL) can resolve multiple MHC-peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE-FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.
View details for DOI 10.1002/asia.201101041
View details for Web of Science ID 000303239700021
View details for PubMedID 22411856
View details for PubMedCentralID PMC3382112
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Combinatorial biosynthesis of polyketides - a perspective
CURRENT OPINION IN CHEMICAL BIOLOGY
2012; 16 (1-2): 117-123
Abstract
Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make 'unnatural' natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms.
View details for DOI 10.1016/j.cbpa.2012.01.018
View details for Web of Science ID 000303640400017
View details for PubMedID 22342766
View details for PubMedCentralID PMC3328608
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Interferon-gamma Activates Transglutaminase 2 via a Phosphatidylinositol-3-Kinase-Dependent Pathway: Implications for Celiac Sprue Therapy
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2012; 341 (1): 104-114
Abstract
The mechanism for activation of extracellular transglutaminase 2 (TG2) in the small intestine remains a fundamental mystery in our understanding of celiac sprue pathogenesis. Using the T84 human enterocytic cell line, we show that interferon-γ (IFN-γ), the predominant cytokine secreted by gluten-reactive T cells in the celiac intestine, activates extracellular TG2 in a dose-dependent manner. IFN-γ mediated activation of TG2 requires phosphatidylinositol-3-kinase (PI3K) activity, but is uninfluenced by a number of other kinases reported to be active in T84 cells. Pharmacological inhibition of PI3K in the presence of IFN-γ prevents TG2 activation as well as the previously characterized increase in transepithelial permeability. Our findings therefore establish PI3K as an attractive target for celiac sprue therapy, a possibility that is underscored by the encouraging safety profiles of several PI3K inhibitors undergoing human clinical trials.
View details for DOI 10.1124/jpet.111.187385
View details for Web of Science ID 000301530100011
View details for PubMedCentralID PMC3310700
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Interferon-? activates transglutaminase 2 via a phosphatidylinositol-3-kinase-dependent pathway: implications for celiac sprue therapy.
journal of pharmacology and experimental therapeutics
2012; 341 (1): 104-114
Abstract
The mechanism for activation of extracellular transglutaminase 2 (TG2) in the small intestine remains a fundamental mystery in our understanding of celiac sprue pathogenesis. Using the T84 human enterocytic cell line, we show that interferon-γ (IFN-γ), the predominant cytokine secreted by gluten-reactive T cells in the celiac intestine, activates extracellular TG2 in a dose-dependent manner. IFN-γ mediated activation of TG2 requires phosphatidylinositol-3-kinase (PI3K) activity, but is uninfluenced by a number of other kinases reported to be active in T84 cells. Pharmacological inhibition of PI3K in the presence of IFN-γ prevents TG2 activation as well as the previously characterized increase in transepithelial permeability. Our findings therefore establish PI3K as an attractive target for celiac sprue therapy, a possibility that is underscored by the encouraging safety profiles of several PI3K inhibitors undergoing human clinical trials.
View details for DOI 10.1124/jpet.111.187385
View details for PubMedID 22228808
View details for PubMedCentralID PMC3310700
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Probing the biosynthesis of the type II polyketide A-74528 through heterologous pathway reconstruction
AMER CHEMICAL SOC. 2012
View details for Web of Science ID 000324475100342
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Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (11): 4110-4115
Abstract
Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein-protein interfaces that are present repetitively along naturally occurring PKS assembly lines.
View details for DOI 10.1073/pnas.1118734109
View details for Web of Science ID 000301426700024
View details for PubMedID 22371562
View details for PubMedCentralID PMC3306671
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Activation and Inhibition of Transglutaminase 2 in Mice
PLOS ONE
2012; 7 (2)
Abstract
Transglutaminase 2 (TG2) is an allosterically regulated enzyme with transamidating, deamidating and cell signaling activities. It is thought to catalyze sequence-specific deamidation of dietary gluten peptides in the small intestines of celiac disease patients. Because this modification has profound consequences for disease pathogenesis, there is considerable interest in the design of small molecule TG2 inhibitors. Although many classes of TG2 inhibitors have been reported, thus far an animal model for screening them to identify promising celiac drug candidates has remained elusive. Using intraperitoneal administration of the toll-like receptor 3 (TLR3) ligand, polyinosinic-polycytidylic acid (poly(I∶C)), we induced rapid TG2 activation in the mouse small intestine. Dose dependence was observed in the activation of TG2 as well as the associated villous atrophy, gross clinical response, and rise in serum concentration of the IL-15/IL-15R complex. TG2 activity was most pronounced in the upper small intestine. No evidence of TG2 activation was observed in the lung mucosa, nor were TLR7/8 ligands able to elicit an analogous response. Introduction of ERW1041E, a small molecule TG2 inhibitor, in this mouse model resulted in TG2 inhibition in the small intestine. TG2 inhibition had no effect on villous atrophy, suggesting that activation of this enzyme is a consequence, rather than a cause, of poly(I∶C) induced enteropathy. Consistent with this finding, administration of poly(I∶C) to TG2 knockout mice also induced villous atrophy. Our findings pave the way for pharmacological evaluation of small molecule TG2 inhibitors as drug candidates for celiac disease.
View details for DOI 10.1371/journal.pone.0030642
View details for Web of Science ID 000301979000009
View details for PubMedID 22319575
View details for PubMedCentralID PMC3271093
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ORAL ENZYME THERAPY FOR CELIAC SPRUE
METHODS IN ENZYMOLOGY, VOL 502: PROTEIN ENGINEERING FOR THERAPEUTICS, PT A
2012; 502: 241-271
Abstract
Celiac sprue is an inflammatory disease of the small intestine caused by dietary gluten and treated by adherence to a life-long gluten-free diet. The recent identification of immunodominant gluten peptides, the discovery of their cogent properties, and the elucidation of the mechanisms by which they engender immunopathology in genetically susceptible individuals have advanced our understanding of the molecular pathogenesis of this complex disease, enabling the rational design of new therapeutic strategies. The most clinically advanced of these is oral enzyme therapy, in which enzymes capable of proteolyzing gluten (i.e., glutenases) are delivered to the alimentary tract of a celiac sprue patient to detoxify ingested gluten in situ. In this chapter, we discuss the key challenges for discovery and preclinical development of oral enzyme therapies for celiac sprue. Methods for lead identification, assay development, gram-scale production and formulation, and lead optimization for next-generation proteases are described and critically assessed.
View details for DOI 10.1016/B978-0-12-416039-2.00013-6
View details for Web of Science ID 000299709600010
View details for PubMedID 22208988
View details for PubMedCentralID PMC3382113
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In vitro and in vivo activity of frenolicin B against Plasmodium falciparum and P berghei
JOURNAL OF ANTIBIOTICS
2011; 64 (12): 799-801
View details for DOI 10.1038/ja.2011.94
View details for Web of Science ID 000298634600008
View details for PubMedID 22008701
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Engineered biosynthesis of the antiparasitic agent frenolicin B and rationally designed analogs in a heterologous host
JOURNAL OF ANTIBIOTICS
2011; 64 (12): 759-762
Abstract
The polyketide antibiotic frenolicin B harbors a biosynthetically intriguing benzoisochromanequinone core, and has been shown to exhibit promising antiparasitic activity against Eimeria tenella. To facilitate further exploration of its chemistry and biology, we constructed a biosynthetic route to frenolicin B in the heterologous host Streptomyces coelicolor CH999, despite the absence of key enzymes in the identified frenolicin gene cluster. Together with our understanding of the underlying polyketide biosynthetic pathway, this heterologous production system was exploited to produce analogs modified at the C15 position. Both the natural product and these analogs inhibited the growth of Toxoplasma gondii in a manner that reveals sensitivity to the length of the C15 substituent. The ability to construct a functional biosynthetic pathway, despite a lack of genetic information, illustrates the feasibility of a modular approach to engineering medicinally relevant polyketide products.
View details for DOI 10.1038/ja.2011.86
View details for Web of Science ID 000298634600001
View details for PubMedID 21934692
View details for PubMedCentralID PMC3245331
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In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (46): 18643-18648
Abstract
Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 μM, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 μM/ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 μM. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 μM; FabH and FabF, which decreased FAS activity at concentrations higher than 1 μM; and holo-ACP and TesA, which gave maximum FAS activity at 30 μM concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels.
View details for DOI 10.1073/pnas.1110852108
View details for Web of Science ID 000297008900026
View details for PubMedID 22042840
View details for PubMedCentralID PMC3219124
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Activation of Extracellular Transglutaminase 2 by Thioredoxin
JOURNAL OF BIOLOGICAL CHEMISTRY
2011; 286 (43): 37866-37873
Abstract
The mechanism of activation of transglutaminase 2 (TG2) in the extracellular matrix remains a fundamental mystery in our understanding of the biology of this multifunctional mammalian enzyme. Earlier investigations have highlighted the role of a disulfide bond formed by vicinal Cys residues in maintaining calcium-bound TG2 in an inactive state. Here, we have shown that the redox potential of this disulfide bond is approximately -190 mV, a high value for a disulfide bond in proteins. Consistent with this observation, TG2 activity in a freshly wounded fibroblast culture depends upon the redox potential of the environment. We sought to identify a physiological mechanism for the activation of oxidized TG2. With a k(cat)/K(m) of 1.6 μm(-1) min(-1), human thioredoxin (Trx) was a highly specific activator of oxidized human TG2. Trx-mediated activation of TG2 was blocked by PX-12, a small molecule Trx inhibitor that is undergoing clinical trials as a cancer chemotherapeutic agent. In a mixed culture containing fibroblasts and monocytic cells, interferon-γ stimulated Trx release from monocytes, which in turn activated TG2 around the fibroblasts. Recombinant human Trx could also activate extracellular TG2 in cryosections of human and mouse small intestinal biopsies. In addition to explaining how TG2 can be activated by dietary gluten in the small intestinal mucosa of celiac sprue patients, our findings reveal a new strategy for inhibiting the undesirable consequences of TG2 activity in this widespread, lifelong disease.
View details for DOI 10.1074/jbc.M111.287490
View details for Web of Science ID 000296542400081
View details for PubMedID 21908620
View details for PubMedCentralID PMC3199528
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Structural and Biochemical Studies of the Hedamycin Type II Polyketide Ketoreductase (HedKR): Molecular Basis of Stereo- and Regiospecificities
BIOCHEMISTRY
2011; 50 (34): 7426-7439
Abstract
Bacterial aromatic polyketides that include many antibiotic and antitumor therapeutics are biosynthesized by the type II polyketide synthase (PKS), which consists of 5-10 stand-alone enzymatic domains. Hedamycin, an antitumor antibiotic polyketide, is uniquely primed with a hexadienyl group generated by a type I PKS followed by coupling to a downstream type II PKS to biosynthesize a 24-carbon polyketide, whose C9 position is reduced by hedamycin type II ketoreductase (hedKR). HedKR is homologous to the actinorhodin KR (actKR), for which we have conducted extensive structural studies previously. How hedKR can accommodate a longer polyketide substrate than the actKR, and the molecular basis of its regio- and stereospecificities, is not well understood. Here we present a detailed study of hedKR that sheds light on its specificity. Sequence alignment of KRs predicts that hedKR is less active than actKR, with significant differences in substrate/inhibitor recognition. In vitro and in vivo assays of hedKR confirmed this hypothesis. The hedKR crystal structure further provides the molecular basis for the observed differences between hedKR and actKR in the recognition of substrates and inhibitors. Instead of the 94-PGG-96 motif observed in actKR, hedKR has the 92-NGG-94 motif, leading to S-dominant stereospecificity, whose molecular basis can be explained by the crystal structure. Together with mutations, assay results, docking simulations, and the hedKR crystal structure, a model for the observed regio- and stereospecificities is presented herein that elucidates how different type II KRs recognize substrates with different chain lengths, yet precisely reduce only the C9-carbonyl group. The molecular features of hedKR important for regio- and stereospecificities can potentially be applied to biosynthesize new polyketides via protein engineering that rationally controls polyketide ketoreduction.
View details for DOI 10.1021/bi2006866
View details for Web of Science ID 000294083600012
View details for PubMedID 21776967
View details for PubMedCentralID PMC3175028
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Analysis of the Ketosynthase-Chain Length Factor Heterodimer from the Fredericamycin Polyketide Synthase
CHEMISTRY & BIOLOGY
2011; 18 (8): 1021-1031
Abstract
The pentadecaketide fredericamycin has the longest carbon chain backbone among polycyclic aromatic polyketide antibiotics whose biosynthetic genes have been sequenced. This backbone is synthesized by the bimodular fdm polyketide synthase (PKS). Here, we demonstrate that the bimodular fdm PKS as well as its elongation module alone synthesize undecaketides and dodecaketides. Thus, unlike other homologs, the fdm ketosynthase-chain length factor (KS-CLF) heterodimer does not exclusively control the backbone length of its natural product. Using sequence- and structure-based approaches, 48 CLF multiple mutants were engineered and analyzed. Unexpectedly, the I134F mutant was unable to turn over but could initiate and partially elongate the polyketide chain. This unprecedented mutant suggests that the KS-CLF heterodimer harbors an as yet uncharacterized chain termination mechanism. Together, our findings reveal fundamental mechanistic differences between the fdm PKS and its well-studied homologs.
View details for DOI 10.1016/j.chembiol.2011.07.015
View details for Web of Science ID 000295203200012
View details for PubMedID 21867917
View details for PubMedCentralID PMC3172819
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Structure and Mechanism of the trans-Acting Acyltransferase from the Disorazole Synthase
BIOCHEMISTRY
2011; 50 (30): 6539-6548
Abstract
The 1.51 Å resolution X-ray crystal structure of the trans-acyltransferase (AT) from the "AT-less" disorazole synthase (DSZS) and that of its acetate complex at 1.35 Å resolution are reported. Separately, comprehensive alanine-scanning mutagenesis of one of its acyl carrier protein substrates (ACP1 from DSZS) led to the identification of a conserved Asp45 residue on the ACP, which contributes to the substrate specificity of this unusual enzyme. Together, these experimental findings were used to derive a model for the selective association of the DSZS AT and its ACP substrate. With a goal of structurally characterizing the AT-ACP interface, a strategy was developed for covalently cross-linking the active site Ser → Cys mutant of the DSZS AT to its ACP substrate and for purifying the resulting AT-ACP complex to homogeneity. The S86C DSZS AT mutant was found to be functional, albeit with a transacylation efficiency 200-fold lower than that of its wild-type counterpart. Our findings provide new insights as well as new opportunities for high-resolution analysis of an important protein-protein interface in polyketide synthases.
View details for DOI 10.1021/bi200632j
View details for Web of Science ID 000293035500003
View details for PubMedID 21707057
View details for PubMedCentralID PMC3144309
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Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase
PROTEIN SCIENCE
2011; 20 (7): 1244-1255
Abstract
The assembly-line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6-deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein-protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃-S-ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein-protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.
View details for DOI 10.1002/pro.652
View details for Web of Science ID 000292257600017
View details for PubMedID 21563224
View details for PubMedCentralID PMC3149197
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Novel chemo-sensitizing agent, ERW1227B, impairs cellular motility and enhances cell death in glioblastomas
JOURNAL OF NEURO-ONCOLOGY
2011; 103 (2): 207-219
Abstract
Glioblastomas display variable phenotypes that include increased drug-resistance associated with enhanced migratory and anti-apoptotic characteristics. These shared characteristics contribute to failure of clinical treatment regimens. Identification of novel compounds that promote cell death and impair cellular motility is a logical strategy to develop more effective clinical protocols. We recently described the ability of the small molecule, KCC009, a tissue transglutaminase (TG2) inhibitor, to sensitize glioblastoma cells to chemotherapy. In the current study, we synthesized a series of related compounds that show variable ability to promote cell death and impair motility in glioblastomas, irrespective of their ability to inhibit TG2. Each compound has a 3-bromo-4,5-dihydroisoxazole component that presumably reacts with nucleophilic cysteine thiol residues in the active sites of proteins that have an affinity to the small molecule. Our studies focused on the effects of the compound, ERW1227B. Treatment of glioblastoma cells with ERW1227B was associated with both down-regulation of the PI-3 kinase/Akt pathway, which enhanced cell death; as well as disruption of focal adhesive complexes and intracellular actin fibers, which impaired cellular mobility. Bioassays as well as time-lapse photography of glioblastoma cells treated with ERW1227B showed cell death and rapid loss of cellular motility. Mice studies with in vivo glioblastoma models demonstrated the ability of ERW1227B to sensitize tumor cells to cell death after treatment with either chemotherapy or radiation. The above findings identify ERW1227B as a potential novel therapeutic agent in the treatment of glioblastomas.
View details for DOI 10.1007/s11060-010-0379-2
View details for Web of Science ID 000290773100004
View details for PubMedID 20824305
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Novel therapies for coeliac disease
JOURNAL OF INTERNAL MEDICINE
2011; 269 (6): 604-613
Abstract
Coeliac disease is a widespread, lifelong disorder for which dietary control represents the only accepted form of therapy. There is an unmet need for nondietary therapies to treat this condition. Most ongoing and emerging drug-discovery programmes are based on the understanding that coeliac disease is caused by an inappropriate T-cell-mediated immune response to dietary gluten proteins. Recent genome-wide association studies lend further support to this pathogenic model. The central role of human leucocyte antigen genes has been validated, and a number of new risk loci have been identified, most of which are related to the biology of T cells and antigen-presenting cells. Here, we review the status of potential nondietary therapies under consideration for coeliac disease. We conclude that future development of novel therapies will be aided considerably by the identification of new, preferably noninvasive, surrogate markers for coeliac disease activity.
View details for DOI 10.1111/j.1365-2796.2011.02376.x
View details for Web of Science ID 000290867900006
View details for PubMedID 21401739
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Acylideneoxoindoles: a new class of reversible inhibitors of human transglutaminase 2.
Bioorganic & medicinal chemistry letters
2011; 21 (9): 2692-2696
Abstract
Inhibitors of human transglutaminase 2 (TG2) are anticipated to be useful in the therapy of a variety of diseases including celiac sprue as well as certain CNS disorders and cancers. A class of 3-acylidene-2-oxoindoles was identified as potent reversible inhibitors of human TG2. Structure-activity relationship analysis of a lead compound led to the generation of several potent, competitive inhibitors. Analogs with significant non-competitive character were also identified, suggesting that the compounds bind at one or more allosteric regulatory sites on this multidomain enzyme. The most active compounds had K(i) values below 1.0 μM in two different kinetic assays for human TG2, and may therefore be suitable for investigations into the role of TG2 in physiology and disease in animals.
View details for DOI 10.1016/j.bmcl.2010.12.037
View details for PubMedID 21215619
View details for PubMedCentralID PMC3081996
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Acylideneoxoindoles: A new class of reversible inhibitors of human transglutaminase 2
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
2011; 21 (9): 2692-2696
Abstract
Inhibitors of human transglutaminase 2 (TG2) are anticipated to be useful in the therapy of a variety of diseases including celiac sprue as well as certain CNS disorders and cancers. A class of 3-acylidene-2-oxoindoles was identified as potent reversible inhibitors of human TG2. Structure-activity relationship analysis of a lead compound led to the generation of several potent, competitive inhibitors. Analogs with significant non-competitive character were also identified, suggesting that the compounds bind at one or more allosteric regulatory sites on this multidomain enzyme. The most active compounds had K(i) values below 1.0 μM in two different kinetic assays for human TG2, and may therefore be suitable for investigations into the role of TG2 in physiology and disease in animals.
View details for DOI 10.1016/j.bmcl.2010.12.037
View details for Web of Science ID 000289773300024
View details for PubMedCentralID PMC3081996
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Chemistry and Biology of Macrolide Antiparasitic Agents
JOURNAL OF MEDICINAL CHEMISTRY
2011; 54 (8): 2792-2804
Abstract
Macrolide antibacterial agents inhibit parasite proliferation by targeting the apicoplast ribosome. Motivated by the long-term goal of identifying antiparasitic macrolides that lack antibacterial activity, we have systematically analyzed the structure-activity relationships among erythromycin analogues and have also investigated the mechanism of action of selected compounds. Two lead compounds, N-benzylazithromycin (11) and N-phenylpropylazithromycin (30), were identified with significantly higher antiparasitic activity and lower antibacterial activity than erythromycin or azithromycin. Molecular modeling based on the cocrystal structure of azithromycin bound to the bacterial ribosome suggested that a substituent at the N-9 position of desmethylazithromycin could improve selectivity because of species-specific interactions with the ribosomal L22 protein. Like other macrolides, these lead compounds display a strong "delayed death phenotype"; however, their early effects on T. gondii replication are more pronounced.
View details for DOI 10.1021/jm101593u
View details for Web of Science ID 000289697800018
View details for PubMedID 21428405
View details for PubMedCentralID PMC3085955
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Dihydroisoxazole Analogs for Labeling and Visualization of Catalytically Active Transglutaminase 2
CHEMISTRY & BIOLOGY
2011; 18 (1): 58-66
Abstract
We report the synthesis and preliminary characterization of "clickable" inhibitors of human transglutaminase 2 (TG2). These inhibitors possess the 3-halo-4,5-dihydroisoxazole warhead along with bioorthogonal groups such as azide or alkyne moieties that enable subsequent covalent modification with fluorophores. Their mechanism for inhibition of TG2 is based on halide displacement, resulting in the formation of a stable imino thioether. Inhibition assays against recombinant human TG2 revealed that some of the clickable inhibitors prepared in this study have comparable specificity as benchmark dihydroisoxazole inhibitors reported earlier. At low micromolar concentrations they completely inhibited transiently activated TG2 in a WI-38 fibroblast scratch assay and could subsequently be used to visualize the active enzyme in situ. The potential use of these inhibitors to probe the role of TG2 in celiac sprue as well as other diseases is discussed.
View details for DOI 10.1016/j.chembiol.2010.11.004
View details for Web of Science ID 000287540300011
View details for PubMedID 21276939
View details for PubMedCentralID PMC3073585
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Improved precursor-directed biosynthesis in E. coli via directed evolution
JOURNAL OF ANTIBIOTICS
2011; 64 (1): 59-64
Abstract
Erythromycin and related macrolide antibiotics are widely used polyketide natural products. We have evolved an engineered biosynthetic pathway in Escherichia coli that yields erythromycin analogs from simple synthetic precursors. Multiple rounds of mutagenesis and screening led to the identification of new mutant strains with improved efficiency for precursor-directed biosynthesis. Genetic and biochemical analysis suggested that the phenotypically relevant alterations in these mutant strains were localized exclusively to the host-vector system, and not to the polyketide synthase. We also demonstrate the utility of this improved system through engineered biosynthesis of a novel alkynyl erythromycin derivative with comparable antibacterial activity to its natural counterpart. In addition to reinforcing the power of directed evolution for engineering macrolide biosynthesis, our studies have identified a new lead substance for investigating structure-function relationships in the bacterial ribosome.
View details for DOI 10.1038/ja.2010.129
View details for Web of Science ID 000287072300009
View details for PubMedID 21081955
View details for PubMedCentralID PMC3030684
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Biosynthetic Engineering of Antibacterial Natural Products
EMERGING TRENDS IN ANTIBACTERIAL DISCOVERY: ANSWERING THE CALL TO ARMS
2011: 171–92
View details for Web of Science ID 000293200400008
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Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (51): 22066-22071
Abstract
Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology. Using both experimental and computational approaches, the molecular basis for KS-ACP interactions in the 6-deoxyerythronolide B synthase has been decoded. Surprisingly, KS-ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases.
View details for DOI 10.1073/pnas.1014081107
View details for Web of Science ID 000285521800027
View details for PubMedID 21127271
View details for PubMedCentralID PMC3009775
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Stereospecificity of the Dehydratase Domain of the Erythromycin Polyketide Synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2010; 132 (42): 14697-14699
Abstract
The dehydratase (DH) domain of module 4 of the 6-deoxyerythronolide B synthase (DEBS) has been shown to catalyze an exclusive syn elimination/syn addition of water. Incubation of recombinant DH4 with chemoenzymatically prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-ACP) gave the dehydration product 3-ACP. Similarly, incubation of DH4 with synthetic 3-ACP resulted in the reverse enzyme-catalyzed hydration reaction, giving an ∼3:1 equilbrium mixture of 2a-ACP and 3-ACP. Incubation of a mixture of propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the DEBS β-ketoacyl synthase-acyl transferase [KS6][AT6] didomain, DEBS ACP6, and the ketoreductase domain from tylactone synthase module 1 (TYLS KR1) generated in situ anti-2a-ACP that underwent DH4-catalyzed syn dehydration to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP, syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7 (RIFS KR7), respectively. Similarly, incubation of a mixture of (2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester (2b-SNAC), methylmalonyl-CoA, and NADPH with DEBS [KS6][AT6], DEBS ACP6, and TYLS KR1 gave anti-(2R,3R)-6-ACP that underwent syn dehydration catalyzed by DEBS DH4 to give (4R,5R)-(E)-2,4-dimethyl-5-hydroxy-hept-2-enoyl-ACP (7-ACP). The structure and stereochemistry of 7 were established by GC-MS and LC-MS comparison of the derived methyl ester 7-Me to a synthetic sample of 7-Me.
View details for DOI 10.1021/ja107344h
View details for Web of Science ID 000283403200003
View details for PubMedID 20925342
View details for PubMedCentralID PMC2959128
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A Balancing Act for Taxol Precursor Pathways in E. coli
SCIENCE
2010; 330 (6000): 44-45
View details for DOI 10.1126/science.1195014
View details for Web of Science ID 000282334500025
View details for PubMedID 20929799
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In Living Color: Bacterial Pigments as an Untapped Resource in the Classroom and Beyond
PLOS BIOLOGY
2010; 8 (10)
View details for DOI 10.1371/journal.pbio.1000510
View details for Web of Science ID 000283495100011
View details for PubMedID 20957190
View details for PubMedCentralID PMC2950131
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Thematic Minireview Series on Antibacterial Natural Products: New Tricks for Old Dogs
JOURNAL OF BIOLOGICAL CHEMISTRY
2010; 285 (36): 27499-27499
View details for DOI 10.1074/jbc.R110.150417
View details for Web of Science ID 000281404100002
View details for PubMedID 20522550
View details for PubMedCentralID PMC2934614
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Redox Regulation of Transglutaminase 2 Activity
JOURNAL OF BIOLOGICAL CHEMISTRY
2010; 285 (33): 25402-25409
Abstract
Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys(230), Cys(370), and Cys(371) that is involved in oxidative inactivation of TG2. Within this triad, Cys(370) was found to participate in disulfide bonds with both Cys(230) and its neighbor, Cys(371). Notably, Ca(2+) was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys(230) appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys(370)-Cys(371) through formation of the Cys(230)-Cys(370) disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys(230) is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.
View details for DOI 10.1074/jbc.M109.097162
View details for Web of Science ID 000280682400033
View details for PubMedID 20547769
View details for PubMedCentralID PMC2919103
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Mechanism and Engineering of Polyketide Chain Initiation in Fredericamycin Biosynthesis
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2010; 132 (26): 8831-?
Abstract
The ability to incorporate atypical primer units through the use of dedicated initiation polyketide synthase (PKS) modules offers opportunities to expand the molecular diversity of polyketide natural products. Here we identify the initiation PKS module responsible for hexadienyl priming of the antibiotic fredericamycin and investigate its biochemical properties. We also exploit this PKS module for the design and in vivo biosynthesis of unusually primed analogues of a representative polyketide product, thereby emphasizing its utility to the metabolic engineer.
View details for DOI 10.1021/ja102517q
View details for Web of Science ID 000279561200009
View details for PubMedID 20540492
View details for PubMedCentralID PMC2904946
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Cloning, Sequencing, Heterologous Expression, and Mechanistic Analysis of A-74528 Biosynthesis
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2010; 132 (26): 9122-9128
Abstract
A-74528 is a recently discovered natural product of Streptomyces sp. SANK 61196 that inhibits 2',5'-oligoadenylate phosphodiesterase (2'-PDE), a key regulatory enzyme of the interferon pathway. Inhibition of 2'-PDE by A-74528 reduces viral replication, and therefore shows promise as a new type of antiviral drug. The complete A-74528 gene cluster, comprising 29 open reading frames, was cloned and sequenced, and shown to possess a type II polyketide synthase (PKS) at its core. Its identity was confirmed by analysis of a mutant generated by targeted disruption of a PKS gene, and by functional expression in a heterologous Streptomyces host. Remarkably, it showed exceptional end-to-end sequence identity to the gene cluster responsible for biosynthesis of fredericamycin A, a structurally unrelated antitumor antibiotic with a distinct mode of action. Whereas the fredericamycin producing strain, Streptomyces griseus, produced undetectable quantities of A-74528, the A-74528 gene cluster was capable of producing both antibiotics. The biosynthetic roles of three genes, including one that represents the only qualitative difference between the two gene clusters, were investigated by targeted gene disruption. The implications for the evolution of antibiotics with different biological activities from the same gene cluster are discussed.
View details for DOI 10.1021/ja102519v
View details for Web of Science ID 000279561200070
View details for PubMedID 20550125
View details for PubMedCentralID PMC2896501
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Quantitative analysis and engineering of fatty acid biosynthesis in E. coli
METABOLIC ENGINEERING
2010; 12 (4): 378-386
Abstract
Fatty acids are central hydrocarbon intermediates in the biosynthesis of diesel from renewable sources. We have engineered an Escherichia coli cell line that produces 4.5 g/L/day total fatty acid in a fed-batch fermentation. However, further enhancement of fatty acid biosynthesis in this cell line proved unpredictable. To develop a more reliable engineering strategy, a cell-free system was developed that enabled direct, quantitative investigation of fatty acid biosynthesis and its regulation in E. coli. Using this system, the strong dependence of fatty acid synthesis on malonyl-CoA availability and several important phenomena in fatty acid synthesis were verified. Results from this cell-free system were confirmed via the generation and analysis of metabolically engineered strains of E. coli. Our quantitative findings highlight the enormous catalytic potential of the E. coli fatty acid biosynthetic pathway, and target specific steps for protein and metabolic engineering to enhance the catalytic conversion of glucose into biodiesel.
View details for DOI 10.1016/j.ymben.2010.02.003
View details for Web of Science ID 000278547700007
View details for PubMedID 20184964
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Characterization of transglutaminase type II role in dendritic cell differentiation and function
JOURNAL OF LEUKOCYTE BIOLOGY
2010; 88 (1): 181-188
Abstract
DCs play an essential role in the endotoxic shock, and their profound depletion occurs in septic patients and septic mice. TG2(-/-) mice are more resistant to the endotoxic shock induced by LPS. Here, we studied the cellular and molecular basis of this effect, analyzing the role of the enzyme in DC maturation and function. We show that TG2 is up-regulated drastically during the final, functional maturation of DCs consequent to LPS treatment. In keeping with this finding, the inhibition of the enzyme cross-linking activity determines the impairment of DC function highlighted by wide phenotypic changes associated with a reduced production of cytokines (IL-10, IL-12) after LPS treatment and a lower ability to induce IFN-gamma production by naïve T cells. The in vivo analysis of DCs obtained from TG2(-/-) mice confirmed that the enzyme ablation leads to an impairment of DC maturation and their reduced responsiveness to LPS treatment. In fact, a marked decrease in DC death, TLR4 down-regulation, and impaired up-regulation of MHCII and CD86 were observed in TG2(-/-) mice. Taken together, these data suggest that TG2 plays an important role in regulating the response of DCs to LPS and could be a candidate target for treating endotoxin-induced sepsis.
View details for DOI 10.1189/jlb.1009691
View details for Web of Science ID 000279356700019
View details for PubMedID 20371597
View details for PubMedCentralID PMC3210574
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Novel aspects of quantitation of immunogenic wheat gluten peptides by liquid chromatography-mass spectrometry/mass spectrometry
JOURNAL OF CHROMATOGRAPHY A
2010; 1217 (25): 4167-4183
Abstract
A novel, specific and sensitive non-immunological liquid chromatography-mass spectrometry (LC-MS) based assay has been developed to detect and quantify trace levels of wheat gluten in food and consumer products. Detection and quantification of dietary gluten is important, because gluten is a principle trigger of a variety of immune diseases including food allergies and intolerances. One such disease, celiac sprue, can cause intestinal inflammation and enteropathy in patients who are exposed to dietary gluten. At present, immunochemistry is the leading analytical method for gluten detection in food. Consequently, enzyme-linked immunosorbent assays (ELISAs), such as the sandwich or competitive type assays, are the only commercially available methods to ensure that food and consumer products are accurately labeled as gluten-free. The availability of a comprehensive, fast and economic alternative to the immunological ELISA may also facilitate research towards the development of new drugs, therapies and food processing technologies to aid patients with gluten intolerances and for gluten-free labeling and certification purposes. LC-MS is an effective and efficient analytical technique for the study of cereal grain proteins and to quantify trace levels of targeted dietary gluten peptides in complex matrices. Initial efforts in this area afforded the unambiguous identification and structural characterization of six unique physiologically relevant wheat gluten peptides. This paper describes the development and optimization of an LC-MS/MS method that attempts to provide the best possible accuracy and sensitivity for the quantitative detection of trace levels of these six peptides in various food and consumer products. The overall performance of this method was evaluated using native cereal grains. Experimental results demonstrated that this method is capable of detecting and quantifying select target peptides in food over a range from 10pg/mg to 100ng/mg (corresponding to approximately 0.01-100ppm). Limits of detection (LOD) and quantification (LOQ) for the six target peptides were determined to range from 1 to 30pg/mg and 10-100pg/mg respectively. Reproducibility of the assay was demonstrated by evaluation of calibration data as well as data collected from the analysis of quality control standards over a period of four consecutive days. The average coefficient of determination (R(2)) for each peptide was consistently found to be >0.995 with residuals ranging from approximately 80% to 110%. Spike recovery data for each peptide in various matrices was evaluated at a concentration level near the approximate LOQ for each, as well as at higher concentration levels (30 and 60ng/mg). The average range of accuracy of detection for all peptides at the lower concentration level was determined to be 90% (+/-11), while accuracy at the 30 and 60ng/mg levels was 98% (+/-5%) and 98% (+/-3%), respectively. The usefulness and capabilities of this method are presented in a practical application to prospectively screen a variety of common commercially available (native and processed) gluten-containing and gluten-free foods and products.
View details for DOI 10.1016/j.chroma.2010.01.067
View details for Web of Science ID 000278779000026
View details for PubMedID 20181349
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Inhibition of Tubulogenesis and of Carcinogen-mediated Signaling in Brain Endothelial Cells Highlight the Antiangiogenic Properties of a Mumbaistatin Analog
CHEMICAL BIOLOGY & DRUG DESIGN
2010; 75 (5): 481-488
Abstract
A better understanding of the metabolic adaptations of the vascular endothelial cells (EC) that mediate tumor vascularization would help the development of new drugs and therapies. Novel roles in cell survival and metabolic adaptation to hypoxia have been ascribed to the microsomal glucose-6-phosphate translocase (G6PT). While antitumorigenic properties of G6PT inhibitors such as chlorogenic acid (CHL) have been documented, those of the G6PT inhibitor and semi-synthetic analog AD4-015 of the polyketide mumbaistatin are not understood. In the present study, we evaluated the in vitro antiangiogenic impact of AD4-015 on human brain microvascular endothelial cells (HBMEC), which play an essential role as structural and functional components in tumor angiogenesis. We found that in vitro HBMEC migration and tubulogenesis were reduced by AD4-015 but not by CHL. The mumbaistatin analog significantly inhibited the phorbol 12-myristate 13-acetate (PMA)-induced matrix-metalloproteinase (MMP)-9 secretion and gene expression as assessed by zymography and RT-PCR. PMA-mediated cell signaling leading to cyclooxygenase (COX)-2 expression and IkappaB downregulation was also inhibited, further confirming AD4-015 as a cell signaling inhibitor in tumor promoting conditions. G6PT functions may therefore account for the metabolic flexibility that enables EC-mediated neovascularization. This process could be specifically targeted within the vasculature of developing brain tumors by G6PT inhibitors.
View details for DOI 10.1111/j.1747-0285.2010.00961.x
View details for Web of Science ID 000275949500006
View details for PubMedID 20486934
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Visualization of Transepithelial Passage of the Immunogenic 33-Residue Peptide from alpha-2 Gliadin in Gluten-Sensitive Macaques
PLOS ONE
2010; 5 (4)
Abstract
Based on clinical, histopathological and serological similarities to human celiac disease (CD), we recently established the rhesus macaque model of gluten sensitivity. In this study, we further characterized this condition based on presence of anti-tissue transglutaminase 2 (TG2) antibodies, increased intestinal permeability and transepithelial transport of a proteolytically resistant, immunotoxic, 33-residue peptide from alpha(2)-gliadin in the distal duodenum of gluten-sensitive macaques.Six rhesus macaques were selected for study from a pool of 500, including two healthy controls and four gluten-sensitive animals with elevated anti-gliadin or anti-TG2 antibodies as well as history of non-infectious chronic diarrhea. Pediatric endoscope-guided pinch biopsies were collected from each animal's distal duodenum following administration of a gluten-containing diet (GD) and again after remission by gluten-free diet (GFD). Control biopsies always showed normal villous architecture, whereas gluten-sensitive animals on GD exhibited histopathology ranging from mild lymphocytic infiltration to villous atrophy, typical of human CD. Immunofluorescent microscopic analysis of biopsies revealed IgG+ and IgA+ plasma-like cells producing antibodies that colocalized with TG2 in gluten-sensitive macaques only. Following instillation in vivo, the Cy-3-labeled 33-residue gluten peptide colocalized with the brush border protein villin in all animals. In a substantially enteropathic macaque with "leaky" duodenum, the peptide penetrated beneath the epithelium into the lamina propria.The rhesus macaque model of gluten sensitivity not only resembles the histopathology of CD but it also may provide a model for studying intestinal permeability in states of epithelial integrity and disrepair.
View details for DOI 10.1371/journal.pone.0010228
View details for Web of Science ID 000276853800018
View details for PubMedID 20419103
View details for PubMedCentralID PMC2856682
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Protein-Protein Recognition between Acyltransferases and Acyl Carrier Proteins in Multimodular Polyketide Synthases
BIOCHEMISTRY
2010; 49 (1): 95-102
Abstract
Acyltransferase (AT) domains of multimodular polyketide synthases are the primary gatekeepers for stepwise incorporation of building blocks into a growing polyketide chain. Each AT domain has two substrates, an alpha-carboxylated CoA thioester (e.g., malonyl-CoA or methylmalonyl-CoA) and an acyl carrier protein (ACP). Whereas the acyl-CoA specificity of AT domains has been extensively investigated, little is known about their ACP specificity. Guided by recent high-resolution structural insights, we have systematically probed the protein-protein interactions between AT domains, ACP domains, and the linkers that flank AT domains. Representative AT domains of the 6-deoxyerythronolide B synthase (DEBS) have greater than 10-fold specificity for their cognate ACP substrates as compared to other ACP domains from the same synthase. Both of the flanking (N- and C-terminal) linkers of an AT domain contributed to the efficiency and specificity of transacylation. As a frame of reference, the activity and specificity of a stand-alone AT domain from the "AT-less" disorazole synthase (DSZS) were also quantified. The activity (k(cat)/K(M)) of this AT was >250-fold higher than the corresponding values for DEBS AT domains. Although the AT from DSZS discriminated modestly against ACP domains from DEBS, it exhibited >40-fold higher activity in trans in the presence of these heterologous substrates than their natural AT domains. Our results highlight the opportunity for regioselective modification of a polyketide backbone by in trans complementation of inactivated AT domains. They also reinforce the need for more careful consideration of protein-protein interactions in the engineering of these assembly line enzymes.
View details for DOI 10.1021/bi901826g
View details for Web of Science ID 000273267300012
View details for PubMedID 19921859
View details for PubMedCentralID PMC2805051
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Genetic Engineering of Escherichia coli for Biofuel Production
ANNUAL REVIEW OF GENETICS, VOL 44
2010; 44: 53-69
Abstract
In order to mitigate climate change without adversely affecting global energy supply, there is growing interest in the possibility of producing transportation fuels from renewable sources via microbial fermentation. Central to this challenge is the design of biocatalysts that can efficiently convert cheap lignocellulosic raw materials into liquid fuels. Owing to the wealth of genetic and metabolic knowledge associated with Escherichia coli, this bacterium is the most convenient starting point for engineering microbial catalysts for biofuel production. Here, we review the range of liquid fuels that can be produced in E. coli and discuss the underlying biochemistry that enables these metabolic products. The fundamental and technological challenges encountered in the development of efficient fermentation processes for biofuel production are highlighted. The example of biodiesel is a particularly illustrative case study and is therefore discussed in detail.
View details for DOI 10.1146/annurev-genet-102209-163440
View details for Web of Science ID 000286042600003
View details for PubMedID 20822440
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The Biochemical Basis for Stereochemical Control in Polyketide Biosynthesis
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2009; 131 (51): 18501-18511
Abstract
One of the most striking features of complex polyketides is the presence of numerous methyl- and hydroxyl-bearing stereogenic centers. To investigate the biochemical basis for the control of polyketide stereochemistry and to establish the timing and mechanism of the epimerization at methyl-bearing centers, a series of incubations was carried out using reconstituted components from a variety of modular polyketide synthases. In all cases the stereochemistry of the product was directly correlated with the intrinsic stereospecificity of the ketoreductase domain, independent of the particular chain elongation domains that were used, thereby establishing that methyl group epimerization, when it does occur, takes place after ketosynthase-catalyzed chain elongation. The finding that there were only minor differences in the rates of product formation observed for parallel incubations using an epimerizing ketoreductase domain and the nonepimerizing ketoreductase domain supports the proposal that the epimerization is catalyzed by the ketoreductase domain itself.
View details for DOI 10.1021/ja908296m
View details for Web of Science ID 000273615800063
View details for PubMedID 19928853
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In Vivo and In Vitro Analysis of the Hedamycin Polyketide Synthase
CHEMISTRY & BIOLOGY
2009; 16 (11): 1197-1207
Abstract
Hedamycin is an antitumor polyketide antibiotic with unusual biosynthetic features. Earlier sequence analysis of the hedamycin biosynthetic gene cluster implied a role for type I and type II polyketide synthases (PKSs). We demonstrate that the hedamycin minimal PKS can synthesize a dodecaketide backbone. The ketosynthase (KS) subunit of this PKS has specificity for both type I and type II acyl carrier proteins (ACPs) with which it collaborates during chain initiation and chain elongation, respectively. The KS receives a C(6) primer unit from the terminal ACP domain of HedU (a type I PKS protein) directly and subsequently interacts with the ACP domain of HedE (a type II PKS protein) during the process of chain elongation. HedE is a bifunctional protein with both ACP and aromatase activity. Its aromatase domain can modulate the chain length specificity of the minimal PKS. Chain length can also be influenced by HedA, the C-9 ketoreductase. While co-expression of the hedamycin minimal PKS and a chain-initiation module from the R1128 PKS yields an isobutyryl-primed decaketide, the orthologous PKS subunits from the hedamycin gene cluster itself are unable to prime the minimal PKS with a nonacetyl starter unit. Our findings provide new insights into the mechanism of chain initiation and elongation by type II PKSs.
View details for DOI 10.1016/j.chembiol.2009.11.005
View details for Web of Science ID 000273207900013
View details for PubMedID 19942143
View details for PubMedCentralID PMC2795786
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Structures and Mechanisms of Polyketide Synthases
JOURNAL OF ORGANIC CHEMISTRY
2009; 74 (17): 6416-6420
Abstract
Nearly a quarter-century ago, the advent of molecular genetic tools in the field of natural product biosynthesis led to the remarkable revelation that the genes responsible for the biosynthesis, regulation, and self-resistance of complex polyketide antibiotics were clustered in the genomes of the bacteria that produced these compounds. This in turn facilitated rapid cloning and sequencing of genes encoding a number of polyketide synthases (PKSs). By now, it is abundantly clear that, notwithstanding extraordinary architectural and biocatalytic diversity, all PKSs are evolutionarily related enzyme assemblies. As such, understanding the molecular logic for the biosynthesis of literally thousands of amazing polyketide natural products made by nature can benefit enormously from detailed investigations into a few "model systems". For nearly the past two decades, our laboratory has focused its efforts on two such PKSs. One of them synthesizes two polyketides in approximately equal ratios, SEK4 and SEK4b, and both shunt products from the pathway that leads to the biosynthesis of the pigmented antibiotic actinorhodin. The other synthesizes 6-deoxyerythronolide B, the first isolable intermediate in the biosynthetic pathway for the widely used antibacterial agent erythromycin. Our present-day knowledge of the structures and mechanisms of these two PKSs is summarized here.
View details for DOI 10.1021/jo9012089
View details for Web of Science ID 000269257800002
View details for PubMedID 19711990
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Noninflammatory Gluten Peptide Analogs as Biomarkers for Celiac Sprue
CHEMISTRY & BIOLOGY
2009; 16 (8): 868-881
Abstract
New tools are needed for managing celiac sprue, a lifelong immune disease of the small intestine. Ongoing drug trials are also prompting a search for noninvasive biomarkers of gluten-induced intestinal change. We have synthesized and characterized noninflammatory gluten peptide analogs in which key Gln residues are replaced by Asn or His. Like their proinflammatory counterparts, these biomarkers are resistant to gastrointestinal proteases, susceptible to glutenases, and permeable across enterocyte barriers. Unlike gluten peptides, however, they are not appreciably recognized by transglutaminase, HLA-DQ2, or disease-specific T cells. In vitro and animal studies show that the biomarkers can detect intestinal permeability changes as well as glutenase-catalyzed gastric detoxification of gluten. Accordingly, controlled clinical studies are warranted to evaluate the use of these peptides as probes for abnormal intestinal permeability in celiac patients and for glutenase efficacy in clinical trials and practice.
View details for DOI 10.1016/j.chembiol.2009.07.009
View details for Web of Science ID 000269718200011
View details for PubMedID 19716477
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Modular Biocatalysts
AICHE JOURNAL
2009; 55 (8): 1926-1929
View details for DOI 10.1002/aic.11981
View details for Web of Science ID 000268413800002
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A Food-Grade Enzyme Preparation with Modest Gluten Detoxification Properties
PLOS ONE
2009; 4 (7)
Abstract
Celiac sprue is a life-long disease characterized by an intestinal inflammatory response to dietary gluten. A gluten-free diet is an effective treatment for most patients, but accidental ingestion of gluten is common, leading to incomplete recovery or relapse. Food-grade proteases capable of detoxifying moderate quantities of dietary gluten could mitigate this problem.We evaluated the gluten detoxification properties of two food-grade enzymes, aspergillopepsin (ASP) from Aspergillus niger and dipeptidyl peptidase IV (DPPIV) from Aspergillus oryzae. The ability of each enzyme to hydrolyze gluten was tested against synthetic gluten peptides, a recombinant gluten protein, and simulated gastric digests of whole gluten and whole-wheat bread. Reaction products were analyzed by mass spectrometry, HPLC, ELISA with a monoclonal antibody that recognizes an immunodominant gluten epitope, and a T cell proliferation assay.ASP markedly enhanced gluten digestion relative to pepsin, and cleaved recombinant alpha2-gliadin at multiple sites in a non-specific manner. When used alone, neither ASP nor DPPIV efficiently cleaved synthetic immunotoxic gluten peptides. This lack of specificity for gluten was especially evident in the presence of casein, a competing dietary protein. However, supplementation of ASP with DPPIV enabled detoxification of moderate amounts of gluten in the presence of excess casein and in whole-wheat bread. ASP was also effective at enhancing the gluten-detoxifying efficacy of cysteine endoprotease EP-B2 under simulated gastric conditions.Clinical studies are warranted to evaluate whether a fixed dose ratio combination of ASP and DPPIV can provide near-term relief for celiac patients suffering from inadvertent gluten exposure. Due to its markedly greater hydrolytic activity against gluten than endogenous pepsin, food-grade ASP may also augment the activity of therapeutically relevant doses of glutenases such as EP-B2 and certain prolyl endopeptidases.
View details for DOI 10.1371/journal.pone.0006313
View details for Web of Science ID 000268147200010
View details for PubMedID 19621078
View details for PubMedCentralID PMC2708912
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Interferon-gamma Released by Gluten-Stimulated Celiac Disease-Specific Intestinal T Cells Enhances the Transepithelial Flux of Gluten Peptides
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2009; 329 (2): 657-668
Abstract
Celiac sprue is a T-cell-mediated enteropathy elicited in genetically susceptible individuals by dietary gluten proteins. To initiate and propagate inflammation, proteolytically resistant gluten peptides must be translocated across the small intestinal epithelium and presented to DQ2-restricted T cells, but the effectors enabling this translocation under normal and inflammatory conditions are not well understood. We demonstrate that a fluorescently labeled antigenic 33-mer gluten peptide is translocated intact across a T84 cultured epithelial cell monolayer and that preincubation of the monolayer with media from gluten-stimulated, celiac patient-derived intestinal T cells enhances the apical-to-basolateral flux of this peptide in a dose-dependent, saturable manner. The permeability-enhancing activity of activated T-cell media is inhibited by blocking antibodies against either interferon-gamma or its receptor and is recapitulated using recombinant interferon-gamma. At saturating levels of interferon-gamma, activated T-cell media does not further increase transepithelial peptide flux, indicating the primacy of interferon-gamma as an effector of increased epithelial permeability during inflammation. Reducing the assay temperature to 4 degrees C reverses the effect of interferon-gamma but does not reduce basal peptide flux occurring in the absence of interferon-gamma, suggesting active transcellular transport of intact peptides is increased during inflammation. A panel of disease-relevant gluten peptides exhibited an inverse correlation between size and transepithelial flux but no apparent sequence constraints. Anti-interferon-gamma therapy may mitigate the vicious cycle of gluten-induced interferon-gamma secretion and interferon-gamma-mediated enhancement of gluten peptide flux but is unlikely to prevent translocation of gluten peptides in the absence of inflammatory conditions.
View details for DOI 10.1124/jpet.108.148007
View details for Web of Science ID 000265444000029
View details for PubMedID 19218531
View details for PubMedCentralID PMC2672868
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Biosynthesis of Aromatic Polyketides in Bacteria
ACCOUNTS OF CHEMICAL RESEARCH
2009; 42 (5): 631-639
Abstract
Natural products, produced chiefly by microorganisms and plants, can be large and structurally complex molecules. These molecules are manufactured by cellular assembly lines, in which enzymes construct the molecules in a stepwise fashion. The means by which enzymes interact and work together in a modular fashion to create diverse structural features has been an active area of research; the work has provided insight into the fine details of biosynthesis. A number of polycyclic aromatic natural products--including several noteworthy anticancer, antibacterial, antifungal, antiviral, antiparasitic, and other medicinally significant substances--are synthesized by polyketide synthases (PKSs) in soil-borne bacteria called actinomycetes. Concerted biosynthetic, enzymological, and structural biological investigations into these modular enzyme systems have yielded interesting mechanistic insights. A core module called the minimal PKS is responsible for synthesizing a highly reactive, protein-bound poly-beta-ketothioester chain. In the absence of other enzymes, the minimal PKS also catalyzes chain initiation and release, yielding an assortment of polycyclic aromatic compounds. In the presence of an initiation PKS module, polyketide backbones bearing additional alkyl, alkenyl, or aryl primer units are synthesized, whereas a range of auxiliary PKS enzymes and tailoring enzymes convert the product of the minimal PKS into the final natural product. In this Account, we summarize the knowledge that has been gained regarding this family of PKSs through recent investigations into the biosynthetic pathways of two natural products, actinorhodin and R1128 (A-D). We also discuss the practical relevance of these fundamental insights for the engineered biosynthesis of new polycyclic aromatic compounds. With a deeper understanding of the biosynthetic process in hand, we can assert control at various stages of molecular construction and thus introduce unnatural functional groups in the process. The metabolic engineer affords a number of new avenues for creating novel molecular structures that will likely have properties akin to their fully natural cousins.
View details for DOI 10.1021/ar8002249
View details for Web of Science ID 000266238500006
View details for PubMedID 19292437
View details for PubMedCentralID PMC2696626
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Revisiting the modularity of modular polyketide synthases
CURRENT OPINION IN CHEMICAL BIOLOGY
2009; 13 (2): 135-143
Abstract
Modularity is a highly sought after feature in engineering design. A modular catalyst is a multi-component system whose parts can be predictably interchanged for functional flexibility and variety. Nearly two decades after the discovery of the first modular polyketide synthase (PKS), we critically assess PKS modularity in the face of a growing body of atomic structural and in vitro biochemical investigations. Both the architectural modularity and the functional modularity of this family of enzymatic assembly lines are reviewed, and the fundamental challenges that lie ahead for the rational exploitation of their full biosynthetic potential are discussed.
View details for DOI 10.1016/j.cbpa.2008.12.018
View details for Web of Science ID 000266345400002
View details for PubMedID 19217343
View details for PubMedCentralID PMC2737389
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Evidence for Transcriptional Regulation of the Glucose-6-Phosphate Transporter by HIF-1 alpha: Targeting G6PT with Mumbaistatin Analogs in Hypoxic Mesenchymal Stromal Cells
STEM CELLS
2009; 27 (3): 489-497
Abstract
Mesenchymal stromal cell (MSC) markers are expressed on brain tumor-initiating cells involved in the development of hypoxic glioblastoma. Given that MSCs can survive hypoxia and that the glucose-6-phosphate transporter (G6PT) provides metabolic control that contributes to MSC mobilization and survival, we investigated the effects of low oxygen (1.2% O(2)) exposure on G6PT gene expression. We found that MSCs significantly expressed G6PT and the glucose-6-phosphatase catalytic subunit beta, whereas expression of the glucose-6-phosphatase catalytic subunit alpha and the islet-specific glucose-6-phosphatase catalytic subunit-related protein was low to undetectable. Analysis of the G6PT promoter sequence revealed potential binding sites for hypoxia inducible factor (HIF)-1alpha and for the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT), AhR:ARNT. In agreement with this, hypoxia and the hypoxia mimetic cobalt chloride induced the expression of G6PT, vascular endothelial growth factor (VEGF), and HIF-1alpha. Gene silencing of HIF-1alpha prevented G6PT and VEGF induction in hypoxic MSCs whereas generation of cells stably expressing HIF-1alpha resulted in increased endogenous G6PT gene expression. A semisynthetic analog of the polyketide mumbaistatin, a potent G6PT inhibitor, specifically reduced MSC-HIF-1alpha cell survival. Collectively, our data suggest that G6PT may account for the metabolic flexibility that enables MSCs to survive under conditions characterized by hypoxia and could be specifically targeted within developing tumors.
View details for DOI 10.1634/stemcells.2008-0855
View details for Web of Science ID 000264706900001
View details for PubMedID 19074414
View details for PubMedCentralID PMC2728688
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THE DIVERSITY OF NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
NATO Advanced Study Institute on Biophysics and the Challenges of Emerging Threats
SPRINGER. 2009: 65–81
View details for Web of Science ID 000267755300005
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Protein engineering of improved prolyl endopeptidases for celiac sprue therapy
PROTEIN ENGINEERING DESIGN & SELECTION
2008; 21 (12): 699-707
Abstract
Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concentration) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clinical relevance. Through two rounds of iterative mutagenesis and analysis, variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, especially in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and analytical cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacologically significant improvements in gluten detoxification activity.
View details for DOI 10.1093/protein/gzn050
View details for Web of Science ID 000260979000002
View details for PubMedID 18836204
View details for PubMedCentralID PMC2583057
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Overproduction of free fatty acids in E. coli: Implications for biodiesel production
METABOLIC ENGINEERING
2008; 10 (6): 333-339
Abstract
Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methyl esters of fatty acids) is exclusively derived from plant oils. Slow cycle times for engineering oilseed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels. By introducing four distinct genetic changes into the E. coli genome, we have engineered an efficient producer of fatty acids. Under fed-batch, defined media fermentation conditions, 2.5 g/L fatty acids were produced by this metabolically engineered E. coli strain, with a specific productivity of 0.024 g/h/g dry cell mass and a peak conversion efficiency of 4.8% of the carbon source into fatty acid products. At least 50% of the fatty acids produced were present in the free acid form.
View details for DOI 10.1016/j.ymben.2008.08.006
View details for Web of Science ID 000261946000006
View details for PubMedID 18812230
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Tissue transgluaminase 2 expression in meningiomas
JOURNAL OF NEURO-ONCOLOGY
2008; 90 (2): 125-132
Abstract
Meningiomas are common intracranial tumors that occur in extra-axial locations, most often over the cerebral convexities or along the skull-base. Although often histologically benign these tumors frequently present challenging clinical problems. Primary clinical management of patients with symptomatic tumors is surgical resection. Radiation treatment may arrest growth or delay recurrence of these tumors, however, meningioma cells are generally resistant to apoptosis after treatment with radiation. Tumor cells are known to alter their expression of proteins that interact in the ECM to provide signals important in tumor progression. One such protein, fibronectin, is expressed in elevated levels in the ECM in a number of tumors including meningiomas. We recently reported that levels of both extracellular fibronectin and tissue transglutaminase 2 (TG2) were increased in glioblastomas. We examined the expression of fibronectin and its association TG2 in meningiomas. Both fibronectin and TG2 were strongly expressed in all meningiomas studied. TG2 activity was markedly elevated in meningiomas, and TG2 was found to co-localize with fibronectin. Treatment of meningiomas with the small molecule TG2 inhibitor, KCC009, inhibited the binding of TG2 to fibronectin and blocked disposition of linear strands of fibronectin in the ECM. KCC009 treatment promoted apoptosis and enhanced radiation sensitivity both in cultured IOMM-Lee meningioma cells and in meningioma tumor explants. These findings support a potential protective role for TG2 in meningiomas.
View details for DOI 10.1007/s11060-008-9642-1
View details for Web of Science ID 000259672100001
View details for PubMedID 18587533
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Stereospecificity of ketoreductase domains 1 and 2 of the tylactone modular polyketide synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2008; 130 (35): 11598-?
Abstract
Tylactone synthase (TYLS) is a modular polyketide synthase that catalyzes the formation of tylactone (1), the parent aglycone precursor of the macrolide antibiotic tylosin. TYLS modules 1 and 2 are responsible for the generation of antidiketide and triketide intermediates, respectively, each bound to an acyl carrier protein (ACP) domain. Each module harbors a ketoreductase (KR) domain. The stereospecificity of TYLS KR1 and TYLS KR2 has been determined by incubating each of the recombinant ketoreductase domains with reconstituted ketosynthase-acyltransferase [KS][AT] and ACP domains from the 6-deoxyerythronolide B synthase (DEBS) in the presence of the N-acetylcysteamine thioester of syn-(2S,3R)-2-methyl-3-hydroxypentanoate (6), methylmalonyl-CoA, and NADPH resulting in the exclusive formation of the ACP-bound (2R,3R,4S,5R)-2,4-methyl-3,5-dihydroxyhepanoyl triketide, as established by GC-MS analysis of the TMS ether of the derived triketide lactone 7. Both TYLS KR1 and KR2 therefore catalyze the stereospecific reduction of the 2-methyl-3-ketoacyl-ACP substrate from the re-face, with specificity for the reduction of the (2R)-methyl (D) diastereomer. The dehydration that is catalyzed by the dehydratase (DH) domains of TYLS module 2 to give the unsaturated (2E,4S,5R)-2,4-dimethyl-5-hydroxyhept-2-enoyl-ACP2 is therefore a syn elimination of water.
View details for DOI 10.1021/ja804453p
View details for Web of Science ID 000258792000018
View details for PubMedID 18693734
View details for PubMedCentralID PMC2654278
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Biochemistry - Fit for an enzyme
NATURE
2008; 454 (7206): 832-833
View details for DOI 10.1038/454832a
View details for Web of Science ID 000258398600019
View details for PubMedID 18704072
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Toward the Assessment of Food Toxicity for Celiac Patients: Characterization of Monoclonal Antibodies to a Main Immunogenic Gluten Peptide
PLOS ONE
2008; 3 (5)
Abstract
Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied.Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin.The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease.The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.
View details for DOI 10.1371/journal.pone.0002294
View details for Web of Science ID 000262268500033
View details for PubMedID 18509534
View details for PubMedCentralID PMC2386552
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Mechanism based protein crosslinking of domains from the 6-deoxyerythronolide B synthase
9th Tetrahedron Symposium on Challenges in Organic and Bioorganic Chemistry
PERGAMON-ELSEVIER SCIENCE LTD. 2008: 3034–38
Abstract
The critical role of protein-protein interactions in the chemistry of polyketide synthases is well established. However, the transient and weak nature of these interactions, in particular those involving the acyl carrier protein (ACP), has hindered efforts to structurally characterize these interactions. We describe a chemo-enzymatic approach that crosslinks the active sites of ACP and their cognate ketosynthase (KS) domains, resulting in the formation of a stable covalent adduct. This process is driven by specific protein-protein interactions between KS and ACP domains. Suitable manipulation of the reaction conditions enabled complete crosslinking of a representative KS and ACP, allowing isolation of a stable, conformationally constrained adduct suitable for high-resolution structural analysis.
View details for DOI 10.1016/j.bmcl.2008.01.073
View details for Web of Science ID 000255954000004
View details for PubMedID 18243693
View details for PubMedCentralID PMC2430738
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Transepithelial Transport and Enzymatic Detoxification of Gluten in Gluten-Sensitive Rhesus Macaques
PLOS ONE
2008; 3 (3)
Abstract
In a previous report, we characterized a condition of gluten sensitivity in juvenile rhesus macaques that is similar in many respects to the human condition of gluten sensitivity, celiac disease. This animal model of gluten sensitivity may therefore be useful toward studying both the pathogenesis and the treatment of celiac disease. Here, we perform two pilot experiments to demonstrate the potential utility of this model for studying intestinal permeability toward an immunotoxic gluten peptide and pharmacological detoxification of gluten in vivo by an oral enzyme drug candidate.Intestinal permeability was investigated in age-matched gluten-sensitive and control macaques by using mass spectrometry to detect and quantify an orally dosed, isotope labeled 33-mer gluten peptide delivered across the intestinal epithelium to the plasma. The protective effect of a therapeutically promising oral protease, EP-B2, was evaluated in a gluten-sensitive macaque by administering a daily gluten challenge with or without EP-B2 supplementation. ELISA-based antibody assays and blinded clinical evaluations of this macaque and of an age-matched control were conducted to assess responses to gluten.Labeled 33-mer peptide was detected in the plasma of a gluten-sensitive macaque, both in remission and during active disease, but not in the plasma of healthy controls. Administration of EP-B2, but not vehicle, prevented clinical relapse in response to a dietary gluten challenge. Unexpectedly, a marked increase in anti-gliadin (IgG and IgA) and anti-transglutaminase (IgG) antibodies was observed during the EP-B2 treatment phase.Gluten-sensitive rhesus macaques may be an attractive resource for investigating important aspects of celiac disease, including enhanced intestinal permeability and pharmacology of oral enzyme drug candidates. Orally dosed EP-B2 exerts a protective effect against ingested gluten. Limited data suggest that enhanced permeability of short gluten peptides generated by gastrically active glutenases may trigger an elevated antibody response, but that these antibodies are not necessarily causative of clinical illness.
View details for DOI 10.1371/journal.pone.0001857
View details for Web of Science ID 000260762400018
View details for PubMedID 18365012
View details for PubMedCentralID PMC2267209
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Extracellular Transglutaminase 2 Is Catalytically Inactive, but Is Transiently Activated upon Tissue Injury
PLOS ONE
2008; 3 (3)
Abstract
Transglutaminase 2 (TG2) is a multifunctional mammalian protein with transamidase and signaling properties. Using selective TG2 inhibitors and tagged nucleophilic amine substrates, we show that the majority of extracellular TG2 is inactive under normal physiological conditions in cell culture and in vivo. However, abundant TG2 activity was detected around the wound in a standard cultured fibroblast scratch assay. To demonstrate wounding-induced activation of TG2 in vivo, the toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly(I:C)), was injected in mice to trigger small intestinal injury. Although no TG2 activity was detected in vehicle-treated mice, acute poly(I:C) injury resulted in rapid TG2 activation in the small intestinal mucosa. Our findings provide a new basis for understanding the role of TG2 in physiology and disease.
View details for DOI 10.1371/journal.pone.0001861
View details for Web of Science ID 000260762400022
View details for PubMedID 18365016
View details for PubMedCentralID PMC2267210
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Evolution of polyketide synthases in bacteria
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2008; 105 (12): 4595-4600
Abstract
The emergence of resistant strains of human pathogens to current antibiotics, along with the demonstrated ability of polyketides as antimicrobial agents, provides strong motivation for understanding how polyketide antibiotics have evolved and diversified in nature. Insights into how bacterial polyketide synthases (PKSs) acquire new metabolic capabilities can guide future laboratory efforts in generating the next generation of polyketide antibiotics. Here, we examine phylogenetic and structural evidence to glean answers to two general questions regarding PKS evolution. How did the exceptionally diverse chemistry of present-day PKSs evolve? And what are the take-home messages for the biosynthetic engineer?
View details for Web of Science ID 000254772700014
View details for PubMedID 18250311
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A Non-Human Primate Model for Gluten Sensitivity
PLOS ONE
2008; 3 (2)
Abstract
Gluten sensitivity is widespread among humans. For example, in celiac disease patients, an inflammatory response to dietary gluten leads to enteropathy, malabsorption, circulating antibodies against gluten and transglutaminase 2, and clinical symptoms such as diarrhea. There is a growing need in fundamental and translational research for animal models that exhibit aspects of human gluten sensitivity.Using ELISA-based antibody assays, we screened a population of captive rhesus macaques with chronic diarrhea of non-infectious origin to estimate the incidence of gluten sensitivity. A selected animal with elevated anti-gliadin antibodies and a matched control were extensively studied through alternating periods of gluten-free diet and gluten challenge. Blinded clinical and histological evaluations were conducted to seek evidence for gluten sensitivity.When fed with a gluten-containing diet, gluten-sensitive macaques showed signs and symptoms of celiac disease including chronic diarrhea, malabsorptive steatorrhea, intestinal lesions and anti-gliadin antibodies. A gluten-free diet reversed these clinical, histological and serological features, while reintroduction of dietary gluten caused rapid relapse.Gluten-sensitive rhesus macaques may be an attractive resource for investigating both the pathogenesis and the treatment of celiac disease.
View details for DOI 10.1371/journal.pone.0001614
View details for Web of Science ID 000260586400007
View details for PubMedID 18286171
View details for PubMedCentralID PMC2229647
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Parallels between pathogens and gluten peptides in celiac sprue
PLOS PATHOGENS
2008; 4 (2)
Abstract
Pathogens are exogenous agents capable of causing disease in susceptible organisms. In celiac sprue, a disease triggered by partially hydrolyzed gluten peptides in the small intestine, the offending immunotoxins cannot replicate, but otherwise have many hallmarks of classical pathogens. First, dietary gluten and its peptide metabolites are ubiquitous components of the modern diet, yet only a small, genetically susceptible fraction of the human population contracts celiac sprue. Second, immunotoxic gluten peptides have certain unusual structural features that allow them to survive the harsh proteolytic conditions of the gastrointestinal tract and thereby interact extensively with the mucosal lining of the small intestine. Third, they invade across epithelial barriers intact to access the underlying gut-associated lymphoid tissue. Fourth, they possess recognition sequences for selective modification by an endogenous enzyme, transglutaminase 2, allowing for in situ activation to a more immunotoxic form via host subversion. Fifth, they precipitate a T cell-mediated immune reaction comprising both innate and adaptive responses that causes chronic inflammation of the small intestine. Sixth, complete elimination of immunotoxic gluten peptides from the celiac diet results in remission, whereas reintroduction of gluten in the diet causes relapse. Therefore, in analogy with antibiotics, orally administered proteases that reduce the host's exposure to the immunotoxin by accelerating gluten peptide destruction have considerable therapeutic potential. Last but not least, notwithstanding the power of in vitro methods to reconstitute the essence of the immune response to gluten in a celiac patient, animal models for the disease, while elusive, are likely to yield fundamentally new systems-level insights.
View details for DOI 10.1371/journal.ppat.0040034
View details for Web of Science ID 000255415900002
View details for PubMedID 18425213
View details for PubMedCentralID PMC2323203
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Transglutaminase 2 undergoes a large conformational change upon activation
PLOS BIOLOGY
2007; 5 (12): 2788-2796
Abstract
Human transglutaminase 2 (TG2), a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-A resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.
View details for DOI 10.1371/journal.pbio.0050327
View details for Web of Science ID 000251874900010
View details for PubMedID 18092889
View details for PubMedCentralID PMC2140088
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Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2007; 129 (44): 13758-13769
Abstract
6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic erythromycin. Individual DEBS modules, which contain the catalytic domains necessary for each step of polyketide chain elongation and chemical modification, can be deconstructed into constituent domains. To better understand the intrinsic stereospecificity of the ketoreductase (KR) domains, an in vitro reconstituted system has been developed involving combinations of ketosynthase (KS)-acyl transferase (AT) didomains with acyl-carrier protein (ACP) and KR domains from different DEBS modules. Incubations with (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (2) and methylmalonyl-CoA plus NADPH result in formation of a reduced, ACP-bound triketide that is converted to the corresponding triketide lactone 4 by either base- or enzyme-catalyzed hydrolysis/cyclization. A sensitive and robust GC-MS technique has been developed to assign the stereochemistry of the resulting triketide lactones, on the basis of direct comparison with synthetic standards of each of the four possible diasteromers 4a-4d. Using the [KS][AT] didomains from either DEBS module 3 or module 6 in combination with KR domains from modules 2 or 6 gave in all cases exclusively (2R,3S,4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-delta-lactone (4a). The same product was also generated by a chimeric module in which [KS3][AT3] was fused to [KR5][ACP5] and the DEBS thioesterase [TE] domain. Reductive quenching of the ACP-bound 2-methyl-3-ketoacyl triketide intermediate with sodium borohydride confirmed that in each case the triketide intermediate carried only an unepimerized d-2-methyl group. The results confirm the predicted stereospecificity of the individual KR domains, while revealing an unexpected configurational stability of the ACP-bound 2-methyl-3-ketoacyl thioester intermediate. The methodology should be applicable to the study of any combination of heterologous [KS][AT] and [KR] domains.
View details for DOI 10.1021/ja0753290
View details for Web of Science ID 000250819200064
View details for PubMedID 17918944
View details for PubMedCentralID PMC2547127
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Cyclic and dimeric gluten peptide analogues inhibiting DQ2-mediated antigen presentation in celiac disease
BIOORGANIC & MEDICINAL CHEMISTRY
2007; 15 (20): 6565-6573
Abstract
Celiac disease is an immune mediated enteropathy elicited by gluten ingestion. The disorder has a strong association with HLA-DQ2. This HLA molecule is involved in the disease pathogenesis by presenting gluten peptides to T cells. Blocking the peptide-binding site of DQ2 may be a way to treat celiac disease. In this study, two types of peptide analogues, modeled after natural gluten antigens, were studied as DQ2 blockers. (a) Cyclic peptides. Cyclic peptides containing the DQ2-alphaI gliadin epitope LQPFPQPELPY were synthesized with flanking cysteine residues introduced and subsequently crosslinked via a disulfide bond. Alternatively, cyclic peptides were prepared with stable polyethylene glycol bridges across internal lysine residues of modified antigenic peptides such as KQPFPEKELPY and LQLQPFPQPEKPYPQPEKPY. The effect of cyclization as well as the length of the spacer in the cyclic peptides on DQ2 binding and T cell recognition was analyzed. Inhibition of peptide-DQ2 recognition by the T cell receptor was observed in T cell proliferation assays. (b) Dimeric peptides. Previously we developed a new type of peptide blocker with much enhanced affinity for DQ2 by dimerizing LQLQPFPQPEKPYPQPELPY through the lysine side chains. Herein, the effect of linker length on both DQ2 binding and T cell inhibition was investigated. One dimeric peptide analogue with an intermediate linker length was found to be especially effective at inhibiting DQ2 mediated antigen presentation. The implications of these findings for the treatment of celiac disease are discussed.
View details for DOI 10.1016/j.bmc.2007.07.001
View details for Web of Science ID 000249713900008
View details for PubMedID 17681795
View details for PubMedCentralID PMC2034199
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Solution structure and proposed domain-domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase
PROTEIN SCIENCE
2007; 16 (10): 2093-2107
Abstract
Polyketides are a medicinally important class of natural products. The architecture of modular polyketide synthases (PKSs), composed of multiple covalently linked domains grouped into modules, provides an attractive framework for engineering novel polyketide-producing assemblies. However, impaired domain-domain interactions can compromise the efficiency of engineered polyketide biosynthesis. To facilitate the study of these domain-domain interactions, we have used nuclear magnetic resonance (NMR) spectroscopy to determine the first solution structure of an acyl carrier protein (ACP) domain from a modular PKS, 6-deoxyerythronolide B synthase (DEBS). The tertiary fold of this 10-kD domain is a three-helical bundle; an additional short helix in the second loop also contributes to the core helical packing. Superposition of residues 14-94 of the ensemble on the mean structure yields an average atomic RMSD of 0.64 +/- 0.09 Angstrom for the backbone atoms (1.21 +/- 0.13 Angstrom for all non-hydrogen atoms). The three major helices superimpose with a backbone RMSD of 0.48 +/- 0.10 Angstrom (0.99 +/- 0.11 Angstrom for non-hydrogen atoms). Based on this solution structure, homology models were constructed for five other DEBS ACP domains. Comparison of their steric and electrostatic surfaces at the putative interaction interface (centered on helix II) suggests a model for protein-protein recognition of ACP domains, consistent with the previously observed specificity. Site-directed mutagenesis experiments indicate that two of the identified residues influence the specificity of ACP recognition.
View details for DOI 10.1110/ps.073011407
View details for PubMedID 17893358
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Structure-based design of alpha-amido aldehyde containing gluten peptide analogues as modulators of HLA-DQ2 and transglutaminase 2
BIOORGANIC & MEDICINAL CHEMISTRY
2007; 15 (18): 6253-6261
Abstract
Complete, life-long exclusion of gluten containing foods from the diet is the only available treatment for celiac sprue, a widespread immune disease of the small intestine. Investigations into the molecular pathogenesis of celiac sprue have identified the major histocompatibility complex protein HLA-DQ2 and the multi-functional enzyme transglutaminase 2 as potential pharmacological targets. Based upon the crystal structure of HLA-DQ2, we rationally designed an aldehyde-functionalized, gluten peptide analogue as a tight-binding HLA-DQ2 ligand. Aldehyde-bearing gluten peptide analogues were also designed as high-affinity, reversible inhibitors of transglutaminase 2. By varying the side-chain length of the aldehyde-functionalized amino acid, we found that the optimal transglutaminase 2 inhibitor was 5 methylene units in length, 2 carbon atoms longer than its natural glutamine substrate.
View details for DOI 10.1016/j.bmc.2007.06.020
View details for Web of Science ID 000249194900027
View details for PubMedID 17590341
View details for PubMedCentralID PMC2041840
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A scaleable manufacturing process for pro-EP-B2, a cysteine protease from barley indicated for Celiac Sprue
BIOTECHNOLOGY AND BIOENGINEERING
2007; 98 (1): 177-185
Abstract
Celiac Sprue is an inflammatory disease of the small intestine triggered by ingestion of dietary gluten, a family of glutamine and proline rich proteins found in common foodgrains such as wheat, rye, and barley. One potential therapy for this lifelong disease anticipates using an oral protease to detoxify gluten in vivo. Recent studies have shown that EP-B2 (endoprotease B, isoform 2) from barley is a promising example of such a glutenase, thus warranting its large-scale production for animal safety and human clinical studies. Here we describe a scaleable fermentation, refolding and purification process for the production of gram to kilogram quantities of pro-EP-B2 (zymogen form of EP-B2) in a lyophilized form. A fed-batch E. coli fermentation system was developed that yields 0.3-0.5 g purified recombinant protein per liter culture volume. Intracellular degradation of pro-EP-B2 during the fermentation was overcome by manipulating the fermentation temperature and duration of protein expression. A simple refolding protocol was developed using a fast dilution method to refold the enzyme at concentrations greater than 0.5 mg/mL. Kinetic analysis showed that pro-EP-B2 refolding is a first-order reaction with an estimated rate constant of 0.15 h(-1). A lyophilization procedure was developed that yielded protein with 85% recoverable activity after 7 weeks of storage at room temperature. The process was successfully scaled up to 100 L with comparable purity and recovery.
View details for DOI 10.1002/bit.21423
View details for Web of Science ID 000248655700018
View details for PubMedID 17385743
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Combination enzyme therapy for gastric digestion of dietary gluten in patients with celiac sprue
GASTROENTEROLOGY
2007; 133 (2): 472-480
Abstract
Celiac sprue is a multifactorial disease characterized by an inflammatory response to ingested gluten in the small intestine. Proteolytically resistant, proline- and glutamine-rich gluten peptides from wheat, rye, and barley persist in the intestinal lumen and elicit an immune response in genetically susceptible persons. We investigated a new combination enzyme product, consisting of a glutamine-specific endoprotease (EP-B2 from barley) and a prolyl endopeptidase (SC PEP from Sphingomonas capsulata), for its ability to digest gluten under gastric conditions.The ability of this combination enzyme to digest and detoxify whole-wheat bread gluten was investigated. In vitro and in vivo (rat) experimental systems were developed to simulate human gastric digestion, and the resulting material was analyzed by high-performance liquid chromatography, enzyme-linked immunoabsorbent assay, and patient-derived T-cell proliferation assays.The analysis revealed that EP-B2 extensively proteolyzes complex gluten proteins in bread, whereas SC PEP rapidly detoxifies the residual oligopeptide products of EP-B2 digestion. In vitro dose variation data suggests that an approximate 1:1 weight ratio of the 2 enzymes should maximize their synergistic potential. The efficacy of this 2-enzyme glutenase was verified in a rat model of gastric gluten digestion.By combining 2 enzymes with gastric activity and complementary substrate specificity, it should be possible to increase the safe threshold of ingested gluten, thereby ameliorating the burden of a highly restricted diet for patients with celiac sprue.
View details for DOI 10.1053/j.gastro.2007.05.028
View details for Web of Science ID 000248585600017
View details for PubMedID 17681168
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Transglutaminase 2 inhibitors and their therapeutic role in disease states
PHARMACOLOGY & THERAPEUTICS
2007; 115 (2): 232-245
Abstract
Transglutaminase 2 (TG2) is a multi-domain, multi-functional enzyme that post-translationally modifies proteins by catalyzing the formation of intermolecular isopeptide bonds between glutamine and lysine side-chains. It plays a role in diverse biological functions, including extracellular matrix formation, integrin-mediated signaling, and signal transduction involving 7-transmembrane receptors. While some of the roles of TG2 under normal physiological conditions remain obscure, the protein is believed to participate in the pathogenesis of several unrelated diseases, including celiac sprue, neurodegenerative diseases, and certain types of cancer. A variety of small molecule and peptidomimetic inhibitors of the TG2 active site have been identified. Here, we summarize the biochemistry, biology, pharmacology and medicinal chemistry of human TG2.
View details for DOI 10.1016/j.pharmthera.2007.05.003
View details for Web of Science ID 000248657000004
View details for PubMedID 17582505
View details for PubMedCentralID PMC1975782
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Structure-activity relationships of semisynthetic mumbaistatin analogs
BIOORGANIC & MEDICINAL CHEMISTRY
2007; 15 (15): 5207-5218
Abstract
Mumbaistatin (1), a new anthraquinone natural product, is one of the most potent known inhibitors of hepatic glucose-6-phosphate translocase, an important target for the treatment of type II diabetes. Its availability, however, has been limited due to its extremely low yield from the natural source. Starting from DMAC (5, 3,8-dihydroxyanthraquinone-2-carboxylic acid), a structurally related polyketide product of engineered biosynthesis, we developed a facile semisynthetic method that afforded a variety of mumbaistatin analogs within five steps. This work was facilitated by the initial development of a DMAC overproduction system. In addition to reinforcing the biological significance of the anthraquinone moiety of mumbaistatin, several semisynthetic analogs were found to have low micromolar potency against the translocase in vitro. Two of them were also active in glucose release assays from primary hepatocytes. The synergistic combination of biosynthesis and synthesis is a promising avenue for the discovery of new bioactive substances.
View details for DOI 10.1016/j.bmc.2007.05.019
View details for Web of Science ID 000247714900017
View details for PubMedID 17524653
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Structural and mechanistic analysis of protein interactions in module 3 of the 6-deoxyerythronolide B synthase
CHEMISTRY & BIOLOGY
2007; 14 (8): 931-943
Abstract
We report the 2.6 A X-ray crystal structure of a 190 kDa homodimeric fragment from module 3 of the 6-deoxyerthronolide B synthase covalently bound to the inhibitor cerulenin. The structure shows two well-organized interdomain linker regions in addition to the full-length ketosynthase (KS) and acyltransferase (AT) domains. Analysis of the substrate-binding site of the KS domain suggests that a loop region at the homodimer interface influences KS substrate specificity. We also describe a model for the interaction of the catalytic domains with the acyl carrier protein (ACP) domain. The ACP is proposed to dock within a deep cleft between the KS and AT domains, with interactions that span both the KS homodimer and AT domain. In conjunction with other recent data, our results provide atomic resolution pictures of several catalytically relevant protein interactions in this remarkable family of modular megasynthases.
View details for DOI 10.1016/j.chembiol.2007.07.012
View details for Web of Science ID 000249257800010
View details for PubMedID 17719492
View details for PubMedCentralID PMC1986752
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Structure-based dissociation of a type I polyketide synthase module
CHEMISTRY & BIOLOGY
2007; 14 (7): 784-792
Abstract
Individual modules of modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) consist of conserved, covalently linked domains separated by unconserved intervening linker sequences. To better understand the protein-protein and enzyme-substrate interactions in modular catalysis, we have exploited recent structural insights to prepare stand-alone domains of selected DEBS modules. When combined in vitro, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and diketide substrate elongation. When added to a minimal PKS, ketoreductase domains from DEBS modules 1, 2, and 6 showed specificity for the beta-ketoacylthioester substrate, but not for either the ACP domain carrying the polyketide substrate or the KS domain that synthesized the substrate. With insights into catalytic efficiency and specificity of PKS modules, our results provide guidelines for constructing optimal hybrid PKS systems.
View details for DOI 10.1016/j.chembiol.2007.05.015
View details for Web of Science ID 000248402400010
View details for PubMedID 17656315
View details for PubMedCentralID PMC1978548
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Enhancement of dietary protein digestion by conjugated bile acids
GASTROENTEROLOGY
2007; 133 (1): 16-23
Abstract
Conjugated bile acids promote absorption of dietary lipids by solubilizing them in mixed micelles. Bile acids are not considered to facilitate the digestion of other nutrients.The effect of conjugated bile acids on the rate of protein hydrolysis by trypsin and chymotrypsin was examined in vitro. Common dietary proteins and 2 bacterial glutenases (proposed oral therapies for celiac sprue) were proteolyzed in the absence or presence of a 10 mmol/L conjugated bile acid mixture, simulating human bile composition. Lipolysis products (monoolein) and fatty acid were also evaluated to simulate postprandial intestinal contents.Conjugated bile acids dramatically enhanced the proteolysis of several dietary proteins, including beta-lactoglobulin, bovine serum albumin, myoglobin, and a commercially available dietary protein supplement. For beta-lactoglobulin, a cow's milk allergen that is resistant to pepsin cleavage, bile acids enhanced its proteolysis by pancreatic proteases even after incubation under gastric conditions. Exposure of prolyl endopeptidases to bile acids made them more susceptible to pancreatic proteases under simulated intestinal conditions. The conjugated bile acid effect was most pronounced in the presence of dihydroxy bile acids and was observable at bile concentrations below the critical micellar concentration but to a much greater extent at concentrations above the critical micellar concentration.We propose that, in addition to promoting lipid absorption, conjugated bile acids affect the digestion and assimilation of dietary proteins by accelerating hydrolysis by pancreatic proteases. These findings have implications for intraluminal protein breakdown and assimilation in the upper small intestine.
View details for DOI 10.1053/j.gastro.2007.04.008
View details for Web of Science ID 000248055400007
View details for PubMedID 17631126
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Transglutaminase 2 inhibitor, KCC009, disrupts fibronectin assembly in the extracellular matrix and sensitizes orthotopic glioblastomas to chemotherapy
ONCOGENE
2007; 26 (18): 2563-2573
Abstract
Transglutaminase 2 (TG2, a.k.a. tissue transglutaminase) belongs to a family of transglutaminase enzymes that stabilize proteins by affecting covalent crosslinking via formation of amide bonds. Cell surface TG2 is directly involved as an adhesive receptor in cell-extracellular matrix (ECM) interactions. Here, we show that TG2 activity is elevated in glioblastomas compared with non-neoplastic brain. Immunofluorescent studies showed increased staining of fibronectin colocalized with TG2 in the ECM in glioblastomas. In addition, small clusters of invading human glioblastoma cells present in non-neoplastic brain parenchyma secrete high levels of TG2 and fibronectin that distinguish them from normal brain stroma. Downregulation of TG2 in U87MG glioblastoma cells with RNAi demonstrated decreased assembly of fibronectin in the ECM. Treatment with KCC009 blocked the remodeling of fibronectin in the ECM in glioblastomas in both in vitro and in vivo studies. KCC009 treatment in mice harboring orthotopic glioblastomas (DBT-FG) sensitized the tumors to N,N'-bis(2-chloroethyl)-N-nitrosourea chemotherapy, as measured by reduced bioluminescence, increased apoptosis and prolonged survival. The ability of KCC009 to interfere with the permissive remodeling of fibronectin in the ECM in glioblastomas suggests a novel target to enhance sensitivity to chemotherapy directed not only at the tumor mass, but also invading glioblastoma cells.
View details for DOI 10.1038/sj.onc.1210048
View details for Web of Science ID 000245831200004
View details for PubMedID 17099729
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Antibiotic production from the ground up
NATURE BIOTECHNOLOGY
2007; 25 (4): 428-429
View details for Web of Science ID 000245651800020
View details for PubMedID 17420748
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Transglutaminase 2 regulates mallory body inclusion formation and injury-associated liver enlargement
GASTROENTEROLOGY
2007; 132 (4): 1515-1526
Abstract
Mallory body (MB) inclusions are a characteristic feature of several liver disorders and share similarities with cytoplasmic inclusions observed in neural diseases and myopathies. MBs consist primarily of keratins 8 and 18 (K8/K18), require a K8-greater-than-K18 ratio for their formation, and contain glutamine-lysine cross-links generated by transglutaminase (TG). We hypothesized that protein transamidation is essential for MB formation.Because TG2 is the most abundant hepatocyte TG, we tested our hypothesis using TG2(-/-) and their wild-type counterpart mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an established MB inducer. Keratin cross-linking was further examined using recombinant proteins or transgenic mice that overexpress K8 or K18.TG2(-/-) livers have markedly reduced TG2 activity as compared with TG2(+/+) livers. The DDC-fed TG2(-/-) mice have dramatic decreases in MB formation and liver hypertrophy response as contrasted with DDC-fed TG2(+/+) mice. Despite similar hepatocellular damage, TG2(-/-) mice had more gallstones, jaundice, and ductal proliferation than wild-type mice. Inhibition of MB formation in TG2(-/-) mice was associated with marked attenuation of ubiquitination and K8-containing protein cross-linking. MB formation and resolution paralleled the generation then disappearance of cross-linked K8, respectively. K8 is a preferential TG2 substrate when compared to K18, as examined in vitro or in DDC-fed transgenic mice that overexpress K8 or K18.We demonstrate an essential role for TG2 in determining injury-mediated liver enlargement and the necessity of K8 and TG2 for generating cross-linked keratins and MBs. The role of TG in inclusion formation might extend to nonkeratin intermediate filament protein-related diseases.
View details for DOI 10.1053/j.gastro.2007.02.020
View details for Web of Science ID 000246020900037
View details for PubMedID 17408647
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Substrate tolerance of module 6 of the epothilone synthetase
BIOCHEMISTRY
2007; 46 (11): 3385-3393
Abstract
The epothilone synthetase is a decamodular megasynthase responsible for the biosynthesis of a class of polyketide natural products with clinically promising antitumor activity. Recently, we developed a system comprised of modules 6-9 of the epothilone synthetase for the precursor-directed biosynthesis of epothilones in Escherichia coli [Boddy, C. N., Hotta, K., Tse, M. L., Watts, R. E., and Khosla, C. (2004) J. Am. Chem. Soc. 126, 7436-7437]. To systematically explore the biosynthetic potential of this system, we have now investigated the ability of the crucial first module in this engineered pathway, EpoD-M6, to accept, elongate, and process unnatural substrates. EpoD-M6 was expressed, purified, and demonstrated to accept both acyl-CoA and acylSNAC substrates. Of the substrates that were tested, octanoylSNAC and 3-octenoylSNAC proved to be excellent substrates in addition to the more complex natural substrate. Thus, this polyketide synthase module showed considerable tolerance, a feature that bodes well for the precursor-directed biosynthesis of epothilone analogues and related complex polyketides.
View details for DOI 10.1021/bi0616448
View details for Web of Science ID 000244854800047
View details for PubMedID 17315981
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Bioassay-Guided evolution of glycosylated macrolide antibiotics in Escherichia coli
PLOS BIOLOGY
2007; 5 (2): 243-250
Abstract
Macrolide antibiotics such as erythromycin are clinically important polyketide natural products. We have engineered a recombinant strain of Escherichia coli that produces small but measurable quantities of the bioactive macrolide 6-deoxyerythromycin D. Bioassay-guided evolution of this strain led to the identification of an antibiotic-overproducing mutation in the mycarose biosynthesis and transfer pathway that was detectable via a colony-based screening assay. This high-throughput assay was then used to evolve second-generation mutants capable of enhanced precursor-directed biosynthesis of macrolide antibiotics. The availability of a screen for macrolide biosynthesis in E. coli offers a fundamentally new approach in dissecting modular megasynthase mechanisms as well as engineering antibiotics with novel pharmacological properties.
View details for DOI 10.1371/journal.pbio.0050045
View details for Web of Science ID 000245243200012
View details for PubMedID 17298179
View details for PubMedCentralID PMC1790958
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Synthesis and biological activity of novel pyranopyrones derived from engineered aromatic polyketides
ACS CHEMICAL BIOLOGY
2007; 2 (2): 104-108
Abstract
The 4-hydroxy-2-pyrone moiety is commonly observed in polyketides generated via biosynthetic engineering of type II polyketide synthases. To explore the synthetic utility of these 2-pyrones, four engineered polyketides (mutactin, SEK4, SEK15, and SEK15b) were isolated from appropriate derivatives of Streptomyces coelicolor CH999. As a test case, we prepared nine novel pyranopyrones through condensation reactions with either citral, 1-cyclohexene-carboxaldehyde, or S-perillaldehyde. Synthetic tricyclic pyranopyrones with simple aromatic substituents are known to possess anticancer properties. We therefore investigated whether pyranopyrone derivatives of aromatic polyketides exhibited bioactivity in a panel of three cancer cell lines. Pyranopyrones generated from SEK4 had activity comparable to that of H10, a pyranopyrone with a 3-pyridyl substituent, whereas other analogues were less active. These results suggest that the diverse library of carbo- and heterocycles available through the genetic engineering of type II polyketide synthases can serve as useful building blocks to generate unique bioactive molecules.
View details for DOI 10.1021/cb600382j
View details for Web of Science ID 000244347600016
View details for PubMedID 17256996
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Prolyl endopeptidases
CELLULAR AND MOLECULAR LIFE SCIENCES
2007; 64 (3): 345-355
Abstract
This review describes the structure and function of prolyl endopeptidase (PEP) enzymes and how they are being evaluated as drug targets and therapeutic agents. The most well studied PEP family has a two-domain structure whose unique seven-blade beta-propeller domain works with the catalytic domain to hydrolyze the peptide bond on the carboxyl side of internal proline residues of an oligopeptide substrate. Structural and functional studies on this protease family have elucidated the mechanism for peptide entry between the two domains. Other structurally unrelated PEPs have been identified, but have not been studied in detail. Human PEP has been evaluated as a pharmacological target for neurological diseases due to its high brain concentration and ability to cleave neuropeptides in vitro. Recently, microbial PEPs have been studied as potential therapeutics for celiac sprue, an inflammatory disease of the small intestine triggered by proline-rich gluten.
View details for DOI 10.1007/s00018-006-6317-y
View details for Web of Science ID 000244390100008
View details for PubMedID 17160352
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Structure and mechanism of the 6-deoxyerythronolide B synthase
ANNUAL REVIEW OF BIOCHEMISTRY
2007; 76: 195-221
Abstract
6-Deoxyerythronolide B, the macrocyclic aglycone of the antibiotic erythromycin, is synthesized by a polyketide synthase (PKS) that has emerged as the prototypical modular megasynthase. A variety of molecular biological, protein chemical, and biosynthetic experiments over the past two decades have yielded insights into its mechanistic features. More recently, high-resolution structural images of portions of the 6-deoxyerythronolide B synthase have provided a platform for interpreting this wealth of biochemical data, while at the same time presenting a fundamentally new basis for the design of more detailed investigations into this remarkable enzyme. For example, the critical roles of domain-domain interactions and nonconserved linkers, as well as large interdomain movements in the structure and function of modular PKSs, have been highlighted. In turn, these insights point the way forward for more sophisticated and efficient biosynthetic engineering of complex polyketide natural products.
View details for DOI 10.1146/annurev.biochem.76.053105.093515
View details for Web of Science ID 000249336800010
View details for PubMedID 17328673
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Structure-activity relationship analysis of the selective inhibition of transglutaminase 2 by dihydroisoxazoles
JOURNAL OF MEDICINAL CHEMISTRY
2006; 49 (25): 7493-7501
Abstract
Human transglutaminase 2 (TG2) is believed to play an important role in the pathogenesis of various human disorders including celiac sprue, certain neurological diseases, and some types of cancer. Selective inhibition of TG2 should therefore enable further investigation of its role in physiology and disease and may lead to effective clinical treatment. Recently we showed that certain 3-halo-4-,5-dihydroisoxazole containing compounds are selective inhibitors of human TG2 with promising pharmacological activities. Here, we present definitive evidence that this class of compounds targets the active site of human TG2. Structure-activity relationship studies have provided insights into the structural prerequisites for selectivity and have led to the discovery of an inhibitor with about 50-fold higher activity than a prototypical dihydroisoxazole inhibitor with good in vivo activity. A method for preparing enantiomerically enriched analogues was also developed. Our studies show that the 5-(S)-dihydroisoxazole is a markedly better inhibitor of human TG2 than its 5-(R) stereoisomer.
View details for DOI 10.1021/jm060839a
View details for Web of Science ID 000242625800024
View details for PubMedID 17149878
View details for PubMedCentralID PMC2526180
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Structural and functional studies on SCO1815: A beta-ketoacyl-acyl carrier protein reductase from Streptomyces coelicolor A3(2)
BIOCHEMISTRY
2006; 45 (47): 14085-14093
Abstract
Aromatic polyketides are medicinally important natural products produced by type II polyketide synthases (PKSs). Some aromatic PKSs are bimodular and include a dedicated initiation module which synthesizes a non-acetate primer unit. Understanding the mechanism of this initiation module is expected to further enhance the potential for regiospecific modification of bacterial aromatic polyketides. A typical initiation module is comprised of a ketosynthase (KS), an acyl carrier protein (ACP), a malonyl-CoA:ACP transacylase (MAT), an acyl-ACP thioesterase, a ketoreductase (KR), a dehydratase (DH), and an enoyl reductase (ER). Thus far, the identities of the ketoreductase, dehydratase, and enoyl reductase remain a mystery because they are not encoded within the PKS biosynthetic gene cluster. Here we report that SCO1815 from Streptomyces coelicolor A3(2), an uncharacterized homologue of a NADPH-dependent ketoreductase, recognizes and reduces the beta-ketoacyl-ACP intermediate from the initiation module of the R1128 PKS. SCO1815 exhibits moderate specificity for both the acyl chain and the thiol donor. The X-ray crystal structure of SCO1815 was determined to 2.0 A. The structure shows that SCO1815 adopts a Rossmann fold and suggests that a conformational change occurs upon cofactor binding. We propose that a positively charged patch formed by three conserved residues is the ACP docking site. Our findings provide new engineering opportunities for incorporating unnatural primer units into novel polyketides and shed light on the biology of yet another cryptic protein in the S. coelicolor genome.
View details for DOI 10.1021/bi061187v
View details for Web of Science ID 000242179100016
View details for PubMedID 17115703
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Effect of barley endoprotease EP-B2 on gluten digestion in the intact rat
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2006; 318 (3): 1178-1186
Abstract
Celiac Sprue is a multifactorial disease characterized by an intestinal inflammatory response to ingested gluten. Proteolytically resistant gluten peptides from wheat, rye, and barley persist in the intestinal lumen and elicit an immune response in genetically susceptible individuals. Here, we demonstrate the in vivo ability of a gluten-digesting protease ("glutenase") to accelerate the breakdown of a gluten-rich solid meal. The proenzyme form of endoprotease B, isoform 2 from Hordeum vulgare (EP-B2), was orally administered to adult rats with a solid meal containing 1 g of gluten. Gluten digestion in the stomach and small intestine was monitored as a function of enzyme dose and time by high-performance liquid chromatography and mass spectrometry. In the absence of supplementary EP-B2, gluten was solubilized and proteolyzed to a limited extent in the stomach and was hydrolyzed and assimilated mostly in the small intestine. In contrast, EP-B2 was remarkably effective at digesting gluten in the rat stomach in a dose- and time-dependent fashion. At a 1:25 EP-B2/gluten dose, the gastric concentration of the highly immunogenic 33-mer gliadin peptide was reduced by more than 50-fold within 90 min with no overt signs of toxicity. Evaluation of EP-B2 as an adjunct to diet control is therefore warranted in celiac patients.
View details for DOI 10.1124/jpet.106.104315
View details for Web of Science ID 000239878900029
View details for PubMedID 16757540
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Production of ansamycin polyketide precursors in Escherichia coli
JOURNAL OF ANTIBIOTICS
2006; 59 (8): 464-470
Abstract
For the heterologous production of ansamycin polyketides such as rifamycin and geldanamycin in Escherichia coli, a number of unusual but important tools must be engineered into the bacterium. Here we demonstrate efficient production of the starter unit 3-amino-5-hydroxybenzoic acid (AHBA) and the methoxymalonyl extender unit in E. coli. Previous work has demonstrated the production of the ansamycin starter unit AHBA in E. coli in low yield. It was shown that the low yield is primarily due to acetylation of AHBA into N-acetyl-AHBA. Three methods for minimizing this side reaction were evaluated. First, a putative N-arylamine-acetyltransferase (NAT) was deleted from the E. coli chromosome, although this did not alter N-acetyl-AHBA production. Next, E. coli grown in media devoid of glucose yielded a nearly equal mixture of AHBA and N-acetyl-AHBA. Lastly, the NAT inhibitor glycyrrhizic acid was shown to further inhibit the acetylation reaction. The entire set of genes for synthesizing the methoxymalonyl extender unit was transferred from the geldanamycin producer Streptomyces hygroscopicus into E. coli. The pathway specific ACP isolated from the resulting recombinant strain was found to predominantly occur as methyoxymalonyl-ACP. Together, these findings set the stage for engineered biosynthesis of ansamycin polyketides in E. coli.
View details for Web of Science ID 000240519800004
View details for PubMedID 17080682
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The 2.7-angstrom crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (30): 11124-11129
Abstract
The x-ray crystal structure of a 194-kDa fragment from module 5 of the 6-deoxyerythronolide B synthase has been solved at 2.7 Angstrom resolution. Each subunit of the homodimeric protein contains a full-length ketosynthase (KS) and acyl transferase (AT) domain as well as three flanking "linkers." The linkers are structurally well defined and contribute extensively to intersubunit or interdomain interactions, frequently by means of multiple highly conserved residues. The crystal structure also reveals that the active site residue Cys-199 of the KS domain is separated from the active site residue Ser-642 of the AT domain by approximately 80 Angstrom. This distance is too large to be covered simply by alternative positioning of a statically anchored, fully extended phosphopantetheine arm of the acyl carrier protein domain from module 5. Thus, substantial domain reorganization appears necessary for the acyl carrier protein to interact successively with both the AT and the KS domains of this prototypical polyketide synthase module. The 2.7-Angstrom KS-AT structure is fully consistent with a recently reported lower resolution, 4.5-Angstrom model of fatty acid synthase structure, and emphasizes the close biochemical and structural similarity between polyketide synthase and fatty acid synthase enzymology.
View details for DOI 10.1073/pnas.0601924103
View details for Web of Science ID 000239353900006
View details for PubMedID 16844787
View details for PubMedCentralID PMC1636687
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Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease
CHEMISTRY & BIOLOGY
2006; 13 (6): 637-647
Abstract
We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/l) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purification and refolding. The zymogen was rapidly autoactivated to its mature form under acidic conditions at a rate independent of proEP-B2 concentration, suggesting a cis mechanism of autoactivation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, alpha2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 A resolution and provided atomic insights into the observed subsite specificity of the endoprotease. Our findings suggest that orally administered proEP-B2 may be especially well suited for treatment of celiac sprue.
View details for DOI 10.1016/j.chembiol.2006.04.008
View details for Web of Science ID 000238723800012
View details for PubMedID 16793521
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Investigating nonribosomal peptide and polyketide biosynthesis by direct detection of intermediates on > 70 kDa polypeptides by using Fourier-transform mass spectrometry
CHEMBIOCHEM
2006; 7 (6): 904-907
View details for DOI 10.1002/cbic.200500416
View details for Web of Science ID 000238171400006
View details for PubMedID 16642537
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Rational design of combination enzyme therapy for celiac sprue
CHEMISTRY & BIOLOGY
2006; 13 (6): 649-658
Abstract
Celiac sprue (also known as celiac disease) is an inheritable, gluten-induced enteropathy of the upper small intestine with an estimated prevalence of 0.5%-1% in most parts of the world. The ubiquitous nature of food gluten, coupled with inadequate labeling regulations in most countries, constantly poses a threat of disease exacerbation and relapse for patients. Here, we demonstrate that a two-enzyme cocktail comprised of a glutamine-specific cysteine protease (EP-B2) that functions under gastric conditions and a PEP, which acts in concert with pancreatic proteases under duodenal conditions, is a particularly potent candidate for celiac sprue therapy. At a gluten:EP-B2:PEP weight ratio of 75:3:1, grocery store gluten is fully detoxified within 10 min of simulated duodenal conditions, as judged by chromatographic analysis, biopsy-derived T cell proliferation assays, and a commercial antigluten antibody test.
View details for DOI 10.1016/j.chembiol.2006.04.009
View details for Web of Science ID 000238723800013
View details for PubMedID 16793522
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Pharmacologic transglutaminase inhibition attenuates drug-primed liver hypertrophy but not Mallory body formation
FEBS LETTERS
2006; 580 (9): 2351-2357
Abstract
Mallory bodies (MBs) are characteristic of several liver disorders, and consist primarily of keratins with transglutaminase-generated keratin crosslinks. We tested the effect of the transglutaminase-2 (TG2) inhibitor KCC009 on MB formation in a mouse model fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). KCC009 decreased DDC-induced liver enlargement without affecting MB formation or extent of liver injury. TG2 protein and activity increased after DDC feeding and localized within and outside hepatocytes. KCC009 inhibited DDC-induced hepatomegaly by affecting hepatocyte cell size rather than proliferation. Hence, TG2 is a potential mediator of injury-induced hepatomegaly via modulation of hepatocyte hypertrophy, and KCC009-mediated TG2 inhibition does not affect mouse MB formation.
View details for DOI 10.1016/j.febslet.2006.03.051
View details for Web of Science ID 000237012800032
View details for PubMedID 16616523
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Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2006; 128 (9): 3067-3074
Abstract
Polyketide synthases (PKSs) catalyze the production of numerous biologically important natural products via repeated decarboxylative condensation reactions. Modular PKSs, such as the 6-deoxyerythronolide B synthase (DEBS), consist of multiple catalytic modules, each containing a unique set of covalently linked catalytic domains. To better understand the engineering opportunities of these assembly lines, the extender unit and acyl carrier protein (ACP) specificity of keto synthase (KS) domains from modules 3 and 6 of DEBS were analyzed. These studies were undertaken with a newly developed didomain [KS][AT] construct, which lacks its own ACP domain and can therefore be interrogated with homologous or heterologous ACP or acyl-ACP substrates. By substituting the natural methylmalonyl extender unit with a malonyl group, a modest role was demonstrated for the KS in recognition of the nucleophilic substrate. The KS domain from module 3 of DEBS was found to exhibit a distinct ACP-recognition profile from the KS domain of module 6. On the basis of the above kinetic insights, a hybrid module was constructed ([KS3][AT3][KR5][ACP5][TE]) which displayed substrate recognition and elongation capabilities consistent with the natural module 3 protein. Unlike module 3, however, which lacks a ketoreductase (KR) domain, the hybrid module was able to catalyze reduction of the beta-ketothioester product of chain elongation. The high expression level and functionality of this hybrid protein demonstrates the usefulness of kinetic analysis for hybrid module design.
View details for DOI 10.1021/ja058093d
View details for Web of Science ID 000235942000060
View details for PubMedID 16506788
View details for PubMedCentralID PMC2532788
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Inhibition of HLA-DQ2-mediated antigen presentation by analogues of a high affinity 33-residue peptide from alpha 2-gliadin
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2006; 128 (6): 1859-1867
Abstract
Human leukocyte antigen DQ2 is a class II major histocompatibility complex protein that plays a critical role in the pathogenesis of Celiac Sprue by binding to epitopes derived from dietary gluten and triggering the inflammatory response of disease-specific T cells. Inhibition of DQ2-mediated antigen presentation in the small intestinal mucosa of Celiac Sprue patients therefore represents a potentially attractive mode of therapy for this widespread but unmet medical need. Starting from a pro-inflammatory, proteolytically resistant, 33-residue peptide, LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF, we embarked upon a systematic effort to dissect the relationships between peptide structure and DQ2 affinity and to translate these insights into prototypical DQ2 blocking agents. Three structural determinants within the first 20 residues of this 33-mer peptide, including a PQPELPYPQ epitope, its N-terminal flanking sequence, and a downstream Glu residue, were found to be important for DQ2 binding. Guided by the X-ray crystal structure of DQ2, the L11 and L18 residues in the truncated 20-mer analogue were replaced with sterically bulky groups so as to retain high DQ2 affinity but abrogate T cell recognition. A dimeric ligand, synthesized by regiospecific coupling of the 20-mer peptide with a bifunctional linker, was identified as an especially potent DQ2 binding agent. Two such ligands were able to attenuate the proliferation of disease-specific T cell lines in response to gluten antigens and, therefore, represent prototypical examples of pharmacologically suitable DQ2 blocking agents for the potential treatment of Celiac Sprue.
View details for DOI 10.1021/ja056423o
View details for Web of Science ID 000235452200025
View details for PubMedID 16464085
View details for PubMedCentralID PMC2597451
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Macrolactonization to 10-deoxymethynolide catalyzed by the recombinant thioesterase of the picromycin/methymycin polyketide synthase
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
2006; 16 (2): 391-394
Abstract
The recombinant thioesterase (TE) domain of the picromycin/methymycin synthase (PICS) catalyzes the macrolactonization of 3, the N-acetylcysteamine thioester of seco-10-deoxymethynolide to generate 10-deoxymethynolide (1) with high efficiency. By contrast, 4, the 7-dihydro derivative of seco-thioester 3, undergoes exclusive hydrolysis by PICS TE to seco-acid 5. The recombinant TE domain of 6-deoxyerythronolide B synthase (DEBS TE) shows the same reaction specificity as PICS TE, but with significantly lower activity.
View details for DOI 10.1016/j.bmcl.2005.09.077
View details for Web of Science ID 000233902200031
View details for PubMedID 16249083
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Trapping transient protein-protein interactions in polyketide biosynthesis
ACS CHEMICAL BIOLOGY
2006; 1 (11): 679-680
Abstract
Transient biomolecular interactions are essential for biological processes, but strategies for studying them have remained elusive. A paper in this issue shows how natural enzymatic activities can be exploited to examine protein-protein interactions in fatty acid synthase.
View details for DOI 10.1021/cb600451d
View details for Web of Science ID 000243895300010
View details for PubMedID 17184129
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Modular polyketide synthases: Investigating intermodular communication using 6 deoxyerythronolide B synthase module 2
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
2006; 16 (1): 213-216
Abstract
A novel variant of 6-deoxyerythronolide B synthase (DEBS) module 2 was constructed to explore the balance between protein-protein-mediated intermodular channeling and intrinsic substrate specificity within DEBS. This construct, termed (N3)Mod2+TE, was co-incubated with a complementary, donor form of the same module, (N5)Mod2(C2), as well as with a mutant of (N5)Mod2(C2) with an inactive ketosynthase domain, in order to determine the extent of intermediate channeling versus substrate diffusion into the downstream module.
View details for DOI 10.1016/j.bmcl.2005.09.017
View details for Web of Science ID 000233516200045
View details for PubMedID 16213712
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Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: Implications for Celiac sprue therapy
BIOTECHNOLOGY AND BIOENGINEERING
2005; 92 (6): 674-684
Abstract
Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat gluten and similar proteins in rye and barley. Proline-rich gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro gluten detoxifying properties of a therapeutically promising prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated capsule containing a pharmacologically useful dose of this enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable enzyme as a dry powder. The dry powder was blended with excipients and encapsulated in a hard gelatin capsule. The resulting capsule was enteric coated using Eudragit L30-D55 polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of trypsin and chymotrypsin). In conjunction with pancreatic enzymes, MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic peptides mixed with whole gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP capsule as an oral therapeutic agent for Celiac Sprue patients.
View details for DOI 10.1002/bit.20643
View details for Web of Science ID 000233711600002
View details for PubMedID 16136593
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Polyketide double bond biosynthesis. Mechanistic analysis of the dehydratase-containing module 2 of the picromycin/methymycin polyketide synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2005; 127 (49): 17393-17404
Abstract
Picromycin/methymycin synthase (PICS) is a modular polyketide synthase (PKS) that is responsible for the biosynthesis of both 10-deoxymethynolide (1) and narbonolide (2), the parent 12- and 14-membered aglycone precursors of the macrolide antibiotics methymycin and picromycin, respectively. PICS module 2 is a dehydratase (DH)-containing module that catalyzes the formation of the unsaturated triketide intermediate using malonyl-CoA as the chain extension substrate. Recombinant PICS module 2+TE, with the PICS thioesterase domain appended to the C-terminus to allow release of polyketide products, was expressed in Escherichia coli. Purified PICS module 2+TE converted malonyl-CoA and 4, the N-acetylcysteamine thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid, to a 1:2 mixture of the triketide acid (4S,5R)-4-methyl-5-hydroxy-2-heptenoic acid (5) and (3S,4S,5R)-3,5-dihydroxy-4-methyl-n-heptanoic acid-delta-lactone (10) with a combined kcat of 0.6 min(-1). The triketide lactone 10 is formed by thioesterase-catalyzed cyclization of the corresponding d-3-hydroxyacyl-SACP intermediate, a reaction which competes with dehydration catalyzed by the dehydratase domain. PICS module 2+TE showed a strong preference for the syn-diketide-SNAC 4, with a 20-fold greater kcat/K(m) than the anti-(2S,3S)-diketide-SNAC 14, and a 40-fold advantage over the syn-(2R,3S)-diketide-SNAC 13. PICS module 2(DH(0))+TE, with an inactivated DH domain, produced exclusively 10, while three PICS module 2(KR(0))+TE mutants, with inactivated KR domains, produced exclusively or predominantly the unreduced triketide ketolactone, (4S,5R)-3-oxo-4-methyl-5-hydroxy-n-heptanoic acid-delta-lactone (7). These studies establish for the first time the structure and stereochemistry of the intermediates of a polyketide chain elongation cycle catalyzed by a DH-containing module, while confirming the importance of key active site residues in both KR and DH domains.
View details for DOI 10.1021/ja055672+
View details for Web of Science ID 000233917500061
View details for PubMedID 16332089
View details for PubMedCentralID PMC2533740
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Orthogonal protein interactions in spore pigment producing and antibiotic producing polyketide synthases
JOURNAL OF ANTIBIOTICS
2005; 58 (10): 663-666
Abstract
The actinomycetes produce antibiotics as well as spore pigments during their life cycle by using Type II polyketide synthases (PKSs). Each PKS minimally consists of a ketosynthase heterodimer and an acyl carrier protein. The acyl carrier protein has been shown to be interchangeable among different antibiotic producing Type II PKSs. Surprisingly, we have discovered a fundamental incompatibility between the ketosynthases and acyl carrier proteins from antibiotic producing pathways and those from spore pigment pathways. Although antibiotic PKSs can interact with acyl carrier proteins from spore pigment pathways, spore pigment PKSs are unable to recognize acyl carrier proteins from polyketide antibiotic pathways. This observation provides an insight into a critical mechanism by which natural product biosynthetic specificity is exercised by members of this bacterial family.
View details for Web of Science ID 000232987100008
View details for PubMedID 16392683
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Engineered biosynthesis of aklanonic acid analogues
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2005; 127 (35): 12254-12262
Abstract
Aklanonic acid, an anthraquinone natural product, is a common advanced intermediate in the biosynthesis of several antitumor polyketide antibiotics, including doxorubicin and aclacinomycin A. Intensive semisynthetic and biosynthetic efforts have been directed toward developing improved analogues of these clinically important compounds. The primer unit of such polyfunctional aromatic polyketides is an attractive site for introducing novel chemical functionality, and attempts have been made to modify the primer unit by precursor-directed biosynthesis or protein engineering of the polyketide synthase (PKS). We have previously demonstrated the feasibility of engineering bimodular aromatic PKSs capable of synthesizing unnatural hexaketides and octaketides. In this report, we extend this ability by preparing analogues of aklanonic acid, a decaketide, and its methyl ester. For example, by recombining the R1128 initiation module with the dodecaketide-specific pradimicin PKS, the isobutyryl-primed analogue of aklanonic acid (YT296b, 10) and its methyl ester (YT299b, 12) were prepared. In contrast, elongation modules from dodecaketide-specific spore pigment PKSs were unable to interact with the R1128 initiation module. Thus, in addition to revealing a practical route to new anthracycline antibiotics, we also observed a fundamental incompatibility between antibiotic and spore pigment biosynthesis in the actinomycetes bacteria.
View details for DOI 10.1021/ja051429z
View details for Web of Science ID 000231637100050
View details for PubMedID 16131203
View details for PubMedCentralID PMC1343498
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Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry
BIOCHEMISTRY
2005; 44 (35): 11836-11842
Abstract
Polyketide natural products are biosynthesized via successive chain-elongation events mediated by elaborate protein assemblies. Facile detection of protein-bound intermediates in these systems will increase our understanding of enzyme reactivity and selectivity. We have developed a tandem proteolysis/mass spectrometric method for monitoring substrate loading and elongation in 6-deoxyerythronolide B synthase (DEBS), responsible for production of the macrolide precursor to erythromycin. Information regarding ketosynthase loading and polyketide unit elongation is readily acquired without need for complex protein or small molecule labels. A panel of structurally related substrates is evaluated through competition experiments and kinetic assays using LC-MS to resolve closely related species. Strong stereochemical effects are observed for ketosynthase substrate specificity. Semiquantitative kinetic analyses allow the resolution of the effects of structural and stereochemical changes on the individual ketosynthase-catalyzed steps of acyl-enzyme formation and polyketide chain extension.
View details for DOI 10.1021/bi0510781
View details for Web of Science ID 000231710000021
View details for PubMedID 16128585
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Identification and analysis of multivalent proteolytically resistant peptides from gluten: Implications for Celiac Sprue
JOURNAL OF PROTEOME RESEARCH
2005; 4 (5): 1732-1741
Abstract
Dietary gluten proteins from wheat, rye, and barley are the primary triggers for the immuno-pathogenesis of Celiac Sprue, a widespread immune disease of the small intestine. Recent molecular and structural analyses of representative gluten proteins, most notably alpha- and gamma-gliadin proteins from wheat, have improved our understanding of these pathogenic mechanisms. In particular, based on the properties of a 33-mer peptide, generated from alpha-gliadin under physiological conditions, a link between digestive resistance and inflammatory character of gluten has been proposed. Here, we report three lines of investigation in support of this hypothesis. First, biochemical and immunological analysis of deletion mutants of alpha-2 gliadin confirmed that the DQ2 restricted T cell response to the alpha-2 gliadin are directed toward the epitopes clustered within the 33-mer. Second, proteolytic analysis of a representative gamma-gliadin led to the identification of another multivalent 26-mer peptide that was also resistant to further gastric, pancreatic and intestinal brush border degradation, and was a good substrate of human transglutaminase 2 (TG2). Analogous to the 33-mer, the synthetic 26-mer peptide displayed markedly enhanced T cell antigenicity compared to monovalent control peptides. Finally, in silico analysis of the gluten proteome led to the identification of at least 60 putative peptides that share the common characteristics of the 33-mer and the 26-mer peptides. Together, these results highlight the pivotal role of physiologically generated, proteolytically stable, TG2-reactive, multivalent peptides in the immune response to dietary gluten in Celiac Sprue patients. Prolyl endopeptidase treatment was shown to abolish the antigenicity of both the 33-mer and the 26-mer peptides, and was also predicted to have comparable effects on other proline-rich putatively immunotoxic peptides identified from other polypeptides within the gluten proteome.
View details for DOI 10.1021/pr050173t
View details for Web of Science ID 000232579100036
View details for PubMedID 16212427
View details for PubMedCentralID PMC1343496
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Tissue transglutaminase 2 inhibition promotes cell death and chemosensitivity in glioblastomas
MOLECULAR CANCER THERAPEUTICS
2005; 4 (9): 1293-1302
Abstract
Tissue transglutaminase 2 belongs to a family of transglutaminase proteins that confers mechanical resistance from proteolysis and stabilizes proteins. Transglutaminase 2 promotes transamidation between glutamine and lysine residues with the formation of covalent linkages between proteins. Transglutaminase 2 also interacts and forms complexes with proteins important in extracellular matrix organization and cellular adhesion. We have identified the novel finding that treatment of glioblastoma cells with transglutaminase 2 inhibitors promotes cell death and enhances sensitivity to chemotherapy. Treatment with either the competitive transglutaminase 2 inhibitor, monodansylcadaverine, or with highly specific small-molecule transglutaminase 2 inhibitors, KCA075 or KCC009, results in induction of apoptosis in glioblastoma cells. Treatment with these transglutaminase 2 inhibitors resulted in markedly decreased levels of the prosurvival protein, phosphorylated Akt, and its downstream targets. These changes promote a proapoptotic profile with altered levels of multiple intracellular proteins that determine cell survival. These changes include decreased levels of the antiapoptotic proteins, survivin, phosphorylated Bad, and phosphorylated glycogen synthetase kinase 3beta (GSK-3beta), and increased levels of the proapoptotic BH3-only protein, Bim. In vivo studies with s.c. murine DBT glioblastoma tumors treated with transglutaminase 2 inhibitors combined with the chemotherapeutic agent, N-N'-bis (2-chloroethyl)-N-nitrosourea (BCNU), decreased tumor size based on weight by 50% compared with those treated with BCNU alone. Groups treated with transglutaminase 2 inhibitors showed an increased incidence of apoptosis determined with deoxynucleotidyl transferase-mediated biotin nick-end labeling staining. These studies identify inhibition of transglutaminase 2 as a potential target to enhance cell death and chemosensitivity in glioblastomas.
View details for DOI 10.1158/1535-7163.MCT-04-0328
View details for Web of Science ID 000231923500002
View details for PubMedID 16170020
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Biological chemistry: Just add chlorine
NATURE
2005; 436 (7054): 1094-1095
View details for DOI 10.1038/4361094a
View details for Web of Science ID 000231416600026
View details for PubMedID 16121161
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Stereochemical assignment of intermediates in the rifamycin biosynthetic pathway by precursor-directed biosynthesis
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2005; 127 (32): 11202-11203
Abstract
Natural and semisynthetic rifamycins are clinically important inhibitors of bacterial DNA-dependent RNA polymerase. Although the polyketide-nonribosomal peptide origin of the naphthalene core of rifamycin B is well established, the absolute and relative configuration of both stereocenters introduced by the first polyketide synthase module is obscured by aromatization of the naphthalene ring. To decode the stereochemistry of the rifamycin polyketide precursor, we synthesized all four diastereomers of the biosynthetic substrate for module 2 of the rifamycin synthetase in the form of their N-acetylcysteamine (SNAC) thioester. Only one diastereomer was turned over in vivo into rifamycin B, thus establishing the absolute and relative configuration of the native biosynthetic intermediates.
View details for DOI 10.1021/ja051430y
View details for Web of Science ID 000231227400002
View details for PubMedID 16089423
View details for PubMedCentralID PMC1360739
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Effect of pretreatment of food gluten with prolyl endopeptidase on gluten-induced malabsorption in celiac sprue
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY
2005; 3 (7): 687-694
Abstract
We sought to determine whether prolyl endopeptidase (PEP) treatment of food gluten would obviate the intestinal dysfunction produced by small amounts of dietary gluten supplement in patients with celiac sprue.Twenty asymptomatic patients with histologically proven celiac sprue completed a randomized, double-blind, cross-over study involving two 14-day stages. Each patient consumed a low dose of a gluten supplement daily (5 g; equivalent to 1 slice of bread) in 1 stage and gluten pretreated with PEP in the other stage. Patients completed a daily symptom questionnaire and a D-xylose urine excretion and a 72-hour quantitative fecal fat were monitored before and after each stage.Despite clinical remission at baseline, 40% of patients had at least 1 abnormal celiac antibody, 20% had an abnormal urine xylose, and 63% had an abnormal fecal fat test result. There was no difference in symptoms as a function of the type of gluten consumed. In response to gluten not treated with PEP, an appreciable proportion of patients developed malabsorption of fat (7 of 17, 41%) or xylose (8 of 14, 57%). When the gluten was pretreated with PEP, fat malabsorption was avoided in 5 of 7 and xylose malabsorption in 4 of 8 of these same patients.A significant proportion of asymptomatic patients with celiac sprue have abnormal celiac antibodies and fat or carbohydrate malabsorption. Pretreatment of gluten with PEP avoided the development of fat or carbohydrate malabsorption in the majority of those patients who developed fat or carbohydrate malabsorption after a 2-week gluten challenge.
View details for DOI 10.1053/S1542-3565(05)00366-6
View details for Web of Science ID 000234105600014
View details for PubMedID 16206502
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Low-dose gluten challenge in celiac sprue: Malabsorptive and antibody responses
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY
2005; 3 (7): 679-686
Abstract
Undiagnosed patients with symptoms of celiac sprue often present to physicians after establishing dietary gluten exclusion. Although they must resume a gluten-containing diet for evaluation, there are no guidelines regarding duration of the gluten challenge, gluten dose, or monitoring parameters. We investigated the effects of a short-term gluten challenge in asymptomatic treated adult celiac patients on intestinal absorption and celiac antibody tests.Eight adult asymptomatic celiac patients consumed either 5 or 10 g of partially hydrolyzed gluten per day in an orange juice mixture for 21 days while maintaining their usual gluten-free diet. A symptom questionnaire, serum antibodies (antigliadin immunoglobulin [Ig]A and antitransglutaminase IgA and IgG), D-xylose urine excretion test, and 72-hour quantitative fecal fat test were monitored.Two patients (25%) had at least 1 abnormal celiac antibody test at baseline. There was no increase in antibodies during gluten exposure compared with baseline for any of the patients (P > .05). At baseline, 1 patient had abnormal urine xylose excretion, and 3 patients had abnormal fecal fat values. At day 15 of gluten challenge, all patients had reduced xylose absorption compared with baseline (P = .0019), and 5 of 8 participants (63%) reduced their xylose excretion to the abnormal range. Seven of 8 patients (88%) had increased fecal fat excretion at day 15 (P = .026), and 6 of these (75%) had steatorrhea by day 15.Short-term gluten challenge in asymptomatic adult celiac patients produces carbohydrate and fat malabsorption but does not increase transglutaminase and antigliadin antibody titers.
View details for DOI 10.1053/S1542-3565(05)00365-4
View details for Web of Science ID 000234105600013
View details for PubMedID 16206501
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Main chain hydrogen bond interactions in the binding of proline-rich gluten peptides to the Celiac disease-associated HLA-DQ2 molecule
JOURNAL OF BIOLOGICAL CHEMISTRY
2005; 280 (23): 21791-21796
Abstract
Binding of peptide epitopes to major histocompatibility complex proteins involves multiple hydrogen bond interactions between the peptide main chain and major histocompatibility complex residues. The crystal structure of HLA-DQ2 complexed with the alphaI-gliadin epitope (LQPFPQPELPY) revealed four hydrogen bonds between DQ2 and peptide main chain amides. This is remarkable, given that four of the nine core residues in this peptide are proline residues that cannot engage in amide hydrogen bonding. Preserving main chain hydrogen bond interactions despite the presence of multiple proline residues in gluten peptides is a key element for the HLA-DQ2 association of celiac disease. We have investigated the relative contribution of each main chain hydrogen bond interaction by preparing a series of N-methylated alphaI epitope analogues and measuring their binding affinity and off-rate constants to DQ2. Additionally, we measured the binding of alphaI-gliadin peptide analogues in which norvaline, which contains a backbone amide hydrogen bond donor, was substituted for each proline. Our results demonstrate that hydrogen bonds at P4 and P2 positions are most important for binding, whereas the hydrogen bonds at P9 and P6 make smaller contributions to the overall binding affinity. There is no evidence for a hydrogen bond between DQ2 and the P1 amide nitrogen in peptides without proline at this position. This is a unique feature of DQ2 and is likely a key parameter for preferential binding of proline-rich gluten peptides and development of celiac disease.
View details for Web of Science ID 000229557900017
View details for PubMedID 15826953
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A new route to designer antibiotics
SCIENCE
2005; 308 (5720): 367-368
View details for DOI 10.1126/science.1111415
View details for Web of Science ID 000228492000039
View details for PubMedID 15831747
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Chemistry and biology of dihydroisoxazole derivatives: Selective inhibitors of human transglutaminase 2
CHEMISTRY & BIOLOGY
2005; 12 (4): 469-475
Abstract
3-halo-4,5-dihydroisoxazoles are attractive warheads for the selective inhibition of nucleophilic active sites in biological systems. A series of 3-bromo-4,5-dihydroisoxazole compounds were prepared and tested for their ability to irreversibly inhibit human transglutaminase 2 (TG2), an enzyme that plays an important role in the pathogenesis of diverse disorders including Celiac Sprue and certain types of cancers. Several compounds showed high specificity for human TG2 (k(inh)/K(I) > 2000 min(-1)M(-1)) but essentially no reactivity (k < 1 min(-1)M(-1)) toward physiological thiols such as glutathione. The pharmacokinetic and pharmacodynamic properties of a prototype dihydroisoxazole inhibitor, 1b, were evaluated; in mice the compound showed good oral bioavailability, short serum half-life and efficient TG2 inhibition in small intestinal tissue, and low toxicity. It also showed excellent synergism with N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU, carmustine) against refractory glioblastoma tumors in mice. A fluorescent dihydroisoxazole inhibitor 5 facilitated microscopic visualization of TG2 endocytosis from the extracellular surface of HCT-116 cells. Together, these findings demonstrate the promise of dihydroisoxazole compounds as probes for the biology of TG2 and its role in human disease.
View details for DOI 10.1016/j.chembiol.2005.02.007
View details for Web of Science ID 000229065700012
View details for PubMedID 15850984
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Equilibrium and kinetic analysis of the unusual binding behavior of a highly immunogenic gluten peptide to HLA-DQ2
BIOCHEMISTRY
2005; 44 (11): 4442-4449
Abstract
HLA-DQ2 predisposes an individual to celiac sprue by presenting peptides from dietary gluten to intestinal CD4(+) T cells. A selectively deamidated multivalent peptide from gluten (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF; underlined residues correspond to posttranslational Q --> E alterations) is a potent trigger of DQ2 restricted T cell proliferation. Here we report equilibrium and kinetic measurements of interactions between DQ2 and (i) this highly immunogenic multivalent peptide, (ii) its individual constituent epitopes, (iii) its nondeamidated precursor, and (iv) a reference high-affinity ligand of HLA-DQ2 that is not recognized by gluten-responsive T cells from celiac sprue patients. The deamidated 33-mer peptide efficiently exchanges with a preloaded peptide in the DQ2 ligand-binding groove at pH 5.5 as well as pH 7.3, suggesting that the peptide can be presented to T cells comparably well through the endocytic pathway or via direct loading onto extracellular HLA-DQ2. In contrast, the monovalent peptides, and the nondeamidated precursor, as well as the tight-binding reference peptide show a much poorer ability to exchange with a preloaded peptide in the DQ2 binding pocket, especially at pH 7.3, suggesting that endocytosis of these peptides is a prerequisite for T cell presentation. At pH 5.5 and 7.3, dissociation of the deamidated 33-mer peptide from DQ2 is much slower than dissociation of its constituent monovalent epitopes or the nondeamidated precursor but faster than dissociation of the reference high-affinity peptide. Oligomeric states involving multiple copies of the DQ2 heterodimer bound to a single copy of the multivalent 33-mer peptide are not observed. Together, these results suggest that the remarkable antigenicity of the 33-mer gluten peptide is primarily due to its unusually efficient ability to displace existing ligands in the HLA-DQ2 binding pocket, rather than an extremely low rate of dissociation.
View details for DOI 10.1021/bi047747c
View details for Web of Science ID 000227736900034
View details for PubMedID 15766274
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Structural and mechanistic analysis of two prolyl endopeptidases: Role of interdomain dynamics in catalysis and specificity
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2005; 102 (10): 3599-3604
Abstract
Prolyl endopeptidases (PEPs) are a unique class of serine proteases with considerable therapeutic potential for the treatment of celiac sprue. The crystal structures of two didomain PEPs have been solved in alternative configurations, thereby providing insights into the mode of action of these enzymes. The structure of the Sphingomonas capsulata PEP, solved and refined to 1.8-A resolution, revealed an open configuration of the active site. In contrast, the inhibitor-bound PEP from Myxococcus xanthus was crystallized (1.5-A resolution) in a closed form. Comparative analysis of the two structures highlights a critical role for the domain interface in regulating interdomain dynamics and substrate specificity. Structure-based mutagenesis of the M. xanthus PEP confirms an important role for several interfacial residues. A salt bridge between Arg-572 and Asp-196/Glu-197 appears to act as a latch for opening or closing the didomain enzyme, and Arg-572 and Ile-575 may also help secure the incoming peptide substrate to the open form of the enzyme. Arg-618 and Asp-145 are responsible for anchoring the invariant proline residue in the active site of this postproline-cleaving enzyme. A model is proposed for the docking of a representative substrate PQPQLPYPQPQLP in the active site, where the N-terminal substrate residues interact extensively with the catalytic domain, and the C-terminal residues stretch into the propeller domain. Given the promise of the M. xanthus PEP as an oral therapeutic enzyme for treating celiac sprue, our results provide a strong foundation for further optimization of the PEP's clinically useful features.
View details for DOI 10.1073/pnas.0408286102
View details for Web of Science ID 000227533100016
View details for PubMedID 15738423
View details for PubMedCentralID PMC553306
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Future therapeutic options for celiac disease
NATURE CLINICAL PRACTICE GASTROENTEROLOGY & HEPATOLOGY
2005; 2 (3): 140-147
Abstract
Celiac disease is a disorder of the small intestine caused by an inappropriate immune response to wheat gluten and similar proteins of barley and rye. At present, the only available treatment is a strict gluten-exclusion diet; hence the need for alternative treatments. Recent advances have improved our understanding of the molecular basis for this disorder and there are several attractive targets for new treatments. Oral enzyme supplementation is designed to accelerate gastrointestinal degradation of proline-rich gluten, especially its proteolytically stable antigenic peptides. Complementary strategies aiming to interfere with activation of gluten-reactive T cells include the inhibition of intestinal tissue transglutaminase activity to prevent selective deamidation of gluten peptides, and blocking the binding of gluten peptides to the HLA-DQ2 or HLA-DQ8 molecules. Other possible treatments include cytokine therapy, and selective adhesion molecule inhibitors that interfere with inflammatory reactions, some of which are already showing promise in the clinic for other gastrointestinal diseases.
View details for Web of Science ID 000229069300009
View details for PubMedID 16265155
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Tissue transglutaminase-mediated formation and cleavage of histamine-gliadin complexes: Biological effects and implications for celiac disease
JOURNAL OF IMMUNOLOGY
2005; 174 (3): 1657-1663
Abstract
Celiac disease is an HLA-DQ2-associated disorder characterized by an intestinal T cell response. The disease-relevant T cells secrete IFN-gamma upon recognition of gluten peptides that have been deamidated in vivo by the enzyme tissue transglutaminase (transglutaminase 2 (TG2)). The celiac intestinal mucosa contains elevated numbers of mast cells, and increased histamine secretion has been reported in celiac patients. This appears paradoxical because histamine typically biases T cell responses in the direction of Th2 instead of the Th1 pattern seen in the celiac lesions. We report that histamine is an excellent substrate for TG2, and it can be efficiently conjugated to gluten peptides through TG2-mediated transamidation. Histamine-peptide conjugates do not exert agonistic effects on histamine receptors, and scavenging of biologically active histamine by gluten peptide conjugation can have physiological implications and may contribute to the mucosal IFN-gamma response in active disease. Interestingly, TG2 is able to hydrolyze the peptide-histamine conjugates when the concentrations of substrates are lowered, thereby releasing deamidated gluten peptides that are stimulatory to T cells.
View details for Web of Science ID 000226571300065
View details for PubMedID 15661929
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Chain elongation, macrolactonization, and hydrolysis of natural and reduced hexaketide substrates by the picromycin/methymycin polyketide synthase
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
2005; 44 (46): 7557-7560
View details for DOI 10.1002/anie.200502246
View details for Web of Science ID 000233694800009
View details for PubMedID 16247819
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Prolyl endopeptidase-mediated destruction of T cell epitopes in whole gluten: Chemical and immunological characterization
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2005; 312 (1): 19-26
Abstract
Celiac Sprue is a widely prevalent immune disease of the small intestine induced by dietary gluten intake in genetically susceptible individuals. It has been suggested that prolyl endopeptidases (PEPs) may be useful catalysts for gluten detoxification. We have investigated this hypothesis using food-grade gluten as the target antigen, and a combination of mass spectrometry and patient-derived T cells as quantitative assay systems. Spectrometric characterization of physiologically proteolyzed gluten revealed a number of 10 to 50 residue peptides containing known T cell epitopes involved in Celiac Sprue pathogenesis. Several of these peptides were multivalent, suggesting they may be potent triggers of the inflammatory response to gluten in celiac patients. Treatment of proteolyzed gluten with recombinant bacterial PEP decreased the number of potentially immunostimulatory peptides. Substantially reduced immunogenicity was also quantified in 12 of 14 intestinal polyclonal T cell lines from celiac patients. Kinetic investigations using eight T cell clones showed rapid destruction of alpha-gliadin epitopes, but less complete processing of gamma-gliadin epitopes. Given the difficulty associated with a strict lifelong gluten-exclusion diet, the ability of a single enzyme to greatly reduce the antigenic burden of grocery store gluten reinforces the case for developing oral peptidase therapy against Celiac Sprue.
View details for DOI 10.1124/jpet.104.073312
View details for Web of Science ID 000225766400003
View details for PubMedID 15358813
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Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase
BIOCHEMISTRY
2004; 43 (51): 16301-16310
Abstract
The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The KS-catalyzed reaction involves two discrete steps consisting of formation of an acyl-enzyme intermediate generated from the incoming acylthioester substrate and an active site cysteine residue, and the conversion of this intermediate to the beta-ketoacyl-acyl carrier protein product by a decarboxylative condensation with a paired methylmalonyl-SACP. We have determined the rate constants for the individual biochemical steps by a combination of protein acylation and transthioesterification experiments. The first-order rate constant (k(2)) for formation of the acyl-enzyme intermediate from [1-(14)C]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (2) and recombinant DEBS module 2 is 5.8 +/- 2.6 min(-)(1), with a dissociation constant (K(S)) of 3.5 +/- 2.8 mM. The acyl-enzyme adduct was formed at a near-stoichiometric ratio of approximately 0.8:1. Transthioesterification between unlabeled diketide-SNAC 2 and N-[1-(14)C-acetyl]cysteamine gave a k(exch) of 0.15 +/- 0.06 min(-)(1), with a K(m) for HSNAC of 5.7 +/- 4.9 mM and a K(m) for 2 of 5.3 +/- 0.9 mM. Under the conditions that were used, k(exch) was equal to k(-)(2), the first-order rate constant for reversal of the acyl-enzyme-forming reaction. Since the rate of the decarboxylative condensation is much greater that the rate of reversion to the starting material (k(3) > k(-)(2)), formation of the acyl-enzyme adduct is effectively irreversible, thereby establishing that the observed value of the specificity constant (k(cat)/K(m)) is solely a reflection of the intrinsic substrate specificity of the KS-catalyzed acyl-enzyme-forming reaction. These findings were also extended to a panel of diketide- and triketide-SNAC analogues, revealing that some substrate analogues that are not converted to product by DEBS module 2 form dead-end acyl-enzyme intermediates.
View details for DOI 10.1021/bi048147g
View details for Web of Science ID 000225937700032
View details for PubMedID 15610024
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Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase
BIOCHEMISTRY
2004; 43 (44): 13892-13898
Abstract
The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions.
View details for DOI 10.1021/bi048418n
View details for Web of Science ID 000224939400002
View details for PubMedID 15518537
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Crystal structure of the beta-subunit of Acyl-CoA carboxylase: Structure-based engineering of substrate specificity
BIOCHEMISTRY
2004; 43 (44): 14027-14036
Abstract
Acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) catalyze the carboxylation of acetyl- and propionyl-CoA to generate malonyl- and methylmalonyl-CoA, respectively. Understanding the substrate specificity of ACC and PCC will (1) help in the development of novel structure-based inhibitors that are potential therapeutics against obesity, cancer, and infectious disease and (2) facilitate bioengineering to provide novel extender units for polyketide biosynthesis. ACC and PCC in Streptomyces coelicolor are multisubunit complexes. The core catalytic beta-subunits, PccB and AccB, are 360 kDa homohexamers, catalyzing the transcarboxylation between biotin and acyl-CoAs. Apo and substrate-bound crystal structures of PccB hexamers were determined to 2.0-2.8 A. The hexamer assembly forms a ring-shaped complex. The hydrophobic, highly conserved biotin-binding pocket was identified for the first time. Biotin and propionyl-CoA bind perpendicular to each other in the active site, where two oxyanion holes were identified. N1 of biotin is proposed to be the active site base. Structure-based mutagenesis at a single residue of PccB and AccB allowed interconversion of the substrate specificity of ACC and PCC. The di-domain, dimeric interaction is crucial for enzyme catalysis, stability, and substrate specificity; these features are also highly conserved among biotin-dependent carboxyltransferases. Our findings enable bioengineering of the acyl-CoA carboxylase (ACCase) substrate specificity to provide novel extender units for the combinatorial biosynthesis of polyketides.
View details for DOI 10.1021/bi049065v
View details for Web of Science ID 000224939400016
View details for PubMedID 15518551
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Engineered biosynthesis of polyketides in heterologous hosts
18th International Symposium on Chemical Reaction Engineering
PERGAMON-ELSEVIER SCIENCE LTD. 2004: 4693–4701
View details for DOI 10.1016/j.ces.2004.09.026
View details for Web of Science ID 000226044200005
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Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue
BIOCHEMICAL JOURNAL
2004; 383: 311-318
Abstract
Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye. We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX). These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis. In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes. All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa. Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity. SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX. Whereas the FM enzyme showed a preference for Pro-Gln bonds, SC cleaved both Pro-Gln and Pro-Tyr bonds with comparable efficiency, and MX had a modest preference for Pro-(Tyr/Phe) sites over Pro-Gln sites. While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.
View details for DOI 10.1042/BJ20040907
View details for Web of Science ID 000224794200013
View details for PubMedID 15245330
View details for PubMedCentralID PMC1134072
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Effect of prolyl endopeptidase on digestive-resistant gliadin peptides in vivo
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2004; 311 (1): 213-219
Abstract
Many gluten peptides elicit proliferative responses from T cells from Celiac Sprue patients, influencing the pathogenesis of this small intestinal disorder. These peptides are Pro- and Gln-rich in character, suggesting that resistance to proteolysis promotes their toxicity. To test this hypothesis, we analyzed the digestive resistance of a panel of alpha- and gamma-gliadin peptides believed to induce toxicity via diverse mechanisms. Most were highly resistant to gastric and pancreatic protease digestion, but they were digested by intestinal brush-border peptidases. In some instances, there was accumulation of relatively long intermediates. Control peptides from gliadin and myoglobin revealed that digestive resistance depended on factors other than size. Prolyl endopeptidase (PEP) supplementation substantially reduced the concentrations of these peptides. To estimate a pharmacologically useful PEP dose, recombinant PEP was coperfused into rat intestine with the highly digestive-resistant 33-mer peptide LQLQPF(PQPQLPY)(3) PQPQPF (PEP: peptide weight ratio 1:50 to 1:5). PEP dosing experiments indicate significant changes in the average residence time. The in vivo benefit of PEP was verified by coperfusion with a mixture of 33-mer and partially proteolyzed gliadin. These data verify and extend our earlier proposal that gliadin peptides, although resistant to proteolysis, can be processed efficiently by PEP supplementation. Indeed, PEP may be able to treat Celiac Sprue by reducing or eliminating such peptides from the intestine.
View details for DOI 10.1124/jpet.104.068429
View details for Web of Science ID 000223896100025
View details for PubMedID 15143130
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An antibiotic factory caught in action
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2004; 11 (9): 888-893
Abstract
The synthesis of aromatic polyketides, such as actinorhodin, tetracycline and doxorubicin, begins with the formation of a polyketide chain. In type II polyketide synthases (PKSs), chains are polymerized by the heterodimeric ketosynthase-chain length factor (KS-CLF). Here we present the 2.0-A structure of the actinorhodin KS-CLF, which shows polyketides being elongated inside an amphipathic tunnel approximately 17 A in length at the heterodimer interface. The structure resolves many of the questions about the roles of KS and CLF. Although CLF regulates chain length, it does not have an active site; KS must catalyze both chain initiation and elongation. We provide evidence that the first cyclization of the polyketide occurs within the KS-CLF tunnel. The mechanistic details of this central PKS polymerase could guide biosynthetic chemists in designing new pharmaceuticals and polymers.
View details for DOI 10.1038/nsmb808
View details for Web of Science ID 000223540200024
View details for PubMedID 15286722
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Exploring the biosynthetic potential of bimodular aromatic polyketide synthases
TETRAHEDRON
2004; 60 (35): 7659-7671
View details for DOI 10.1016/j.tet.2004.05.118
View details for Web of Science ID 000223259900014
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Reconstitution and characterization of a new desosaminyl transferase, EryCIII, from the erythromycin biosynthetic pathway
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2004; 126 (32): 9924-9925
Abstract
EryCIII converts alpha-mycarosyl erythronolide B into erythromycin D using TDP-d-desosamine as the glycosyl donor. We report the heterologous expression, purification, in vitro reconstitution, and preliminary characterization of EryCIII. Coexpression of EryCIII with the GroEL/ES chaperone complex was found to enhance greatly the expression of soluble EryCIII protein. The enzyme was found to be highly active with a kcat greater than 100 min-1. EryCIII was quite selective for the natural nucleotide sugar donor and macrolide acceptor substrates, unlike several other antibiotic glycosyl transferases with broad specificity such as desVII, oleG2, and UrdGT2. Within detectable limits, neither 6-deoxyerythronolide B nor 10-deoxymethynolide were found to be glycosylated by EryCIII. Furthermore, TDP-d-mycaminose, which only differs from TDP-d-desosamine at the C4 position, could not be transferred to alphaMEB. These studies lay the groundwork for detailed structural and mechanistic analysis of an important member of the desosaminyl transferase family of enzymes.
View details for DOI 10.1021/ja048836f
View details for Web of Science ID 000223279300025
View details for PubMedID 15303858
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Antigen presentation to celiac lesion-derived T cells of a 33-mer gliadin peptide naturally formed by gastrointestinal digestion
JOURNAL OF IMMUNOLOGY
2004; 173 (3): 1757-1762
Abstract
Celiac disease is an HLA-DQ2-associated disorder characterized by intestinal T cell responses to ingested wheat gluten proteins. A peptide fragment of 33 residues (alpha(2)-gliadin 56-88) produced by normal gastrointestinal proteolysis contains six partly overlapping copies of three T cell epitopes and is a remarkably potent T cell stimulator after deamidation by tissue transglutaminase (TG2). This 33-mer is rich in proline residues and adopts the type II polyproline helical conformation in solution. In this study we report that after deamidation, the 33-mer bound with higher affinity to DQ2 compared with other monovalent peptides harboring gliadin epitopes. We found that the TG2-treated 33-mer was presented equally effectively by live and glutaraldehyde-fixed, EBV-transformed B cells. The TG2-treated 33-mer was also effectively presented by glutaraldehyde-fixed dendritic cells, albeit live dendritic cells were the most effective APCs. A strikingly increased T cell stimulatory potency of the 33-mer compared with a 12-mer peptide was also seen with fixed APCs. The 33-mer showed binding maximum to DQ2 at pH 6.3, higher than maxima found for other high affinity DQ2 binders. The 33-mer is thus a potent T cell stimulator that does not require further processing within APC for T cell presentation and that binds to DQ2 with a pH profile that promotes extracellular binding.
View details for Web of Science ID 000222807100032
View details for PubMedID 15265905
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The acyltransferase homologue from the initiation module of the R1128 polyketide synthase is an acyl-ACP thioesterase that edits acetyl primer units
BIOCHEMISTRY
2004; 43 (29): 9546-9555
Abstract
Type II polyketide synthases (PKSs) synthesize polyfunctional aromatic polyketides through iterative condensations of malonyl extender units. The biosynthesis of most aromatic polyketides is initiated through an acetate unit derived from decarboxylation of malonyl-acyl carrier protein (ACP). Modification of this primer unit represents a powerful method of generating novel polyketides. We have demonstrated that recombination of the initiation module from the R1128 PKS with heterologous elongation modules afforded regioselectively modified polyketides containing alternative primer units. With the exception of the role of the acyltransferase homologue ZhuC, the catalytic cycle of the initiation module has been well explored. ZhuC, along with the ketosynthase III homologue ZhuH and the ACP(p) ZhuG, is essential for the in vivo biosynthesis of aromatic polyketides derived from non-acetate primer units. Here we have studied the role of ZhuC using PKS proteins reconstituted in vitro. We show that the tetracenomycin (tcm) minimal PKS can be directly primed with non-acetate acyl groups. In the presence of approximately 10 microM hexanoyl-ZhuG or approximately 100 microM hexanoyl-CoA, the tcm minimal PKS synthesized hexanoyl-primed analogues of octaketides SEK4 and SEK4b, as well as acetate-primed decaketides SEK15 and SEK15b at comparable levels. Addition of ZhuC abolished synthesis of the acetate-primed decaketides, resulting in exclusive synthesis of the hexanoyl-primed octaketides. In the absence of alternative acyl donors, ZhuC severely retarded the activity of the tcm minimal PKS. The editing capabilities of ZhuC were directly revealed by demonstrating that ZhuC has 100 times greater specificity for acetyl- and propionyl-ACP as compared to hexanoyl- and octanoyl-ACP. Thus, by purging the acetate primer units that otherwise dominate polyketide chain initiation, ZhuC (and presumably its homologues in other PKSs such as the doxorubicin and frenolicin PKSs) allows alternative primer units to be utilized by the elongation module in vivo. The abilities of other alkylacyl primer units to prime the tcm minimal PKS were also investigated in this report.
View details for DOI 10.1021/bi049157k
View details for Web of Science ID 000222964900027
View details for PubMedID 15260498
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Precursor-directed biosynthesis of epothilone in Escherichia coli
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2004; 126 (24): 7436-7437
Abstract
Engineered biosynthetic pathways provide a powerful method for generating complex molecules. Precursor-directed biosynthesis, which combines chemical synthesis and enzymatic transformations, allows non-native starting materials to be incorporated into biosynthetic pathways. Using this approach, we achieved the production of the anticancer agent epothilone C in Escherichia coli. An E. coli strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosynthetic enzymes was obtained by chemical synthesis. Under high-density cell culture conditions, the E. coli strain processed exogenously fed synthetic substrate into epothilone C at levels comparable to the native host (1 mg/L) and at higher levels than other heterologous hosts. Importantly, this precursor-directed approach will allow chemical modifications to be introduced into the polyketide backbone and may ultimately provide access to epothilone analogues with improved pharmacological properties in quantities sufficient for clinical development.
View details for DOI 10.1021/ja048108s
View details for Web of Science ID 000222120900012
View details for PubMedID 15198579
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Kinetic study of the ketoacyl-ACP synthase of the erythromycin polyketide synthase.
227th National Meeting of the American-Chemical Society
AMER CHEMICAL SOC. 2004: U241–U241
View details for Web of Science ID 000223655600882
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Structural basis for HLA-DQ2-mediated presentation of gluten epitopes in celiac disease
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2004; 101 (12): 4175-4179
Abstract
Celiac disease, also known as celiac sprue, is a gluten-induced autoimmune-like disorder of the small intestine, which is strongly associated with HLA-DQ2. The structure of DQ2 complexed with an immunogenic epitope from gluten, QLQPFPQPELPY, has been determined to 2.2-A resolution by x-ray crystallography. The glutamate at P6, which is formed by tissue transglutaminase-catalyzed deamidation, is an important anchor residue as it participates in an extensive hydrogen-bonding network involving Lys-beta71 of DQ2. The gluten peptide-DQ2 complex retains critical hydrogen bonds between the MHC and the peptide backbone despite the presence of many proline residues in the peptide that are unable to participate in amide-mediated hydrogen bonds. Positioning of proline residues such that they do not interfere with backbone hydrogen bonding results in a reduction in the number of registers available for gluten peptides to bind to MHC class II molecules and presumably impairs the likelihood of establishing favorable side-chain interactions. The HLA association in celiac disease can be explained by a superior ability of DQ2 to bind the biased repertoire of proline-rich gluten peptides that have survived gastrointestinal digestion and that have been deamidated by tissue transglutaminase. Finally, surface-exposed proline residues in the proteolytically resistant ligand were replaced with functionalized analogs, thereby providing a starting point for the design of orally active agents for blocking gluten-induced toxicity.
View details for DOI 10.1073/pnas.0306885101
View details for Web of Science ID 000220472200035
View details for PubMedID 15020763
View details for PubMedCentralID PMC384714
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Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases.
PLoS biology
2004; 2 (2): E31-?
Abstract
Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs). The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.
View details for PubMedID 14966533
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Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases
PLOS BIOLOGY
2004; 2 (2): 227-238
Abstract
Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs). The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.
View details for DOI 10.1371/journal.pbio.0020031
View details for Web of Science ID 000189314400015
View details for PubMedCentralID PMC340942
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Manipulation and analysis of polyketide synthases
PROTEIN ENGINEERING
2004; 388: 269-293
View details for Web of Science ID 000223657700023
View details for PubMedID 15289078
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Timeline - Metabolic engineering for drug discovery and development
NATURE REVIEWS DRUG DISCOVERY
2003; 2 (12): 1019-1025
View details for DOI 10.1038/nrd1256
View details for Web of Science ID 000186893200019
View details for PubMedID 14654799
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Crystal structure of an Acyl-ACP dehydrogenase from the FK520 polyketide Biosynthetic pathway: Insights into extender unit biosynthesis
JOURNAL OF MOLECULAR BIOLOGY
2003; 334 (3): 435-444
Abstract
Polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as the immunosuppressants FK520 and FK506. Understanding polyketide biosynthesis at atomic resolution could present new opportunities for chemo-enzymatic synthesis of complex molecules. The crystal structure of FkbI, an enzyme involved in the biosynthesis of the methoxymalonyl extender unit of FK520, was solved to 2.1A with an R(crys) of 24.4%. FkbI has a similar fold to acyl-CoA dehydrogenases. Notwithstanding this similarity, the surface and substrate-binding site of FkbI reveal key differences from other acyl-CoA dehydrogenases, suggesting that FkbI may recognize an acyl-ACP substrate rather than an acyl-CoA substrate. This structural observation coincided the genetic experiment done by Carroll et al. J. Am. Chem. Soc., 124 (2002) 4176. Although an in vitro assay for FkbI remains elusive, the structural basis for the substrate specificity of FkbI is analyzed by a combination of sequence comparison, docking simulations and structural analysis. A biochemical mechanism for the role of FkbI in the biosynthesis of methoxymalonyl-ACP is proposed.
View details for DOI 10.1016/j.jmb.2003.10.021
View details for Web of Science ID 000186710600007
View details for PubMedID 14623185
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Enhancing the modularity of the modular polyketide synthases: Transacylation in modular polyketide synthases catalyzed by malonyl-CoA : ACP transacylase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2003; 125 (47): 14307-14312
Abstract
Selective incorporation of extender units in modular polyketide synthases is primarily controlled by acyl transferase (AT) domains. The AT domains catalyze transacylation of the extender unit from acyl-CoA to the phosphopantetheine arm of an acyl carrier protein (ACP) domain in the same module. New methods that can modulate the extender unit specificity of individual modules with minimal structural or kinetic perturbations in the engineered module are desirable for the efficient biosynthesis of novel natural product analogues. We have demonstrated that transacylation of malonyl groups onto an AT-null form of a mutant modular polyketide synthase by malonyl-CoA:ACP transacylase is an effective strategy for the engineered biosynthesis of site specifically modified polyketides. Using this strategy, 6-deoxyerythronolide B synthase was engineered to exclusively produce 2-desmethyl-6-deoxyerythronolide B. The productivity of the modified system was comparable to that of the wild-type synthase in vitro and in vivo.
View details for DOI 10.1021/ja037429l
View details for Web of Science ID 000186722200046
View details for PubMedID 14624579
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A switch for the transfer of substrate between nonribosomal peptide and polyketide modules of the rifamycin synthetase assembly line
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2003; 125 (45): 13664-13665
Abstract
A nonribosomal peptide synthetase (NRPS) loading module and a polyketide synthase (PKS) elongation module catalyze the preliminary steps in the biosynthesis of the rifamycin antibiotics. A benzoate molecule is covalently attached to the phosphopantetheine arm of the thiolation domain of the loading module when its reaction partner methylmalonyl-CoA is absent. Occupancy of the thiolation domain of the elongation module by a methylmalonyl moiety appears to trigger intermodular transfer of benzoate to the ketosynthase domain of the elongation module. This transthiolation event is fast relative to the initial loading of benzoate onto the loading module. It will be of interest to determine if these results are generally true for intermodular acyl transfer in other NRPS-PKS and PKS assembly lines.
View details for DOI 10.1021/ja0379060
View details for Web of Science ID 000186424800021
View details for PubMedID 14599196
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Precursor-directed polyketide biosynthesis in Escherichia coli
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
2003; 13 (21): 3701-3704
Abstract
Precursor-directed polyketide biosynthesis was demonstrated in the heterologous host Escherichia coli. Several diketide and triketide substrates were fed to a recombinant E. coli strain containing a variant form of deoxyerythronolide B synthase (DEBS) from which the first elongation module was deleted resulting in successful macrolactone formation from the diketide, but not the triketide, substrates.
View details for DOI 10.1016/j.bmcl.2003.08.008
View details for Web of Science ID 000185992400014
View details for PubMedID 14552761
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Biosynthesis of yersiniabactin, a complex polyketide-nonribosomal peptide, using Escherichia coli as a heterologous host
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
2003; 69 (11): 6698-6702
Abstract
The medicinal value associated with complex polyketide and nonribosomal peptide natural products has prompted biosynthetic schemes dependent upon heterologous microbial hosts. Here we report the successful biosynthesis of yersiniabactin (Ybt), a model polyketide-nonribosomal peptide hybrid natural product, using Escherichia coli as a heterologous host. After introducing the biochemical pathway for Ybt into E. coli, biosynthesis was initially monitored qualitatively by mass spectrometry. Next, production of Ybt was quantified in a high-cell-density fermentation environment with titers reaching 67 +/- 21 (mean +/- standard deviation) mg/liter and a volumetric productivity of 1.1 +/- 0.3 mg/liter-h. This success has implications for basic and applied studies on Ybt biosynthesis and also, more generally, for future production of polyketide, nonribosomal peptide, and mixed polyketide-nonribosomal peptide natural products using E. coli.
View details for DOI 10.1128/AEM.69.11.6698-6702.2003
View details for Web of Science ID 000186427800046
View details for PubMedID 14602630
View details for PubMedCentralID PMC262314
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Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases
JOURNAL OF BIOLOGICAL CHEMISTRY
2003; 278 (43): 42020-42026
Abstract
Modular polyketide biosynthesis can be harnessed to generate rationally designed complex natural products through bioengineering. A detailed understanding of the features that govern transfer and processing of polyketide biosynthetic intermediates is crucial to successfully engineer new polyketide pathways. Previous studies have shown that substrate stereochemistry and protein-protein interactions between polyketide synthase modules are both important factors in this process. Here we investigated the substrate tolerance of different polyketide modules and assessed the relative importance of inter-module chain transfer versus chain elongation activity of some of these modules. By constructing a variety of hybrid modular polyketide synthase systems and assaying their ability to generate polyketide products, it was determined that the substrate tolerance of each individual ketosynthase domain is an important parameter for the successful recombination of polyketide synthase modules. Surprisingly, however, failure by a module to process a candidate substrate was not due to its inability to bind to it. Rather, it appeared to result from a blockage in carbon-carbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzyme-catalyzed decarboxylative condensation reaction.
View details for DOI 10.1074/jbc.M305339200
View details for Web of Science ID 000185989500057
View details for PubMedID 12923197
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Polyketide chain length control by chain length factor
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2003; 125 (42): 12708-12709
Abstract
Bacterial aromatic polyketides are pharmacologically important natural products. A critical parameter that dictates product structure is the carbon chain length of the polyketide backbone. Systematic manipulation of polyketide chain length represents a major unmet challenge in natural product biosynthesis. Polyketide chain elongation is catalyzed by a heterodimeric ketosynthase. In contrast to homodimeric ketosynthases found in fatty acid synthases, the active site cysteine is absent from the one subunit of this heterodimer. The precise role of this catalytically silent subunit has been debated over the past decade. We demonstrate here that this subunit is the primary determinant of polyketide chain length, thereby validating its designation as chain length factor. Using structure-based mutagenesis, we identified key residues in the chain length factor that could be manipulated to convert an octaketide synthase into a decaketide synthase and vice versa. These results should lead to novel strategies for the engineered biosynthesis of hitherto unidentified polyketide scaffolds.
View details for DOI 10.1021/ja0378759
View details for Web of Science ID 000185990300021
View details for PubMedID 14558809
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Structure-based mutagenesis of the Malonyl-CoA : Acyl carrier protein transacylase from Streptomyces coelicolor
BIOCHEMISTRY
2003; 42 (37): 11057-11064
Abstract
Malonyl-CoA:acyl carrier protein transacylase (MAT) provides acyl-ACP thioesters for the biosynthesis of aromatic polyketides, and thus is the primary gatekeeper of substrate specificity in type II PKS. A recent report described the X-ray crystal structure of the Streptomyces coelicolor MAT and suggested active site residues which may be important for substrate selectivity [Keatinge-Clay, A. T., et al. (2003) Structure 11, 147-154]. Mutants were made to test the proposed roles of these residues, and the enzymes were characterized kinetically with respect to native and non-native substrates. The activity of the MAT was observed to be greatly attenuated in many of the observed mutants; however, the K(m) for malonyl-CoA was only modestly affected. Our results suggest the MAT uses an active site that is rigorously ordered around the acyl-thioester moiety of the acyl-CoA to facilitate rapid and efficient transacylation to an ACP. Our results also suggest that the MAT does not discriminate against alpha-substituted acyl-CoA thioesters solely on the basis of substrate size.
View details for DOI 10.1021/bi0349672
View details for Web of Science ID 000185447100022
View details for PubMedID 12974642
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Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2003; 100 (17): 9774-9778
Abstract
Ansamycins such as rifamycin, ansamitocin, and geldanamycin are an important class of polyketide natural products. Their biosynthetic pathways are especially complex because they involve the formation of 3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid nonribosomal peptide synthetase/polyketide synthase. We have reconstituted the ability to synthesize 2,6-dimethyl-3,5,7-trihydroxy-7-(3'-amino-5'-hydroxyphenyl)-2,4-heptadienoic acid (P8/1-OG), an intermediate in rifamycin biosynthesis, in an extensively manipulated strain of Escherichia coli. The parent strain, BAP1, contains the sfp phosphopantetheinyl transferase gene from Bacillus subtilis, which posttranslationally modifies polyketide synthase and nonribosomal peptide synthetase modules. AHBA biosynthesis in this host required introduction of seven genes from Amycolatopsis mediterranei, which produces rifamycin, and Actinosynnema pretiosum, which produces ansamitocin. Because the four-module RifA protein (530 kDa) from the rifamycin synthetase could not be efficiently produced in an intact form in E. coli, it was genetically split into two bimodular proteins separated by matched linker pairs to facilitate efficient inter-polypeptide transfer of a biosynthetic intermediate. A derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon, the bicistronic RifA construct and the pccB and accA1 genes from Streptomyces coelicolor, which enable methylmalonyl-CoA biosynthesis. Fermentation of this strain of E. coli yielded P8/1-OG, an N-acetyl P8/1-OG analog, and AHBA. In addition to providing a fundamentally new route to shikimate and ansamycin-type compounds, this result enables further genetic manipulation of AHBA-derived polyketide natural products with unprecedented power.
View details for DOI 10.1073/pnas.1632167100
View details for Web of Science ID 000184926000028
View details for PubMedID 12888623
View details for PubMedCentralID PMC187841
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A specific role of the Saccharopolyspora erythraea thioesterase II gene in the function of modular polyketide synthases
MICROBIOLOGY-SGM
2003; 149: 2213-2225
Abstract
Bacterial modular polyketide synthase (PKS) genes are commonly associated with another gene that encodes a thioesterase II (TEII) believed to remove aberrantly loaded substrates from the PKS. Co-expression of the Saccharopolyspora erythraea ery-ORF5 TEII and eryA genes encoding 6-deoxyerythronolide B synthase (DEBS) in Streptomyces hosts eliminated or significantly lowered production of 8,8'-deoxyoleandolide [15-nor-6-deoxyerythronolide B (15-nor-6dEB)], which arises from an acetate instead of a propionate starter unit. Disruption of the TEII gene in an industrial Sac. erythraea strain caused a notable amount of 15-norerythromycins to be produced by utilization of an acetate instead of a propionate starter unit and also resulted in moderately lowered production of erythromycin compared with the amount produced by the parental strain. A similar behaviour of the TEII gene was observed in Escherichia coli strains that produce 6dEB and 15-methyl-6dEB. Direct biochemical analysis showed that the ery-ORF5 TEII enzyme favours hydrolysis of acetyl groups bound to the loading acyl carrier protein domain (ACP(L)) of DEBS. These results point to a clear role of the TEII enzyme, i.e. removal of a specific type of acyl group from the ACP(L) domain of the DEBS1 loading module.
View details for DOI 10.1099/mic.0.26015-0
View details for Web of Science ID 000184846300028
View details for PubMedID 12904561
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Ketosynthases in the initiation and elongation modules of aromatic polyketide synthases have orthogonal acyl carrier protein specificity
BIOCHEMISTRY
2003; 42 (21): 6588-6595
Abstract
Many bacterial aromatic polyketides are synthesized by type II polyketide synthases (PKSs) which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT). This minimal PKS initiates polyketide biosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leads to incorporation of an acetate starter unit. In non-acetate-primed PKSs, such as the frenolicin (fren) PKS and the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternative acyl groups. Elucidation of these unusual priming pathways could lead to the engineered biosynthesis of polyketides containing novel starter units. Unique to some non-acetate-primed PKSs is a second catalytic module comprised of a dedicated homodimeric KS, an additional ACP, and a MAT. This initiation module is responsible for starter-unit selection and catalysis of the first chain elongation step. To elucidate the protein-protein recognition features of this dissociated multimodular PKS system, we expressed and purified two priming and two elongation KSs, a set of six ACPs from diverse sources, and a MAT. In the presence of the MAT, each ACP was labeled with malonyl-CoA rapidly. In the presence of a KS-CLF and MAT, all ACPs from minimal PKSs supported polyketide synthesis at comparable rates (k(cat) between 0.17 and 0.37 min(-1)), whereas PKS activity was attenuated by at least 50-fold in the presence of an ACP from an initiation module. In contrast, the opposite specificity pattern was observed with priming KSs: while ACPs from initiation modules were good substrates, ACPs from minimal PKSs were significantly poorer substrates. Our results show that KS-CLF and KSIII recognize orthogonal sets of ACPs, and the additional ACP is indispensable for the incorporation of non-acetate primer units. Sequence alignments of the two classes of ACPs identified a tyrosine residue that is unique to priming ACPs. Site-directed mutagenesis of this amino acid in the initiation and elongation module ACPs of the R1128 PKS confirmed the importance of this residue in modulating interactions between KSs and ACPs. Our study provides new biochemical insights into unusual chain initiation mechanisms of bacterial aromatic PKSs.
View details for DOI 10.1021/bi0341962
View details for Web of Science ID 000183210900031
View details for PubMedID 12767243
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Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2003; 125 (19): 5671-5676
Abstract
Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products. The substrate specificity of PICS modules 5+TE and 6+TE was investigated using N-acetylcysteamine thioesters of 2-methyl-3-hydroxy-pentanoic acid as diketide analogues of the natural polyketide chain elongation substrates. PICS module 5+TE could catalyze the chain elongation of only the syn diketide (2S,3R)-4, while PICS module 6+TE processed both syn diastereomers, (2S,3R)-4 and (2R,3S)-5, with a 2.5:1 preference in k(cat)/K(m) for 5 but did not turn over either of the two anti diketides. The observed substrate specificity patterns are in contrast to the 15-100:1 preference for 4 over 5 previously established for several modules of the closely related erythromycin PKS, 6-deoxyerythronolide B synthase (DEBS).
View details for DOI 10.1021/ja034574q
View details for Web of Science ID 000182769400038
View details for PubMedID 12733905
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Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2003; 125 (18): 5366-5374
Abstract
Many polyketides are synthesized by a class of multifunctional enzymes called type I modular polyketide synthases (PKSs). Several reports have described the power of predictively altering polyketide structure by replacing individual PKS domains with homologues from other PKSs. For example, numerous erythromycin analogues have been generated by replacing individual methylmalonyl-specific acyl transferase (AT) domains of the 6-deoxyerythronolide B synthase (DEBS) with malonyl-, ethylmalonyl-, or methoxymalonyl-specific domains. However, the construction of hybrid PKS modules often attenuates product formation both kinetically and distributively. The molecular basis for this mechanistic imperfection is not understood. We have systematically analyzed the impact of replacing an AT domain of DEBS on acyl-AT formation, acyl-CoA:HS-NAc acyl transferase activity, acyl-CoA:ACP acyl transferase activity (nucleophile charging), acyl-SNAc:ketosynthase acyl transferase activity (electrophile charging), and beta-ketoacyl ACP synthase activity (condensation). As usual, domain junctions were located in interdomain regions flanking the AT domain. Kinetic analysis of hybrid modules containing either malonyl transferase or methylmalonyl transferase domains revealed a 15-20-fold decrease in overall turnover numbers of the hybrid modules as compared to the wild-type module. In contrast, both the activity and the specificity of the heterologous AT domains remained unaffected. Moreover, no defects could be detected in the ability of the heterologous AT domains to catalyze acyl-CoA:ACP acyl transfer. Single turnover studies aimed at directly probing the ketosynthase-catalyzed reaction led to two crucial findings. First, wild-type modules catalyzed chain elongation with comparable efficiency regardless of whether methylmalonyl-ACP or malonyl-ACP were the nucleophilic substrates. Second, chain elongation in all hybrid modules tested was seriously attenuated relative to the wild-type module. Our data suggest that, as currently practiced, the most deleterious impact of AT domain swapping is not on the substrate specificity. Rather, it is due to the impaired ability of the KS and ACP domains in the hybrid module to catalyze chain elongation. Consistent with this proposal, limited proteolysis of wild-type and hybrid modules showed major differences in cleavage patterns, especially in the region between the KR and ACP domains.
View details for DOI 10.1021/ja029539i
View details for Web of Science ID 000182682700035
View details for PubMedID 12720450
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Solution structure and backbone dynamics of the holo form of the frenolicin acyl carrier protein
BIOCHEMISTRY
2003; 42 (16): 4648-4657
Abstract
During polyketide biosynthesis, acyl carrier proteins (ACPs) perform the central role of transferring polyketide intermediates between active sites of polyketide synthase. The 4'-phosphopantetheine prosthetic group of a holo-ACP is a long and flexible arm that can reach into different active sites and provide a terminal sulfhydryl group for the attachment of acyl groups through a thioester linkage. We have determined the solution structure and characterized backbone dynamics of the holo form of the frenolicin acyl carrier protein (fren holo-ACP) by nuclear magnetic resonance (NMR). Unambiguous assignments were made for 433 hydrogen atoms, 333 carbon atoms, and 84 nitrogen atoms, representing a total of 94.6% of the assignable atoms in this protein. From 879 meaningful NOEs and 45 angle constraints, a family of 24 structures has been calculated. The solution structure is composed of three major alpha-helices packed in a bundle with three additional short helices in intervening loops; one of the short helices slowly exchanges between two conformations. Superposition of the major helical regions on the mean structure yields average atomic rmsd values of 0.49 +/- 0.09 and 0.91 +/- 0.08 A for backbone and non-hydrogen atoms, respectively. Although the three-helix bundle fold is conserved among acyl carrier proteins involved in fatty acid synthases and polyketide synthases, a detailed comparison revealed that ACPs from polyketide biosynthetic pathways are more related to each other in tertiary fold than to their homologues from fatty acid biosynthetic pathways. Comparison of the free form of ACPs (NMR structures of fren ACP and the Bacillus subtilis ACP) with the substrate-bound form of ACP (crystal structure of butyryl-ACP from Escherichia coli) suggests that conformational exchange plays a role in substrate binding.
View details for DOI 10.1021/bi0274120
View details for PubMedID 12705828
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Intermodular communication in modular polyketide synthases: Structural and mutational analysis of linker mediated protein-protein recognition
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2003; 125 (14): 4097-4102
Abstract
Modular polyketide synthases (PKSs) present an attractive scaffold for the engineered biosynthesis of novel polyketide products via recombination of naturally occurring enzyme modules with desired catalytic properties. Recent studies have highlighted the pivotal role of short intermodular "linker pairs" in the selective channeling of biosynthetic intermediates between adjacent PKS modules. Using a combination of computer modeling, NMR spectroscopy, cross-linking, and site-directed mutagenesis, we have investigated the mechanism by which a linker pair from the 6-deoxyerythronolide B synthase promotes chain transfer. Our studies support a "coiled-coil" model in which the individual peptides comprising this linker pair adopt helical conformations that associate through a combination of hydrophobic and electrostatic interactions in an antiparallel fashion. Given the important contribution of such linker pair interactions to the kinetics of chain transfer between PKS modules, the ability to rationally modulate linker pair affinity by site-directed mutagenesis could be useful in the construction of optimized hybrid PKSs.
View details for DOI 10.1021/ja0297537
View details for Web of Science ID 000182003500035
View details for PubMedID 12670230
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Building-block selectivity of polyketide synthases
CURRENT OPINION IN CHEMICAL BIOLOGY
2003; 7 (2): 279-284
Abstract
For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.
View details for DOI 10.1016/S1367-5931(03)000016-4
View details for Web of Science ID 000182739700017
View details for PubMedID 12714062
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Epothilone C macrolactonization and hydrolysis are catalyzed by the isolated thioesterase domain of epothilone polyketide synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2003; 125 (12): 3428-3429
Abstract
Epothilone C is produced by the combined action of one nonribosomal peptide synthetase (NRPS) and nine polyketide synthase (PKS) modules in a multienzyme system. The final step in the biosynthesis is the thioesterase (TE)-catalyzed cyclorelease of epothilone from the EpoF protein. It has been unclear whether isolated PKS TE domains could exhibit macrolactonization activity. Here we demonstrate that the excised epothilone TE domain can catalyze the efficient cyclization of the N-acetylcysteamine thioester of seco-epothilone C to generate epothilone C (kcat/KM = 0.41 +/- 0.03 min-1 mM-1). The TE domain also catalyzes the hydrolysis of both the N-acetylcysteamine thioester of seco-epothilone C (kcat = 0.087 +/- 0.005 min-1, KM = 291 +/- 53 muM) and that of the epothilone C (kcat = 0.67 +/- 0.01 min-1, KM = 117 +/- 5 muM) to form seco-epothilone C.
View details for DOI 10.1021/ja0298646
View details for Web of Science ID 000181755800014
View details for PubMedID 12643694
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Design, synthesis, and evaluation of gluten peptide analogs as selective inhibitors of human tissue transglutaminase
CHEMISTRY & BIOLOGY
2003; 10 (3): 225-231
Abstract
Recent studies have implicated a crucial role for tissue transglutaminase (TG2) in the pathogenesis of Celiac Sprue, a disorder of the small intestine triggered in genetically susceptible individuals by dietary exposure to gluten. Proteolytically stable peptide inhibitors of human TG2 were designed containing acivicin or alternatively 6-diazo-5-oxo-norleucine (DON) as warheads. In biochemical and cell-based assays, the best of these inhibitors, Ac-PQP-(DON)-LPF-NH(2), was considerably more potent and selective than other TG2 inhibitors reported to date. Selective pharmacological inhibition of extracellular TG2 should be useful in exploring the mechanistic implications of TG2-catalyzed modification of dietary gluten, a phenomenon of considerable relevance in Celiac Sprue.
View details for DOI 10.1016/S1074-5521(03)00045-0
View details for Web of Science ID 000184092400008
View details for PubMedID 12670536
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Catalysis, specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA : ACP transacylase
STRUCTURE
2003; 11 (2): 147-154
Abstract
Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.
View details for Web of Science ID 000180905000006
View details for PubMedID 12575934
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Quantitative analysis of loading and extender acyltransferases of modular polyketide synthases
BIOCHEMISTRY
2003; 42 (1): 200-207
Abstract
The acyltransferase (AT) domains of modular polyketide synthases (PKSs) are the primary determinants of building block specificity in polyketide biosynthesis and are therefore attractive targets for protein engineering. Thus far, investigations into the fundamental biochemical properties of AT domains have been hampered by the inability to produce these enzymes as self-standing polypeptides. Here we describe an alternative, generally applicable strategy for overexpression and analysis of AT domains from modular PKSs as truncated didomain proteins (approximately 60 kDa). Recently, we reported the expression and reconstitution of the loading didomain of 6-deoxyerythronolide B synthase (Lau, J., Cane, D. E., and Khosla, C. (2000) Biochemistry 39, 10514-20). By replacing the AT domain of this protein with a methylmalonyl-CoA specific AT domain from module 6 of the 6-deoxyerythronolide B synthase, or alternatively a malonyl-CoA specific AT domain from module 2 of the rapamycin synthase, each of these extender unit AT domains could be overproduced and purified to homogeneity. Using acyl-CoA substrates as acyl group donors and N-acetylcysteamine as the thiol acceptor, we devised a steady-state kinetic assay to probe the properties of these three didomain proteins and selected mutants. Propionyl-CoA was the preferred substrate of the loading didomain, although acetyl- and butyryl-CoA were also accepted with approximately 40-fold-lower specificity. In contrast to the relatively relaxed specificity of the loading AT domain, the methylmalonyl- and malonyl-specific AT domains had high specificity (>1000-fold) toward their natural substrates. The acyl transfer reaction was inhibited by coenzyme A (CoASH) with both a competitive and a noncompetitive component. Use of an exogenous holo-acyl carrier protein (ACP) as an acceptor thiol did not increase the rate of acyl transfer relative to the reaction involving N-acetylcysteamine, suggesting that either the on-rate of the acyl group is rate-limiting or that the apo-ACP component of the didomain protein precludes effective docking of a second ACP onto the AT active site. Mutation of Trp-222 in the loading AT domain to an Arg residue that is universally conserved in all extender unit AT domains failed to enable the loading AT domain to accept methylmalonyl-CoA as an alternative substrate. In contrast, mutation of the equivalent Arg residue in an extender AT domain resulted in a protein with no activity. Together, these results provide a foundation for future structural and mechanistic investigations into the properties of AT domains of modular PKSs.
View details for DOI 10.1021/bi0268100
View details for Web of Science ID 000180324300023
View details for PubMedID 12515555
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Precursor-directed biosynthesis: Stereospecificity for branched-chain diketides of the beta-ketoacyl-ACP synthase domain 2 of 6-deoxyerythronolide B synthase
HELVETICA CHIMICA ACTA
2003; 86 (12): 3889-3907
View details for Web of Science ID 000188095500001
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Circular dichroism and nuclear magnetic resonance spectroscopic analysis of immunogenic gluten peptides and their analogs
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (47): 45572-45578
Abstract
Celiac Sprue, or gluten-sensitive enteropathy, is an inheritable human disease of the small intestine that is triggered by the dietary intake of gluten. Recently, several Pro- and Gln-rich peptide sequences (most notably PQPQLPY and analogs) have been identified from gluten with potent immunogenic activity toward CD4(+) T cells from small intestinal biopsies of Celiac Sprue patients. These peptides have three unusual properties. First, they are relatively stable toward further proteolysis by gastric, pancreatic, and intestinal enzymes. Second, they are recognized and deamidated by human tissue transglutaminase (tTGase) with high selectivity. Third, tTGase-catalyzed deamidation enhances their affinity for HLA-DQ2, the disease-specific class II major histocompatibility complex heterodimer. In an attempt to seek a mechanistic explanation for these properties, we undertook secondary structural studies on PQPQLPY and its analogs. Circular dichroism studies on a series of monomeric and dimeric analogs revealed a strong polyproline II helical propensity in a subset of them. Two-dimensional nuclear magnetic resonance spectroscopic analysis confirmed a polyproline II conformation of PQPQLPY, and was also used to elucidate the secondary structure of the most helical variant, (D-P)QPQLPY. Remarkably, a strong correlation was observed between polyproline II content of naturally occurring gluten peptides and the specificity of human tTGase toward these substrates. Analogs with up to two D-amino acid residues retained both polyproline II helical content and transglutaminase affinity. Since the Michaelis constant (K(m)) is the principal determinant of tTGase specificity for naturally occurring gluten peptides and their analogs, our results suggest that the tTGase binding site may have a preference for polyproline II helical substrates. If so, these insights could be exploited for the design of selective small molecule inhibitors of this pharmacologically important enzyme.
View details for DOI 10.1074/jbc.M207606200
View details for Web of Science ID 000179404800123
View details for PubMedID 12324465
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Crystal structure of the priming beta-ketosynthase from the R1128 polyketide biosynthetic pathway
STRUCTURE
2002; 10 (11): 1559-1568
Abstract
ZhuH is a priming ketosynthase that initiates the elongation of the polyketide chain in the biosynthetic pathway of a type II polyketide, R1128. The crystal structure of ZhuH in complex with the priming substrate acetyl-CoA reveals an extensive loop region at the dimer interface that appears to affect the selectivity for the primer unit. Acetyl-CoA is bound in a 20 A-long channel, which placed the acetyl group against the catalytic triad. Analysis of the primer unit binding site in ZhuH suggests that it can accommodate acyl chains that are two to four carbons long. Selectivity and primer unit size appear to involve the side chains of three residues on the loops close to the dimer interface that constitute the bottom of the substrate binding pocket.
View details for Web of Science ID 000179175300014
View details for PubMedID 12429097
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Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases
BIOCHEMISTRY
2002; 41 (42): 12598-12606
Abstract
Modular polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as erythromycin and picromycin. Understanding PKSs at high resolution could present new opportunities for chemoenzymatic synthesis of complex molecules. The crystal structures of macrocycle-forming thioesterase (TE) domains from the picromycin synthase (PICS) and 6-deoxyerythronolide B synthase (DEBS) were determined to 1.8-3.0 A with an R(crys) of 19.2-24.4%, including three structures of PICS TE (crystallized at pH 7.6, 8.0, and 8.4) and a second crystal form of DEBS TE. As predicted by the previous work on DEBS TE [Tsai, S. C., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14808-14813], PICS TE contains an open substrate channel and a hydrophobic dimer interface. Notwithstanding their similarity, the dimer interfaces and substrate channels of DEBS TE and PICS TE reveal key differences. The structural basis for the divergent substrate specificities of DEBS TE and PICS TE is analyzed. The size of the substrate channel increases with increasing pH, presumably due to electrostatic repulsion in the channel at elevated pH. Together, these structures support previous predictions that macrocycle-forming thioesterases from PKSs share the same protein fold, an open substrate channel, a similar catalytic mechanism, and a hydrophobic dimer interface. They also provide a basis for the design of enzymes capable of catalyzing regioselective macrocyclization of natural or synthetic substrates. A series of high-resolution snapshots of a protein channel at different pHs is presented alongside analysis of channel residues, which could help in the redesign of the protein channel architecture.
View details for DOI 10.1021/bi0260177
View details for Web of Science ID 000178694000005
View details for PubMedID 12379102
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Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase
BIOCHEMISTRY
2002; 41 (42): 12590-12597
Abstract
The thioesterase (TE) domain of the methymycin/picromycin synthase (PICS) was functionally expressed in Escherichia coli, and the optimal N-terminal boundary of the recombinant TE was determined. A series of diketide-N-acetylcysteamine (SNAC) thioesters were tested as substrates. PICS TE showed a strong preference for the 2-methyl-3-ketopentanoyl-SNAC substrate 5 over the stereoisomers of the reduced diketides 1-4, with an approximately 1.6:1 preference for the (2R,3S)-2-methyl-3-hydroxy diastereomer 2 over the (2S,3R)-diketide 1. The closely related DEBS TE, the thioesterase from the 6-deoxyerythronolide B synthase, showed a more marked 4.4:1 preference for 2 over 1, with only a slightly greater preference for the 3-ketoacyl-SNAC substrate 5. The roles of several active site residues in PICS TE were examined by site-directed mutagenesis. Serine 148, which is part of the apparent catalytic triad consisting of S148, H268, and D176, was found to be essential for thioesterase activity, while replacement of D176 with asparagine (D176N) gave a mutant thioesterase that retained substantial, albeit reduced, hydrolytic activity toward diketide-SNAC substrates. Mutation of E187 and R191, each of which is thought to play a role in substrate binding, had only minor effects on the relative specificity for diketide substrates 1, 2, and 5. Finally, when PICS TE was fused to the C-terminus of DEBS module 3, the resultant chimeric protein converted diketide 1 with methylmalonyl-CoA to triketide ketolactone 6 with improved catalytic efficiency compared to that of the previously developed DEBS module 3-(DEBS)TE construct.
View details for DOI 10.1021/bi026006d
View details for Web of Science ID 000178694000004
View details for PubMedID 12379101
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Intestinal digestive resistance of immunodominant gliadin peptides
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
2002; 283 (4): G996-G1003
Abstract
Two recently identified immunodominant epitopes from alpha-gliadin account for most of the stimulatory activity of dietary gluten on intestinal and peripheral T lymphocytes in patients with celiac sprue. The proteolytic kinetics of peptides containing these epitopes were analyzed in vitro using soluble proteases from bovine and porcine pancreas and brush-border membrane vesicles from adult rat intestine. We showed that these proline-glutamine-rich epitopes are exceptionally resistant to enzymatic processing. Moreover, as estimated from the residual peptide structure and confirmed by exogenous peptidase supplementation, dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I were identified as the rate-limiting enzymes in the digestive breakdown of these peptides. A similar conclusion also emerged from analogous studies with brush-border membrane from a human intestinal biopsy. Supplementation of rat brush-border membrane with trace quantities of a bacterial prolyl endopeptidase led to the rapid destruction of the immunodominant epitopes in these peptides. These results suggest a possible enzyme therapy strategy for celiac sprue, for which the only current therapeutic option is strict exclusion of gluten-containing food.
View details for DOI 10.1152/ajpgi.00136.2002
View details for Web of Science ID 000177916600020
View details for PubMedID 12223360
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Structural basis for gluten intolerance in Celiac sprue
SCIENCE
2002; 297 (5590): 2275-2279
Abstract
Celiac Sprue, a widely prevalent autoimmune disease of the small intestine, is induced in genetically susceptible individuals by exposure to dietary gluten. A 33-mer peptide was identified that has several characteristics suggesting it is the primary initiator of the inflammatory response to gluten in Celiac Sprue patients. In vitro and in vivo studies in rats and humans demonstrated that it is stable toward breakdown by all gastric, pancreatic, and intestinal brush-border membrane proteases. The peptide reacted with tissue transglutaminase, the major autoantigen in Celiac Sprue, with substantially greater selectivity than known natural substrates of this extracellular enzyme. It was a potent inducer of gut-derived human T cell lines from 14 of 14 Celiac Sprue patients. Homologs of this peptide were found in all food grains that are toxic to Celiac Sprue patients but are absent from all nontoxic food grains. The peptide could be detoxified in in vitro and in vivo assays by exposure to a bacterial prolyl endopeptidase, suggesting a strategy for oral peptidase supplement therapy for Celiac Sprue.
View details for Web of Science ID 000178222000051
View details for PubMedID 12351792
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Engineering of molecular and cellular biocatalysts: Selected contributions by James E. Bailey
BIOTECHNOLOGY AND BIOENGINEERING
2002; 79 (5): 490-495
Abstract
James (Jay) E. Bailey was a pioneer in biotechnology and biochemical engineering. During his 30 years in academia he made seminal contributions to many fields of chemical engineering science, including catalysis and reaction engineering, bioprocess engineering, mathematical modeling of cellular processes, recombinant DNA technology, enzyme engineering, and metabolic engineering. This article celebrates some of his contributions to the engineering of molecular and cellular biocatalysts, and identifies the influence he had on current and future research in biotechnology.
View details for DOI 10.1002/bit.10404
View details for Web of Science ID 000177483600003
View details for PubMedID 12209820
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Kinetic and structural analysis of a new group of acyl-CoA carboxylases found in Streptomyces coelicolor A3(2)
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (34): 31228-31236
Abstract
Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated alpha subunit, AccA2. The beta and the epsilon subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the beta and epsilon components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding epsilon subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionyl-CoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the epsilon subunits were found to specifically interact with their carboxyltransferase component forming a beta-epsilon subcomplex; this appears to facilitate the further interaction of these subunits with the alpha component. The epsilon subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.
View details for DOI 10.1074/jbc.M203263200
View details for Web of Science ID 000177579800104
View details for PubMedID 12048195
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Process and metabolic strategies for improved production of Escherichia coli-derived 6-deoxyetythronolide B
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
2002; 68 (7): 3287-3292
Abstract
Recently, the feasibility of using Escherichia coli for the heterologous biosynthesis of complex polyketides has been demonstrated. In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of complex polyketides is described. The effects of various physiological conditions on the productivity and titers of 6-deoxyerythronolide B (6dEB; the macrocyclic core of the antibiotic erythromycin) in recombinant cultures of E. coli were studied in shake flask cultures. The resulting data were used as a foundation to develop a high-cell-density fermentation procedure by building upon procedures reported earlier for recombinant protein production in E. coli. The fermentation strategy employed consistently produced approximately 100 mg of 6dEB per liter, whereas shake flask conditions generated between 1 and 10 mg per liter. The utility of an accessory thioesterase (TEII from Saccharopolyspora erythraea) for enhancing the productivity of 6dEB in E. coli was also demonstrated (increasing the final titer of 6dEB to 180 mg per liter). In addition to reinforcing the potential for using E. coli as a heterologous host for wild-type- and engineered-polyketide biosynthesis, the procedures described in this study may be useful for the production of secondary metabolites that are difficult to access by other routes.
View details for DOI 10.1128/AEM.68.7.3287-3292.2002
View details for Web of Science ID 000176631600013
View details for PubMedID 12089005
View details for PubMedCentralID PMC126764
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Biochemistry-engineering interface in biochemical engineering
AICHE JOURNAL
2002; 48 (7): 1366-1368
View details for Web of Science ID 000176822400001
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Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli
BIOCHEMISTRY
2002; 41 (16): 5193-5201
Abstract
A barrier to heterologous production of complex polyketides in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a common extender substrate for the biosynthesis of complex polyketides by modular polyketide synthases. One biosynthetic route to (2S)-methylmalonyl-CoA involves the sequential actions of two enzymes, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase, which convert succinyl-CoA to (2R)- and then to (2S)-methylmalonyl-CoA. As reported [McKie, N., et al. (1990) Biochem. J. 269, 293-298; Haller, T., et al. (2000) Biochemistry 39, 4622-4629], when genes encoding coenzyme B(12)-dependent methylmalonyl-CoA mutases were expressed in E. coli, the inactive apo-enzyme was produced. However, when cells harboring the mutase genes from Propionibacterium shermanii or E. coli were treated with the B12 precursor hydroxocobalamin, active holo-enzyme was isolated, and (2R)-methylmalonyl-CoA represented approximately 10% of the intracellular CoA pool. When the E. coli BAP1 cell line [Pfeifer, B. A., et al. (2001) Science 291, 1790-1792] harboring plasmids that expressed P. shermanii methylmalonyl-CoA mutase, Streptomyces coelicolor methylmalonyl-CoA epimerase, and the polyketide synthase DEBS (6-deoxyerythronolide B synthase) was fed propionate and hydroxocobalamin, the polyketide 6-deoxyerythronolide B (6-dEB) was produced. Isotopic labeling studies using [(13)C]propionate showed that the starter unit for polyketide synthesis was derived exclusively from exogenous propionate, while the extender units stemmed from methylmalonyl-CoA via the mutase-epimerase pathway. Thus, the introduction of an engineered mutase-epimerase pathway in E. coli enabled the uncoupling of carbon sources used to produce starter and extender units of polyketides.
View details for DOI 10.1021/bi015593k
View details for Web of Science ID 000175223400015
View details for PubMedID 11955068
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The loading and initial elongation modules of rifamycin synthetase collaborate to produce mixed aryl ketide products-1
BIOCHEMISTRY
2002; 41 (16): 5313-5324
Abstract
Rifamycin synthetase assembles the chemical backbone that members of the rifamycin family of antibiotics have in common. The synthetase contains a mixed biosynthetic interface between its loading module, which uses a nonribosomal peptide synthetase mechanism, and its initial elongation module, which uses a polyketide synthase mechanism. Biochemical studies of the loading and initial elongation modules of rifamycin synthetase reveal that this bimodular protein (LM-M1) catalyzes the formation of the phenyl ketide 3-hydroxy-2-methyl-3-phenylpropionate via a series of reactions that require benzoate, Mg.ATP, methylmalonyl-CoA, and NADPH. The overall rate of phenyl ketide production appears to be determined by the covalent loading of benzoate onto LM-M1, rather than by subsequent steps such as intermodular transfer of benzoate or condensation of benzoate and methylmalonate. Substituted benzoates that have previously been shown to be substrates for the loading module alone can also be incorporated into the corresponding aryl ketides by LM-M1, suggesting that the bimodular protein has a broad substrate tolerance. Discrimination between the substituted benzoates appears to reside in the benzoate loading reaction, and preincubation of LM-M1 with substituted benzoates and Mg.ATP allows faster downstream reactions to be unmasked. LM-M1 may be a useful biochemical system for exploring interactions between nonribosomal peptide synthetase and polyketide synthase modules.
View details for DOI 10.1021/bi0200312
View details for Web of Science ID 000175223400029
View details for PubMedID 11955082
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Quantitative analysis of the relative contributions of donor acyl carrier proteins, acceptor ketosynthases, and linker regions to intermodular transfer of intermediates in hybrid polyketide synthases
BIOCHEMISTRY
2002; 41 (15): 5056-5066
Abstract
6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB, the aglycon core of the antibiotic erythromycin. The biosynthesis of 6-dEB proceeds in an assembly-line fashion through the six modules of DEBS, each of which catalyzes a dedicated set of reactions, such that the structure of the final product is determined by the arrangement of modules along the assembly line. This transparent relationship between protein sequence and enzyme function is common to all modular PKSs and makes these enzymes an attractive scaffold for protein engineering through module swapping. One of the fundamental issues relating to module swapping that still needs to be addressed is the mechanism by which intermediates are channeled from one module to the next. While it has been previously shown that short linker regions at the N- and C-termini of adjacent polypeptides play an important role in mediating intermodular transfer, the contributions of other protein-protein interactions have not yet been probed. Here, we investigate the roles of the linker interactions as well as the interactions between the donor acyl carrier protein (ACP) domain and the downstream ketosynthase (KS) domain in various contexts. Linker interactions and ACP-KS interactions make relatively equal contributions at the module 2-module 3 and the module 4-module 5 interfaces in DEBS. In contrast, modules 2 and 6 are more tolerant toward substrates presented by nonnatural ACP domains. This tolerance was exploited for engineering hybrid PKS-PKS and PKS-NRPS (nonribosomal peptide synthetase) junctions and suggests fundamental ground rules for engineering novel chimeric PKSs in the future.
View details for DOI 10.1021/bi012086u
View details for Web of Science ID 000175012900035
View details for PubMedID 11939803
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Structural and mechanistic studies on the interactions between human tissue transglutaminase and immunodominant peptides: Implications for Celiac Sprue
W B SAUNDERS CO. 2002: A15
View details for Web of Science ID 000175366600075
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High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: Implications for Celiac Sprue
BIOCHEMISTRY
2002; 41 (1): 386-393
Abstract
Celiac Sprue is an HLA DQ2 (or DQ8)-associated autoimmune disorder of the human small intestine that is induced by dietary exposure to wheat gliadin and related proteins from barley, rye, and possibly other food grains. Recently, tissue transglutaminase (tTGase)-catalyzed deamidation of gliadin peptides has been shown to increase their potency for activating patient-derived, gliadin-specific T cells, suggesting that tTGase plays a causative role in the onset of an inflammatory response to toxic food grains. To dissect the molecular recognition features of tTGase for gluten derived peptides, the regioselectivity and steady-state kinetics of tTGase-catalyzed deamidation of known immunogenic peptides were investigated. The specificity of recombinant human tTGase for all immunogenic peptides tested was comparable to and, in some cases, appreciably higher than the specificity for its natural substrate. Although each peptide was glutamine-rich, tTGase exhibited a high degree of regioselectivity for a particular glutamine residue in each peptide. This selectivity correlated well with Q --> E substitutions that have earlier been shown to enhance the immunogenicity of the corresponding gliadin peptides. The specificity of tTGase toward homologues of PQPQLPY, a sequence motif found in immunodominant gliadin peptides, was analyzed in detail. Remarkably, the primary amino acid sequences of wheat-, rye-, and barley-derived proteins included many single-residue variants of this sequence that were high-affinity substrates of tTGase, whereas the closest homologues of this sequence found in rice, corn, or oat proteins were much poorer substrates of tTGase. (Rice, corn, and oats are nontoxic ingredients of the Celiac diet.) No consensus sequence for a high-affinity substrate of tTGase could be derived from our data, suggesting that the secondary structures of these food-grain peptides were important in their recognition by tTGase. Finally, under steady-state turnover conditions, a significant fraction of the tTGase active site was covalently bound to a representative high-affinity immunogenic gliadin peptide, suggesting a common mechanism by which cells responsible for immune surveillance of the intestinal tract recognize and generate an antibody response against both gliadin and tTGase. In addition to providing a quantitative framework for understanding the role of tTGase in Celiac Sprue, our results lay the groundwork for the design of small molecule mimetics of gliadin peptides as selective inhibitors of tTGase.
View details for DOI 10.1021/bi011715x
View details for Web of Science ID 000173216500043
View details for PubMedID 11772038
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Precursor-directed biosynthesis: Biochemical basis of the remarkable selectivity of the erythromycin polyketide synthase toward unsaturated triketides
CHEMISTRY & BIOLOGY
2002; 9 (1): 131-142
Abstract
The structural basis for the striking stereochemical discrimination among triketide analogs has been investigated by incubating a series of N-acetyl cysteamine (-SNAC) esters of unsaturated triketides with DEBS module 2+TE. The triketide analogs were first screened under a standard set of short-term incubation conditions in the presence of the extender substrate methylmalonyl-CoA and NADPH. For those triketide analogs that served as substrates for module 2+TE, the relative specificity, represented by the k(cat)/K(M) values, was quantitated. Triketide diastereomers that were converted in precursor-directed biosynthesis experiments to unsaturated 16-membered ring macrolides by DEBS(KS1(0)) were good to excellent substrates for DEBS module 2+TE, whereas analogs that were converted to the 14-membered ring analogs of 10,11-dehydro-6-deoxyerythronolide B by DEBS(KS1(0)) were not turned over at all by module 2+TE.
View details for Web of Science ID 000173696100013
View details for PubMedID 11841945
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Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: Versatility from a unique substrate channel
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2001; 98 (26): 14808-14813
Abstract
As the first structural elucidation of a modular polyketide synthase (PKS) domain, the crystal structure of the macrocycle-forming thioesterase (TE) domain from the 6-deoxyerythronolide B synthase (DEBS) was solved by a combination of multiple isomorphous replacement and multiwavelength anomalous dispersion and refined to an R factor of 24.1% to 2.8-A resolution. Its overall tertiary architecture belongs to the alpha/beta-hydrolase family, with two unusual features unprecedented in this family: a hydrophobic leucine-rich dimer interface and a substrate channel that passes through the entire protein. The active site triad, comprised of Asp-169, His-259, and Ser-142, is located in the middle of the substrate channel, suggesting the passage of the substrate through the protein. Modeling indicates that the active site can accommodate and orient the 6-deoxyerythronolide B precursor uniquely, while at the same time shielding the active site from external water and catalyzing cyclization by macrolactone formation. The geometry and organization of functional groups explain the observed substrate specificity of this TE and offer strategies for engineering macrocycle biosynthesis. Docking of a homology model of the upstream acyl carrier protein (ACP6) against the TE suggests that the 2-fold axis of the TE dimer may also be the axis of symmetry that determines the arrangement of domains in the entire DEBS. Sequence conservation suggests that all TEs from modular polyketide synthases have a similar fold, dimer 2-fold axis, and substrate channel geometry.
View details for Web of Science ID 000172848800016
View details for PubMedID 11752428
View details for PubMedCentralID PMC64940
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In vitro reconstitution and analysis of the chain initiating enzymes of the R1128 polyketide synthase
BIOCHEMISTRY
2001; 40 (49): 14855-14861
Abstract
Biosynthesis of the carbon chain backbone of the R1128 substances is believed to involve the activity of a ketosynthase/chain length factor (ZhuB/ZhuA), an additional ketosynthase (ZhuH), an acyl transferase (ZhuC), and two acyl carrier proteins (ACPs; ZhuG and ZhuN). A subset of these proteins initiate chain synthesis via decarboxylative condensation between an acetyl-, propionyl-, isobutyryl-, or butyryl-CoA derived primer unit and a malonyl-CoA derived extender unit to yield an acetoacetyl-, beta-ketopentanoyl-, 3-oxo-4-methylpentanoyl-, or beta-ketohexanoyl-ACP product, respectively. To investigate the precise roles of ZhuH, ZhuC, ZhuG, and ZhuN, each protein was expressed in Escherichia coli and purified to homogeneity. Although earlier reports had proposed that ZhuC and its homologues played a role in primer unit selection, direct in vitro analysis of ZhuC showed that it was in fact a malonyl-CoA:ACP malonyltransferase (MAT). The enzyme could catalyze malonyl transfer but not acetyl- or propionyl-transfer onto R1128 ACPs or onto ACPs from other biosynthetic pathways, suggesting that ZhuC has broad substrate specificity with respect to the holo-ACP substrate but is specific for malonyl-CoA. Thus, ZhuC supplies extender units to both the initiating and elongating ketosynthases from this pathway. To interrogate the primer unit specificity of ZhuH, the kinetics of beta-ketoacyl-ACP formation in the presence of various acyl-CoAs and malonyl-ZhuG were measured. Propionyl-CoA and isobutyryl-CoA were the two most preferred substrates of ZhuH, although acetyl-CoA and butyryl-CoA could also be accepted and elongated. This specificity is not only consistent with earlier reports demonstrating that R1128B and R1128C are the major products of the R1128 pathway in vivo, but is also in good agreement with the properties of the ZhuH substrate binding pocket, as deduced from a recently solved crystal structure of the enzyme. Finally, to investigate the molecular logic for the occurrence of not one but two ACP genes within the R1128 gene cluster, the inhibition of ZhuH-catalyzed formation of beta-ketopentanoyl-ACP was quantified in the presence of apo-ZhuG or apo-ZhuN. Both apo-proteins were comparable inhibitors of the ZhuH catalyzed reaction, suggesting that the corresponding apo-proteins can be used interchangeably during chain initiation. Together, these results provide direct biochemical insights into the mechanism of chain initiation of an unusual bacterial aromatic PKS.
View details for Web of Science ID 000172608100015
View details for PubMedID 11732905
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Malonyl-CoA : ACP transacylase from Streptomyces coelicolor has two alternative catalytically active nucleophiles
BIOCHEMISTRY
2001; 40 (41): 12407-12411
Abstract
Fatty acids and polyketides are synthesized by mechanistically and evolutionarily related multienzyme systems. Their carbon chain backbones are synthesized via repeated decarboxylative condensations of alpha-carboxylated building blocks onto a growing acyl chain. These alpha-carboxylated building blocks are transferred from the corresponding coenzyme A thioesters onto the phosphopantetheine arm of an acyl carrier protein (ACP) by acyl transferases, which operate by a ping-pong mechanism involving an acyl-O-serine intermediate. In the course of our studies on the malonyl-CoA:ACP transacylase (MAT) from Streptomyces coelicolor, we observed that an active-site Ser (97) --> Ala mutant retains activity as well as the ability to be covalently labeled by (14)C malonyl-CoA. Here we demonstrate that an alternative, catalytically competent nucleophile exists in the active site of this enzyme. Next to the active-site serine is a histidine residue that is conserved in some, but not all acyl transferases. The H96A mutant is also active and can be labeled, but an H96A/S97A double mutant is inactive and cannot be labeled. The ability of H96 to form a malonyl-imidazole adduct was confirmed by proteolysis, followed by radio-HPLC and mass spectrometric analysis of the S97A mutant enzyme. Kinetic analysis revealed that the k(cat) of the S97A mutant was within 10-fold that of the wild-type enzyme, whereas the K(M)s of the two enzymes were comparable. Sequence comparison with the E. coli MAT (whose X-ray structure is known) led to the identification of H201 as the putative base in the serine-histidine catalytic dyad of the S. coelicolor enzyme. The absence of MAT activity in the H201A mutant and the detection of weak activity in the H201Q mutant was consistent with this proposal. The implications of this unexpected finding are discussed.
View details for Web of Science ID 000171601700021
View details for PubMedID 11591161
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Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2001; 98 (15): 8548-8553
Abstract
Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein-protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an alpha-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or alpha-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.
View details for Web of Science ID 000169967000063
View details for PubMedID 11447274
View details for PubMedCentralID PMC37473
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Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2001; 123 (27): 6465-6474
Abstract
6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) that catalyzes the biosynthesis of 6-deoxyerythronolide B (6-dEB), the aglycon precursor of the antibiotic erythromycin. The biosynthesis of 6-dEB exemplifies the extraordinary substrate- and stereo-selectivity of this family of multifunctional enzymes. Paradoxically, DEBS has been shown to be an attractive scaffold for combinatorial biosynthesis, indicating that its constituent modules are also very tolerant of unnatural substrates. By interrogating individual modules of DEBS with a panel of diketides activated as N-acetylcysteamine (NAC) thioesters, it was recently shown that individual modules have a marked ability to discriminate among certain diastereomeric diketides. However, since free NAC thioesters were used as substrates in these studies, the modules were primed by a diffusive process, which precluded involvement of the covalent, substrate-channeling mechanism by which enzyme-bound intermediates are directly transferred from one module to the next in a multimodular PKS. Recent evidence pointing to a pivotal role for protein-protein interactions in the substrate-channeling mechanism has prompted us to develop novel assays to reassess the steady-state kinetic parameters of individual DEBS modules when primed in a more "natural" channeling mode by the same panel of diketide substrates used earlier. Here we describe these assays and use them to quantify the kinetic benefit of linker-mediated substrate channeling in a modular PKS. This benefit can be substantial, especially for intrinsically poor substrates. Examples are presented where the k(cat) of a module for a given diketide substrate increases >100-fold when the substrate is presented to the module in a channeling mode as opposed to a diffusive mode. However, the substrate specificity profiles for individual modules are conserved regardless of the mode of presentation. By highlighting how substrate channeling can allow PKS modules to effectively accept and process intrinsically poor substrates, these studies provide a rational basis for examining the enormous untapped potential for combinatorial biosynthesis via module rearrangement.
View details for DOI 10.1021/ja010219t
View details for Web of Science ID 000169835300001
View details for PubMedID 11439032
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Enhancing the atom economy of polyketide biosynthetic processes through metabolic engineering
BIOTECHNOLOGY PROGRESS
2001; 17 (4): 612-617
Abstract
Polyketides, a large family of bioactive natural products, are synthesized from building blocks derived from alpha-carboxylated Coenzyme A thioesters such as malonyl-CoA and (2S)-methylmalonyl-CoA. The productivity of polyketide fermentation processes in natural and heterologous hosts is frequently limited by the availability of these precursors in vivo. We describe a metabolic engineering strategy to enhance both the yield and volumetric productivity of polyketide biosynthesis. The genes matB and matC from Rhizobium trifolii encode a malonyl-CoA synthetase and a putative dicarboxylate transport protein, respectively. These proteins can directly convert exogenous malonate and methylmalonate into their corresponding CoA thioesters with an ATP requirement of 2 mol per mol of acyl-CoA produced. Heterologous expression of matBC in a recombinant strain of Streptomyces coelicolor that produces the macrolactone 6-deoxyerythronolide B results in a 300% enhancement of macrolactone titers. The unusual efficiency of the bioconversion is illustrated by the fact that approximately one-third of the methylmalonate units added to the fermentation medium are converted into macrolactones. The direct conversion of inexpensive feedstocks such as malonate and methylmalonate into polyketides represents the most carbon- and energy-efficient route to these high value natural products and has implications for cost-effective fermentation of numerous commercial and development-stage small molecules.
View details for Web of Science ID 000170338000004
View details for PubMedID 11485419
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Remarkably broad substrate tolerance of Malonyl-CoA synthetase, an enzyme capable of intracellular synthesis of polyketide precursors
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2001; 123 (24): 5822-5823
View details for PubMedID 11403625
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Erythromycin biosynthesis. The 4-pro-S hydride of NADPH is utilized for ketoreduction by both module 5 and module 6 of the 6-deoxyerythronolide B synthase
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
2001; 11 (12): 1477-1479
Abstract
Incubation of chirally deuterated NADPH with 6-deoxyerythronolide B synthase (DEBS) modules 5 and module 6 and analysis of the derived triketide lactones established that the two ketoreductase domains, KR5 and KR6, are both specific for the 4-pro-S hydride of the nicotinamide cofactor.
View details for Web of Science ID 000169365500001
View details for PubMedID 11412964
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The loading module of rifamycin synthetase is an adenylation-thiolation didomain with substrate tolerance for substituted benzoates
BIOCHEMISTRY
2001; 40 (20): 6116-6123
Abstract
The rifamycin synthetase is primed with a 3-amino-5-hydroxybenzoate starter unit by a loading module that contains domains homologous to the adenylation and thiolation domains of nonribosomal peptide synthetases. Adenylation and thiolation activities of the loading module were reconstituted in vitro and shown to be independent of coenzyme A, countering literature proposals that the loading module is a coenzyme A ligase. Kinetic parameters for covalent arylation of the loading module were measured directly for the unnatural substrates benzoate and 3-hydroxybenzoate. This analysis was extended through competition experiments to determine the relative rates of incorporation of a series of substituted benzoates. Our results show that the loading module can accept a variety of substituted benzoates, although it exhibits a preference for the 3-, 5-, and 3,5-disubstituted benzoates that most closely resemble its biological substrate. The considerable substrate tolerance of the loading module of rifamycin synthetase suggests that the module has potential as a tool for generating substituted derivatives of natural products.
View details for Web of Science ID 000168932900031
View details for PubMedID 11352749
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Intellectual border: Two-way traffic
CHEMICAL & ENGINEERING NEWS
2001; 79 (13): 149-149
View details for Web of Science ID 000167717100090
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Precursor-directed biosynthesis of 16-membered macrolides by the erythromycin polyketide synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2001; 123 (11): 2495-2502
Abstract
Streptomyces coelicolor CH999/pJRJ2 harbors a plasmid encoding DEBS(KS1 degrees ), a mutant form of 6-deoxyerythronolide B synthase that is blocked in the formation of 6-deoxyerythronolide B (1, 6-dEB) due to a mutation in the active site of the ketosynthase (KS1) domain that normally catalyzes the first polyketide chain elongation step of 6-dEB biosynthesis. Administration of (2E,4S,5R)-2,4-dimethyl-5-hydroxy-2-heptenoic acid, N-acetylcysteamine thioester (6) an unsaturated triketide analogue of the natural triketide chain elongation intermediate to cultures of S. coelicolor CH999/pJRJ2 results in formation of a 16-membered macrolactone, which is isolated in the hemiketal form 33. The formation of the octaketide 33 indicates that the triketide substrate has been processed by DEBS module 2 as if it were a diketide analogue. The substrate specificity of this novel reaction has been explored by the incubation of three additional analogues of the unsaturated triketide 6, compounds 18, 31, and 32, with S. coelicolor CH999/pJRJ2, resulting in the formation of the corresponding macrolactones 34, 35, and 36. By contrast, the unsaturated triketide 10, lacking a methyl group at C-2, did not give rise to any detectable macrolactone product when incubated with S. coelicolor CH999/pJRJ2.
View details for DOI 10.1021/ja0041391
View details for Web of Science ID 000167632900004
View details for PubMedID 11456917
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Biosynthesis of complex polyketides in a metabolically engineered strain of E-coli
SCIENCE
2001; 291 (5509): 1790-1792
Abstract
The macrocyclic core of the antibiotic erythromycin, 6-deoxyerythronolide B (6dEB), is a complex natural product synthesized by the soil bacterium Saccharopolyspora erythraea through the action of a multifunctional polyketide synthase (PKS). The engineering potential of modular PKSs is hampered by the limited capabilities for molecular biological manipulation of organisms (principally actinomycetes) in which complex polyketides have thus far been produced. To address this problem, a derivative of Escherichia coli has been genetically engineered. The resulting cellular catalyst converts exogenous propionate into 6dEB with a specific productivity that compares well with a high-producing mutant of S. erythraea that has been incrementally enhanced over decades for the industrial production of erythromycin.
View details for Web of Science ID 000167320600060
View details for PubMedID 11230695
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Biosynthesis of polyketides in heterologous hosts
MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS
2001; 65 (1): 106-?
Abstract
Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed.
View details for Web of Science ID 000167343200004
View details for PubMedID 11238987
View details for PubMedCentralID PMC99020
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Intermodular communication in polyketide syntheses: Comparing the role of protein-protein interactions to those in other multidomain proteins
BIOCHEMISTRY
2001; 40 (8): 2317-2325
Abstract
Although the role of protein-protein interactions in transducing signals within biological systems has been extensively explored, their relevance to the channeling of intermediates in metabolism is not widely appreciated. Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are two related families of modular megasynthases that channel covalently bound intermediates from one active site to the next. Recent biochemical studies have highlighted the importance of protein-protein interactions in these chain transfer processes. The information available on this subject is reviewed, and its possible mechanistic implications are placed in context by comparisons with selected well-studied multicomponent protein systems.
View details for DOI 10.1021/bi002462v
View details for Web of Science ID 000167121500001
View details for PubMedID 11327851
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Selective protein-protein interactions direct channeling of intermediates between polyketide synthase modules
BIOCHEMISTRY
2001; 40 (8): 2326-2331
Abstract
Polyketide synthases (PKSs) have represented fertile targets for rational manipulation via protein engineering ever since their modular architecture was first recognized. However, the mechanistic principles by which biosynthetic intermediates are sequentially channeled between modules remain poorly understood. Here we demonstrate the importance of complementarity in a remarkably simple, repetitive structural motif within these megasynthases that has been implicated to affect intermodular chain transfer [Gokhale, R. S., et al. (1999) Science 284, 482]. The C- and N-terminal ends of adjacent PKS polypeptides are capped by short peptides of 20-40 residues. Mismatched sequences abolish intermodular chain transfer without affecting the activity of individual modules, whereas matched sequences can facilitate the channeling of intermediates between ordinarily nonconsecutive modules. Thus, in addition to substrate-PKS interactions and domain-domain interactions, these short interpolypeptide sequences represent a third determinant of selective chain transfer that must be taken into consideration in the protein engineering of PKSs. Preliminary biophysical studies on synthetic peptide mimics of these linkers suggest that they may adopt coiled-coil conformations.
View details for Web of Science ID 000167121500002
View details for PubMedID 11327852
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Structure-activity relationships within a family of selectively cytotoxic macrolide natural products
ORGANIC LETTERS
2001; 3 (1): 57-59
Abstract
[figure: see text] We describe a semi-synthetic deglycosylated derivative of apoptolidin that retains considerable activity against the mitochondrial ATPase but has greatly reduced cellular cytotoxicity. We also demonstrate that a related antifungal natural product, cytovaricin, inhibits the same molecular target. Structural comparison of these macrolides provides insights into their conserved features that are presumably important for biological activity and identifies promising avenues at the interface of organic synthesis and biosynthesis for the generation of new selective cytotoxic agents.
View details for DOI 10.1021/ol006767d
View details for Web of Science ID 000166360000016
View details for PubMedID 11429871
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Modular enzymes
NATURE
2001; 409 (6817): 247-252
Abstract
Although modular macromolecular devices are encountered frequently in a variety of biological situations, their occurrence in biocatalysis has not been widely appreciated. Three general classes of modular biocatalysts can be identified: enzymes in which catalysis and substrate specificity are separable, multisubstrate enzymes in which binding sites for individual substrates are modular, and multienzyme systems that can catalyse programmable metabolic pathways. In the postgenomic era, the discovery of such systems can be expected to have a significant impact on the role of enzymes in synthetic and process chemistry.
View details for Web of Science ID 000166316200056
View details for PubMedID 11196653
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Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase
CHEMISTRY & BIOLOGY
2001; 8 (1): 71-80
Abstract
Apoptolidin is a macrolide originally identified on the basis of its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells. The goal of this study was to identify the molecular target of this newly discovered natural product.Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action. Cell killing induced by apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9. PARP was completely cleaved in the presence of 1 microM apoptolidin within 6 h in a mouse lymphoma cell line. Together these results suggested that apoptolidin might target a mitochondrial protein. Structural comparisons between apoptolidin and other macrolides revealed significant similarity between the apoptolidin aglycone and oligomycin, a known inhibitor of mitochondrial F0F1-ATP synthase. The relevance of this similarity was established by demonstrating that apoptolidin is a potent inhibitor of the F0F1-ATPase activity in intact yeast mitochondria as well as Triton X-100-solubilized ATPase preparations. The K(i) for apoptolidin was 4-5 microM. The selectivity of apoptolidin in the NCI-60 cell line panel was found to correlate well with that of several known anti-fungal natural products that inhibit the eukaryotic mitochondrial F0F1-ATP synthase.Although the anti-fungal activities of macrolide inhibitors of the mitochondrial F0F1-ATP synthase such as oligomycin, ossamycin and cytovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated. The recent discovery of apoptolidin, followed by the demonstration that it is an inhibitor of the mitochondrial F0F1-ATP synthase, highlights the potential relevance of these natural products as small molecules to modulate apoptotic pathways. The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is discussed.
View details for Web of Science ID 000167275800008
View details for PubMedID 11182320
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Process development and metabolic engineering for the overproduction of natural and unnatural polyketides.
Advances in biochemical engineering/biotechnology
2001; 73: 31-52
Abstract
Polyketide natural products are a rich source of bioactive substances that have found considerable use in human health and agriculture. Their complex structures require that they be produced via fermentation processes. This review describes the strategies and challenges used to develop practical fermentation strains and processes for polyketide production. Classical strain improvement procedures, process development methods, and metabolic engineering approaches are described. The elucidation of molecular mechanisms that underlie polyketide biosynthesis has played an important role in each of these areas over the past few years.
View details for PubMedID 11816811
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Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F0F1-ATPase
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2000; 97 (26): 14766-14771
Abstract
Recently, a family of polyketide inhibitors of F(0)F(1)-ATPase, including apoptolidin, ossamycin, and oligomycin, were shown to be among the top 0.1% most cell line selective cytotoxic agents of 37, 000 molecules tested against the 60 human cancer cell lines of the National Cancer Institute. Many cancer cells maintain a high level of anaerobic carbon metabolism even in the presence of oxygen, a phenomenon that is historically known as the Warburg effect. A mechanism-based strategy to sensitize such cells to this class of potent small molecule cytotoxic agents is presented. These natural products inhibit oxidative phosphorylation by targeting the mitochondrial F(0)F(1) ATP synthase. Evaluation of gene expression profiles in a panel of leukemias revealed a strong correlation between the expression level of the gene encoding subunit 6 of the mitochondrial F(0)F(1) ATP synthase (known to be the binding site of members of this class of macrolides) and their sensitivity to these natural products. Within the same set of leukemia cell lines, comparably strong drug-gene correlations were also observed for the genes encoding two key enzymes involved in central carbon metabolism, pyruvate kinase, and aspartate aminotransferase. We propose a simple model in which the mitochondrial apoptotic pathway is activated in response to a shift in balance between aerobic and anaerobic ATP biosynthesis. Inhibitors of both lactate formation and carbon flux through the Embden-Meyerhof pathway significantly sensitized apoptolidin-resistant tumors to this drug. Nine different cell lines derived from human leukemias and melanomas, and colon, renal, central nervous system, and ovarian tumors are also sensitized to killing by apoptolidin.
View details for Web of Science ID 000165993700138
View details for PubMedID 11121076
View details for PubMedCentralID PMC18993
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Natural product biosynthesis: A new interface between enzymology and medicine
JOURNAL OF ORGANIC CHEMISTRY
2000; 65 (24): 8127-8133
View details for Web of Science ID 000165548900001
View details for PubMedID 11101363
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Dissecting the chain length specificity in bacterial aromatic polyketide synthases using chimeric genes
TETRAHEDRON
2000; 56 (48): 9401-9408
View details for Web of Science ID 000165485300002
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Cloning, nucleotide sequence, and heterologous expression of the biosynthetic gene cluster for R1128, a non-steroidal estrogen receptor antagonist - Insights into an unusual priming mechanism
JOURNAL OF BIOLOGICAL CHEMISTRY
2000; 275 (43): 33443-33448
Abstract
R1128 substances are anthraquinone natural products that were previously reported as non-steroidal estrogen receptor antagonists with in vitro and in vivo potency approaching that of tamoxifen. From a biosynthetic viewpoint, these polyketides possess structurally interesting features such as an unusual primer unit that are absent in the well studied anthracyclic and tetracyclic natural products. The entire R1128 gene cluster was cloned and expressed in Streptomyces lividans, a genetically well developed heterologous host. In addition to R1128C, a novel optically active natural product, designated HU235, was isolated. Nucleotide sequence analysis of the biosynthetic gene cluster revealed genes encoding two ketosynthases, a chain length factor, an acyl transferase, three acetyl-CoA carboxylase subunits, two cyclases, two oxygenases, an amidase, and remarkably, two acyl carrier proteins. Feeding studies indicate that the unusual 4-methylvaleryl side chain of R1128C is derived from valine. Together with the absence of a dedicated ketoreductase, dehydratase, or enoylreductase within the R1128 gene cluster, this suggests a functional link between fatty acid biosynthesis and R1128 biosynthesis in the engineered host. Specifically, we propose that the R1128 synthase recruits four subunits from the endogenous fatty acid synthase during the biosynthesis of this family of pharmacologically significant natural products.
View details for Web of Science ID 000090104600038
View details for PubMedID 10931852
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Substrate specificity of the loading didomain of the erythromycin polyketide synthase
BIOCHEMISTRY
2000; 39 (34): 10514-10520
Abstract
The priming of many modular polyketide synthases is catalyzed by a loading acyltransferase-acyl carrier protein (AT(L)-ACP(L)) didomain which initiates polyketide biosynthesis by transferring a primer unit to the ketosynthase domain of the first module. Because the AT(L) domain influences the choice of the starter unit incorporated into the polyketide backbone, its specificity is of considerable interest. The AT(L)-ACP(L) didomain of the 6-deoxyerythronolide B synthase (DEBS) was functionally expressed in Escherichia coli. Coexpression of the Sfp phosphopantetheinyl transferase from Bacillus subtilis in E. coli leads to efficient posttranslational modification of the ACP(L) domain with a phosphopantetheine moiety. Competition experiments were performed with the holo-protein to determine the relative rates of incorporation of a variety of unnatural substrates in the presence of comparable concentrations of labeled acetyl-CoA. Our results showed that the loading didomain of DEBS can accept a surprisingly broad range of substrates, although it exhibits a preference for unbranched alkyl chain substrates over branched alkyl chain, polar, aromatic, and charged substrates. In particular, its tolerance toward acetyl- and butyryl-CoA is unexpectedly strong. The studies described here present an attractive prototype for the expression, analysis, and engineering of acyltransferase domains in modular polyketide synthases.
View details for DOI 10.1021/bi000602v
View details for Web of Science ID 000089066600019
View details for PubMedID 10956042
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Analysis of the molecular recognition features of individual modules derived from the erythromycin polyketide synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2000; 122 (20): 4847-4852
View details for Web of Science ID 000087266700001
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Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum
GENE
2000; 249 (1-2): 153-160
Abstract
The epothilone biosynthetic gene cluster was isolated from Sorangium cellulosum strain SMP44. The gene cluster contains seven genes and spans approx. 56kb. The genes encoding the PKS, epoA, epoC, epoD, epoE, and epoF, are divided into nine modules. The EpoB protein is a non-ribosomal peptide synthetase (NRPS) that catalyzes formation of the thiazole found in the epothilones. EpoK is a P450 enzyme responsible for the epoxidation of epothilones C and D to epothilones A and B, respectively. EpoK was expressed in Escherichia coli, and the purified protein was shown to convert epothilone D to epothilone B in vitro.
View details for Web of Science ID 000087398000016
View details for PubMedID 10831849
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Directed transfer of large DNA fragments between Streptomyces species
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
2000; 66 (5): 2274-2277
Abstract
The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically amenable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene cluster from one Streptomyces species to another. The method takes advantage of tra gene-mediated conjugal transfer of chromosomal DNA between actinomycetes. As proof of principle, we demonstrate transfer of the entire approximately 22-kb actinorhodin gene cluster, and also the high-frequency cotransfer of two loci that are 150 to 200 kb apart, from Streptomyces coelicolor to an engineered derivative of Streptomyces lividans.
View details for Web of Science ID 000086805500080
View details for PubMedID 10788417
View details for PubMedCentralID PMC101490
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Studies oh the substrate specificity of loading end extender unit acyltransferase domains in the erythromycin polyketide synthase.
AMER CHEMICAL SOC. 2000: U158–U158
View details for Web of Science ID 000087246100735
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Mechanistic analysis of a type II polyketide synthase. Role of conserved residues in the beta-ketoacyl synthase-chain length factor heterodimer
BIOCHEMISTRY
2000; 39 (8): 2088-2095
Abstract
Type II polyketide synthases (PKSs) are a family of multienzyme systems that catalyze the biosynthesis of polyfunctional aromatic natural products such as actinorhodin, frenolicin, tetracenomycin, and doxorubicin. A central component in each of these systems is the beta-ketoacyl synthase-chain length factor (KS-CLF) heterodimer. In the presence of an acyl carrier protein (ACP) and a malonyl-CoA:ACP malonyl transferase (MAT), this enzyme synthesizes a polyketide chain of defined length from malonyl-CoA. We have investigated the role of the actinorhodin KS-CLF in priming, elongation, and termination of its octaketide product by subjecting the wild-type enzyme and selected mutants to assays that probe key steps in the overall catalytic cycle. Under conditions reflecting steady-state turnover of the PKS, a unique acyl-ACP intermediate is detected that carries a long, possibly full-length, acyl chain. This species cannot be synthesized by the C169S, H309A, K341A, and H346A mutants of the KS, all of which are blocked in early steps in the PKS catalytic cycle. These four residues are universally conserved in all known KSs. Malonyl-ACP alone is sufficient for kinetically and stoichiometrically efficient synthesis of polyketides by the wild-type KS-CLF, but not by heterodimers that carry the mutations listed above. Among these mutants, C169S is an efficient decarboxylase of malonyl-ACP, but the H309A, K341A, and H346A mutants are unable to catalyze decarboxylation. Transfer of label from [(14)C]malonyl-ACP to the nucleophile at position 169 in the KS can be detected for the wild-type enzyme and for the C169S and K341A mutants, but not for the H309A mutant and only very weakly for the H346A mutant. A model is proposed for decarboxylative priming and extension of a polyketide chain by the KS, where C169 and H346 form a catalytic dyad for acyl chain attachment, H309 positions the malonyl-ACP in the active site and supports carbanion formation by interacting with the thioester carbonyl, and K341 enhances the rate of malonyl-ACP decarboxylation via electrostatic interaction. Our data also suggest that the ACP and the KS dissociate after each C-C bond forming event, and that the newly extended acyl chain is transferred back from the ACP pantetheine to the KS cysteine before dissociation can occur. Chain termination is most likely the rate-limiting step in polyketide biosynthesis. Within the act CLF, neither the universally conserved S145 residue nor Q171, which aligns with the active site cysteine of the ketosynthase, is essential for PKS activity. The results described here provide a basis for a better understanding of the catalytic cycle of type II PKSs and fatty acid synthases.
View details for Web of Science ID 000085640500022
View details for PubMedID 10684659
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Role of linkers in communication between protein modules
CURRENT OPINION IN CHEMICAL BIOLOGY
2000; 4 (1): 22-27
Abstract
Multidomain proteins are common in a variety of cellular processes. Their domains are interconnected through short stretches of amino acid residues referred to as linkers. Recent studies on many systems have provided compelling evidence that linkers are more than simple covalent connectors. They also perform the important task of establishing communication between the different functional modules that exist within such proteins.
View details for Web of Science ID 000085289500003
View details for PubMedID 10679375
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Cloning and heterologous expression of the epothilone gene cluster
SCIENCE
2000; 287 (5453): 640-642
Abstract
The polyketide epothilone is a potential anticancer agent that stabilizes microtubules in a similar manner to Taxol. The gene cluster responsible for epothilone biosynthesis in the myxobacterium Sorangium cellulosum was cloned and completely sequenced. It encodes six multifunctional proteins composed of a loading module, one nonribosomal peptide synthetase module, eight polyketide synthase modules, and a P450 epoxidase that converts desoxyepothilone into epothilone. Concomitant expression of these genes in the actinomycete Streptomyces coelicolor produced epothilones A and B. Streptomyces coelicolor is more amenable to strain improvement and grows about 10-fold as rapidly as the natural producer, so this heterologous expression system portends a plentiful supply of this important agent.
View details for Web of Science ID 000084989400044
View details for PubMedID 10649995
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Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase
CHEMISTRY & BIOLOGY
1999; 6 (9): 607-615
Abstract
Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC).The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate.Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.
View details for Web of Science ID 000082542000005
View details for PubMedID 10467128
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A host-vector system for analysis and manipulation of rifamycin polyketide biosynthesis in Amycolatopsis mediterranei
MICROBIOLOGY-SGM
1999; 145: 2335-2341
Abstract
Modular polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for the biosynthesis of numerous bacterial natural products such as erythromycin and rifamycin. Advanced genetic analysis of these remarkable systems is often seriously hampered by the large size (>40 kb) of PKS gene clusters, and, notwithstanding their considerable fundamental and biotechnological significance, by the lack of suitable methods for engineering non-selectable modifications in chromosomally encoded PKS genes. The development of a facile host-vector strategy for genetic engineering of the rifamycin PKS in the producing organism, Amycolatopsis mediterranei S699, is described here. The genes encoding all 10 modules of the rifamycin PKS were replaced with a hygromycin-resistance marker gene. In a similar construction, only the first six modules of the PKS were replaced. The deletion hosts retained the ability to synthesize the primer unit 3-amino-5-hydroxybenzoic acid (AHBA), as judged by co-synthesis experiments with a mutant strain lacking AHBA synthase activity. Suicide plasmids carrying a short fragment from the 5' flanking end of the engineered deletion, an apramycin-resistance marker gene, and suitably engineered PKS genes could be introduced via electroporation into the deletion hosts, resulting in the integration of PKS genes and biosynthesis of a reporter polyketide in quantities comparable to those produced by the wild-type organism. Since this strategy for engineering recombinant PKSs in A. mediterranei requires only a selectable single crossover and eliminates the need for tedious non-selectable double-crossover experiments, it makes rifamycin PKS an attractive target for extensive genetic manipulation.
View details for Web of Science ID 000082829000016
View details for PubMedID 10517586
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Kinetic analysis of the actinorhodin aromatic polyketide synthase
JOURNAL OF BIOLOGICAL CHEMISTRY
1999; 274 (35): 25108-25112
Abstract
Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [(14)C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.
View details for Web of Science ID 000082193400090
View details for PubMedID 10455191
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Harnessing the biochemical potential of natural product biosynthetic pathways
FEDERATION AMER SOC EXP BIOL. 1999: A1334
View details for Web of Science ID 000082033400080
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Dissecting and exploiting intermodular communication in polyketide synthases
SCIENCE
1999; 284 (5413): 482-485
Abstract
Modular polyketide synthases catalyze the biosynthesis of medicinally important natural products through an assembly-line mechanism. Although these megasynthases display very precise overall selectivity, we show that their constituent modules are remarkably tolerant toward diverse incoming acyl chains. By appropriate engineering of linkers, which exist within and between polypeptides, it is possible to exploit this tolerance to facilitate the transfer of biosynthetic intermediates between unnaturally linked modules. This protein engineering strategy also provides insights into the evolution of modular polyketide synthases.
View details for Web of Science ID 000079792200046
View details for PubMedID 10205055
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Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase.
Metabolic engineering
1999; 1 (2): 180-187
Abstract
6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase from Penicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes--a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction. MSAS has recently been functionally expressed in Escherichia coli and Saccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts. These developments have set the stage for detailed mechanistic studies of this model system.A three--step purification procedure was developed to obtain >95% pure MSAS from extracts of E. coli. As reported earlier for the enzyme isolated from P. patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH. Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo. The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA. Interestingly, MSAS readily accepted the N-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate.NAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks. The ability to produce 4-hydroxy-6-methyl-2-pyrone in both E. coli and yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides. Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.
View details for PubMedID 10935930
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Tolerance and Specificity of Recombinant 6-Methylsalicylic Acid Synthase
METABOLIC ENGINEERING
1999; 1 (2): 180-187
Abstract
6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase from Penicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes--a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction. MSAS has recently been functionally expressed in Escherichia coli and Saccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts. These developments have set the stage for detailed mechanistic studies of this model system.A three--step purification procedure was developed to obtain >95% pure MSAS from extracts of E. coli. As reported earlier for the enzyme isolated from P. patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH. Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo. The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA. Interestingly, MSAS readily accepted the N-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate.NAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks. The ability to produce 4-hydroxy-6-methyl-2-pyrone in both E. coli and yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides. Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.
View details for Web of Science ID 000209415000006
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Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units
BIOCHEMISTRY
1999; 38 (5): 1643-1651
Abstract
Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzyme complexes that are organized into modules, where each module carries the domains needed to catalyze the condensation of an extender unit onto a growing polyketide chain. Each module also dictates the stereochemistry of the chiral centers introduced into the backbone during the chain elongation process. Here we used domain mutagenesis to investigate the role of the acyl transferase (AT) domains of individual modules in the choice and stereochemical fate of extender units. Our results indicate that the AT domains of DEBS do not influence epimerization of the (2S)-methylmalonyl-CoA extender units. Hence, stereochemical control of the methyl-branched centers generated by DEBS most likely resides in the ketosynthase (KS) domains of the individual modules. In contrast, several recent studies have demonstrated that extender unit specificity can be altered by AT domain substitution. In some of these examples, the resulting polyketide was produced at considerably lower titers than the corresponding natural product. We analyzed one such attenuated mutant of DEBS, in which the methylmalonyl transferase domain of module 2 was replaced with a malonyl transferase domain. As reported earlier, the resulting PKS produced only small quantities of the expected desmethyl analogue of 6-deoxyerythronolide B. However, when the same hybrid module was placed as the terminal module in a truncated 2-module PKS, it produced nearly normal quantities of the expected desmethyl triketide lactone. These results illustrate the limits to modularity of these multifunctional enzymes. To dissect the role of specific amino acids in controlling AT substrate specificity, we exchanged several segments of amino acids between selected malonyl and methylmalonyl transferases, and found that a short (23-35 amino acid) C-terminal segment present in all AT domains is the principal determinant of their substrate specificity. Interestingly, its length and amino acid sequence vary considerably among the known AT domains. We therefore suggest that the choice of extender units by the PKS modules is influenced by a "hypervariable region", which could be manipulated via combinatorial mutagenesis to generate novel AT domains possessing relaxed or altered substrate specificity.
View details for Web of Science ID 000078836700030
View details for PubMedID 9931032
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Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase
CHEMISTRY & BIOLOGY
1999; 6 (2): 117-125
Abstract
Polyketides are important compounds with antibiotic and anticancer activities. Several modular polyketide synthases (PKSs) contain a terminal thioesterase (TE) domain probably responsible for the release and concomitant cyclization of the fully processed polyketide chain. Because the TE domain influences qualitative aspects of product formation by engineered PKSs, its mechanism and specificity are of considerable interest.The TE domain of the 6-deoxyerythronolide B synthase was overexpressed in Escherichia coli. When tested against a set of N-acetyl cysteamine thioesters the TE domain did not act as a cyclase, but showed significant hydrolytic specificity towards substrates that mimic important features of its natural substrate. Also the overall rate of polyketide chain release was strongly enhanced by a covalent connection between the TE domain and the terminal PKS module (by as much as 100-fold compared with separate TE and PKS 'domains').The inability of the TE domain alone to catalyze cyclization suggests that macrocycle formation results from the combined action of the TE domain and a PKS module. The chain-length and stereochemical preferences of the TE domain might be relevant in the design and engineered biosynthesis of certain novel polyketides. Our results also suggest that the TE domain might loop back to catalyze the release of polyketide chains from both terminal and pre-terminal modules, which may explain the ability of certain naturally occurring PKSs, such as the picromycin synthase, to generate both 12-membered and 14-membered macrolide antibiotics.
View details for Web of Science ID 000082564800008
View details for PubMedID 10021418
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Precursor directed biosynthesis of novel 6-deoxyerythronolide B analogs containing non-natural oxygen substituents and reactive functionalities
TETRAHEDRON LETTERS
1999; 40 (4): 635-638
View details for Web of Science ID 000078111100016
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Tolerance and specificity of polyketide synthases
ANNUAL REVIEW OF BIOCHEMISTRY
1999; 68: 219-253
Abstract
Polyketide synthases catalyze the assembly of complex natural products from simple precursors such as propionyl-CoA and methylmalonyl-CoA in a biosynthetic process that closely parallels fatty acid biosynthesis. Like fatty acids, polyketides are assembled by successive decarboxylative condensations of simple precursors. But whereas the intermediates in fatty acid biosynthesis are fully reduced to generate unfunctionalized alkyl chains, the intermediates in polyketide biosynthesis may be only partially processed, giving rise to complex patterns of functional groups. Additional complexity arises from the use of different starter and chain extension substrates, the generation of chiral centers, and further functional group modifications, such as cyclizations. The structural and functional modularity of these multienzyme systems has raised the possibility that polyketide biosynthetic pathways might be rationally reprogrammed by combinatorial manipulation. An essential prerequisite for harnessing this biosynthetic potential is a better understanding of the molecular recognition features of polyketide synthases. Within this decade, a variety of genetic, biochemical, and chemical investigations have yielded insights into the tolerance and specificity of several architecturally different polyketide synthases. The results of these studies, together with their implications for biosynthetic engineering, are summarized in this review.
View details for Web of Science ID 000082693200008
View details for PubMedID 10872449
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Synthesis and incorporation of an N-acetylcysteamine analogue of methylmalonyl-CoA by a modular polyketide synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1998; 120 (43): 11206-11207
View details for Web of Science ID 000076855000032
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Biochemistry - Harnessing the biosynthetic code: Combinations, permutations, and mutations
SCIENCE
1998; 282 (5386): 63-68
Abstract
Polyketides and non-ribosomal peptides are two large families of complex natural products that are built from simple carboxylic acid or amino acid monomers, respectively, and that have important medicinal or agrochemical properties. Despite the substantial differences between these two classes of natural products, each is synthesized biologically under the control of exceptionally large, multifunctional proteins termed polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) that contain repeated, coordinated groups of active sites called modules, in which each module is responsible for catalysis of one complete cycle of polyketide or polypeptide chain elongation and associated functional group modifications. It has recently become possible to use molecular genetic methodology to alter the number, content, and order of such modules and, in so doing, to alter rationally the structure of the resultant products. This review considers the promise and challenges inherent in the combinatorial manipulation of PKS and NRPS structure in order to generate entirely "unnatural" products.
View details for Web of Science ID 000076294900037
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Dissecting the evolutionary relationship between 14-membered and 16-membered macrolides
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1998; 120 (35): 9096-9097
View details for Web of Science ID 000075860100036
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Dissecting and manipulating substrate specificity of the acyltransferase domains of modular polyketide synthases.
AMER CHEMICAL SOC. 1998: U252–U252
View details for Web of Science ID 000075235100765
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Engineered biosynthesis of novel polyketides from Streptomyces spore pigment polyketide synthases
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1998; 120 (31): 7749-7759
View details for Web of Science ID 000075420100010
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Precursor-directed biosynthesis of 12-ethyl erythromycin
BIOORGANIC & MEDICINAL CHEMISTRY
1998; 6 (8): 1171-1177
Abstract
A precursor-directed method for the biosynthesis of novel 6-deoxyerythronolide B derivatives has been extended to allow alteration of the functionality at C-12. We recently described a simple and practical method for harnessing the biosynthetic potential of the erythromycin pathway to generate novel molecules of natural product-like complexity by feeding designed synthetic molecules to an engineered mutant strain having an altered 6-deoxyerythronolide B synthase (DEBS). Our initial applications of this technique focused on alteration of the ethyl side chain of 6-dEB (C14-C15). We now report the extension of this approach to modification of the C-12 substituent. An appropriately designed substrate is shown to incorporate into 6-dEB biosynthesis, yielding a 6-dEB analogue bearing a 12-ethyl group. This 6-dEB analogue is a substrate for post-polyketide tailoring enzymes, and is converted into the corresponding analogue of erythromycin C. These results show that many of the downstream active sites are tolerant of this unnatural functionality and suggest that a wide variety of erythromycin derivatives should be accessible by this approach or by total biosynthesis via genetic engineering.
View details for Web of Science ID 000075803700005
View details for PubMedID 9784859
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Erythromycin biosynthesis: The beta-ketoreductase domains catalyze the stereospecific transfer of the 4-pro-S hydride of NADPH
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1998; 120 (13): 3267-3268
View details for Web of Science ID 000073072700042
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Spontaneous priming of a downstream module in 6-deoxyerythronolide B synthase leads to polyketide biosynthesis
BIOCHEMISTRY
1998; 37 (14): 4928-4934
Abstract
Modular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products by repetitive condensation of simple carboxylic acid monomers. The synthase can be divided into groups of domains, called "modules", each of which is responsible for one cycle of chain extension and processing. The modular nature of these enzymes suggests that the biosynthetic pathway might be rationally reprogrammed by manipulation of synthases at the domain level. Although, several examples of successful engineering of DEBS have been reported, a critical issue which has not been well-studied is the tolerance of "downstream" active sites to nonnatural substrates. Here, we report that the terminal modules of DEBS, which normally process highly functionalized intermediates, are competent to carry out their natural functions on smaller, more simple substrates. Expressed in the absence of other DEBS proteins, the DEBS3 protein, which normally carries out the final two extension cycles in the synthesis of 6-deoxyerythronolide B (6-dEB), is spontaneously primed with a C3 carboxylic acid. This substrate is then extended through two condensation cycles to form a triketide. Tolerance of the "shortened" intermediates in the biosynthesis of this triketide, in combination with results reported elsewhere [Jacobsen, J. R., Hutchinson, C. R., Cane, D. E., and Khosla, C. (1997) Science 277, 367-369], suggests that relaxed substrate specificity may be a common feature of modular polyketide synthases. Interestingly, priming of DEBS3 appears to proceed, not by acyltransfer from propionyl-CoA, but by decarboxylation of an enzyme-bound methylmalonyl extender unit. This is the second example of decarboxylative priming within DEBS [see also Pieper, R., Gokhale, R. S., Luo, G., Cane, D. E., and Khosla, C. (1997) Biochemistry 36, 1846-1851] and suggests that, in the absence of an acceptable primer (or transferred intermediate), decarboxylative priming of ketosynthase domains may be a general property of modular polyketide synthases.
View details for Web of Science ID 000073279100029
View details for PubMedID 9538011
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Alcohol stereochemistry in polyketide backbones is controlled by the beta-ketoreductase domains of modular polyketide synthases
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1998; 120 (10): 2478-2479
View details for Web of Science ID 000072624500032
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Functional orientation of the acyltransferase domain in a module of the erythromycin polyketide synthase
BIOCHEMISTRY
1998; 37 (8): 2524-2528
Abstract
Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multienzymes are organized into a series of functional units known as modules. Each dimeric module contains two catalytically independent clusters of active sites homologous to those of vertebrate fatty acid synthases. Earlier studies have shown that modules consist of head-to-tail homodimers in which ketosynthase (KS) and acyl carrier protein (ACP) domains are contributed by opposite subunits to form a catalytic center. Here, we probe the functional topology of the acyltransferase (AT) domain which transfers the methylmalonyl moiety of methylmalonyl-CoA onto the phosphopantetheine arm of the ACP domain. Using a bimodular derivative of DEBS, the AT domain of module 2 (AT2) was inactivated by site-directed mutagenesis. Heterodimeric protein pairs were generated in vitro between the inactivated AT2 (AT2 degrees) polypeptide and an inactive KS1 (KS1 degrees) or KS2 (KS2 degrees) protein. Both of these hybrid proteins supported polyketide synthesis, suggesting that AT2 can perform its function from either subunit. The apparent catalytic rate constants for each of the two hybrid protein pairs, KS1 degrees/AT2 degrees and KS2 degrees/AT2 degrees, were identical, indicating that no significant kinetic preference exists for a particular AT2-ACP2 combination. These results suggest that the AT domain can be shared between the two clusters of active sites within the same dimeric module. Such a novel structural organization might provide a functional advantage for the efficient biosynthesis of polyketides.
View details for Web of Science ID 000072299900049
View details for PubMedID 9485401
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Purification and in vitro reconstitution of the essential protein components of an aromatic polyketide synthase
BIOCHEMISTRY
1998; 37 (8): 2084-2088
Abstract
A minimal set of proteins which catalyze the synthesis of aromatic polketides from malonyl CoA has been purified and partially characterized. Plasmid-encoded actinorhodin (act) ketosynthase/chain-length factor (KS/CLF) complex was purified from Streptomyces coelicolor CH999/pSEK38, and assayed with purified aromatic PKS holo-ACPs which were overproduced and purified from Escherichia coli and phosphopantetheinylated in vitro using purified E. coli holo-ACP synthase. When highly purified preparations of KS/CLF, and holo-ACP failed to catalyze polyketide biosynthesis, a fourth protein was sought and purified from the S. coelicolor CH999 host on the basis of its ability to complement KS, CLF, and holo-ACP in polyketide synthesis. N-terminal sequencing identified this protein as the fatty acid synthase (fabD) malonyl CoA:ACP transacylase (MAT), recruited from primary metabolism. A alpha2/beta2 structure was shown for the act KS/CLF complex, and three malonyl-enzyme biosynthetic intermediates were identified, defining an escorted path followed by malonyl groups en route from CoA to polyketide.
View details for Web of Science ID 000072299900002
View details for PubMedID 9518007
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Primer unit specificity in rifamycin biosynthesis principally resides in the later stages of the biosynthetic pathways
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1998; 120 (5): 1092-1093
View details for Web of Science ID 000072002600037
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New directions in metabolic engineering
CURRENT OPINION IN CHEMICAL BIOLOGY
1998; 2 (1): 133-137
Abstract
Metabolic engineering is a rapidly evolving field. The term typically refers to the genetic modification of cellular biochemistry to introduce new properties or modify existing ones. Recent progress in genetics, molecular biology, microbiology and chemistry are driving advances in this field. Many well-studied areas continue to yield exciting results and new problems and technologies are constantly being developed.
View details for Web of Science ID 000072701000017
View details for PubMedID 9667907
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Gain of function mutagenesis of the erythromycin polyketide synthase .2. Engineered biosynthesis of eight-membered ring tetraketide lactone
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1997; 119 (46): 11339-11340
View details for Web of Science ID A1997YG90100033
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Molecular recognition of diketide substrates by a beta-ketoacyl-acyl carrier protein synthase domain within a bimodular polyketide synthase
CHEMISTRY & BIOLOGY
1997; 4 (10): 757-766
Abstract
Modular polyketide synthases (PKSs) are large multifunctional proteins that catalyze the biosynthesis of structurally complex bioactive products. The modular organization of PKSs has allowed the application of a combinatorial approach to the synthesis of novel polyketides via the manipulation of these biocatalysts at the genetic level. The inherent specificity of PKSs for their natural substrates, however, may place limits on the spectrum of molecular diversity that can be achieved in polyketide products. With the aim of further understanding PKS specificity, as a route to exploiting PKSs in combinatorial synthesis, we chose to examine the substrate specificity of a single intact domain within a bimodular PKS to investigate its capacity to utilize unnatural substrates.We used a blocked mutant of a bimodular PKS in which formation of the triketide product could occur only via uptake and processing of a synthetic diketide intermediate. By introducing systematic changes in the native diketide structure, by means of the synthesis of unnatural diketide analogs, we have shown that the ketosynthase domain of module 2 (KS2 domain) in 6-deoxyerythronolide B synthase (DEBS) tolerates a broad range of variations in substrate structure, but it strongly discriminates against some others.Defining the boundaries of substrate recognition within PKS domains is crucial to the rationally engineered biosynthesis of novel polyketide products, many of which could be prepared only with great difficulty, if at all, by direct chemical synthesis or semi-synthesis. Our results suggest that the KS2 domain of DEBS1 has a relatively relaxed specificity that can be exploited for the design and synthesis of medicinally important polyketide products.
View details for Web of Science ID A1997YG16200007
View details for PubMedID 9375254
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Utilization of enzymatically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase
BIOCHEMISTRY
1997; 36 (39): 11757-11761
Abstract
The functional reconstitution of two purified proteins of an aromatic polyketide synthase pathway, the acyl carrier protein (ACP) and holo-ACP synthase (ACPS), is described. Holo-ACPs were enzymatically synthesized from coenzyme A and apo-ACPs using Escherichia coli ACPS. Frenolicin and granaticin holo-ACPs formed in this manner were shown to be fully functional together with the other components of the minimal actinorhodin polyketide synthase (act PKS), resulting in synthesis of the same aromatic polyketides as those formed by the act PKS in vivo. ACPS also catalyzed the transfer of acetyl-, propionyl-, butyryl-, benzoyl-, phenylacetyl-, and malonylphosphopantetheines to apo-ACPs from their corresponding coenzyme As, as detected by electrophoresis and/or mass spectrometry. A steady state kinetic study showed that acetyl-coenzyme A is as efficient an ACPS substrate as coenzyme A, with kcat and Km values of 20 min-1 and 25 microM, respectively. In contrast to acetyl-coenzyme A, enzymatically synthesized acetyl-ACPs were shown to be efficient substrates for the act PKS, indicating that acetyl-ACP is a chemically competent intermediate of aromatic polyketide biosynthesis. Together, these methods provide a valuable tool for dissecting the mechanisms and molecular recognition features of polyketide biosynthesis.
View details for Web of Science ID A1997XY95300023
View details for PubMedID 9305965
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Engineered intermodular and intramodular polyketide synthase fusions
CHEMISTRY & BIOLOGY
1997; 4 (9): 667-674
Abstract
Modular polyketide synthases (PKSs) are very large multifunctional enzyme complexes that synthesize a number of medicinally important natural products. The modular arrangement of active sites has made these enzyme systems amenable to combinatorial manipulation for the biosynthesis of novel polyketides. Here, we investigate the involvement of subunit interactions in hybrid and artificially linked PKSs with several series of intermodular and intramodular fusions using the erythromycin (6-deoxyerythronolide B synthase; DEBS) and rapamycin (RAPS) PKSs.Several two-module and three-module derivatives of DEBS were constructed by fusing module 6 to either module 2 or module 3 at varying junctions. Polyketide production by these intramodular fusions indicated that the core set of active sites remained functional in these hybrid modules, although the ketoreductase domain of module 6 was unable to recognize unnatural triketide and tetraketide substrates. Artificial trimodular PKS subunits were also engineered by covalently linking modules 2 and 3 of DEBS, thereby demonstrating the feasibility of constructing single-chain PKSs. Finally, a series of fusions containing DEBS and RAPS domains in module 2 of an engineered trimodular PKS revealed the structural and functional tolerance for hybrid modules created from distinct PKS gene clusters.The general success of the intermodular and intramodular fusions described here demonstrates significant structural tolerance among PKS modules and subunits and suggests that substrate specificity, rather than protein-protein interactions, is the primary determinant of molecular recognition features of PKSs. Furthermore, the ability to artificially link modules may considerably simplify the heterologous expression of modular PKSs in higher eukaryotic systems.
View details for Web of Science ID A1997YA84900005
View details for PubMedID 9331407
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Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase
SCIENCE
1997; 277 (5324): 367-369
Abstract
A genetic block was introduced in the first condensation step of the polyketide biosynthetic pathway that leads to the formation of 6-deoxyerythronolide B (6-dEB), the macrocyclic precursor of erythromycin. Exogenous addition of designed synthetic molecules to small-scale cultures of this null mutant resulted in highly selective multimilligram production of unnatural polyketides, including aromatic and ring-expanded variants of 6-dEB. Unexpected incorporation patterns were observed, illustrating the catalytic versatility of modular polyketide synthases. Further processing of some of these scaffolds by postpolyketide enzymes of the erythromycin pathway resulted in the generation of novel antibacterials with in vitro potency comparable to that of their natural counterparts.
View details for Web of Science ID A1997XL35800045
View details for PubMedID 9219693
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Domain analysis of the molecular recognition features of aromatic polyketide synthase subunits
JOURNAL OF BIOLOGICAL CHEMISTRY
1997; 272 (26): 16184-16188
Abstract
Bacterial aromatic polyketide synthases (PKSs) are a family of homologous multienzyme assemblies that catalyze the biosynthesis of numerous polyfunctional aromatic natural products. In the absence of direct insights into their structures, the use of gene fusions can be a powerful tool for understanding the structural basis for their properties. A series of truncated and hybrid proteins were constructed and analyzed within a family of PKS subunits, designated aromatases/cyclases (ARO/CYCs). When expressed alone, neither the N-terminal nor the C-terminal domain of the actinorhodin (act) or the griseusin (gris) ARO/CYC exhibited substantial aromatase activity. However, in the presence of each other, the half proteins were active. Furthermore, analysis of a set of hybrid proteins derived from the act and gris ARO/CYCs allowed us to localize the chain length dependence of this aromatase activity to their N-terminal domains. Unexpectedly, however, when the C-terminal domain of the gris ARO/CYC was expressed in a context where aromatase activity was absent, it could modulate the chain length specificity of the tetracenomycin (tcm) minimal PKS, leading to the formation of a novel 18-carbon product in addition to the expected 20-carbon one. It was also found that monodomain ARO/CYCs such as tcmN cannot substitute for the the N-terminal domain of didomain ARO/CYCs, even though they exhibit high sequence similarity with the N-terminal domain. Together, these results illustrate the utility of protein engineering approaches for dissecting the structure-function relationships of PKS subunits and for the generation of mutant alleles with novel biosynthetic properties.
View details for Web of Science ID A1997XG01900021
View details for PubMedID 9195917
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Gain-of-function mutagenesis of a modular polyketide synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1997; 119 (18): 4309-4310
View details for Web of Science ID A1997WX98900031
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Structure, function and engineering of modular polyketide synthases
AMER CHEMICAL SOC. 1997: 77-BIOT
View details for Web of Science ID A1997WP18500671
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Purification and characterization of bimodular and trimodular derivatives of the erythromycin polyketide synthase
BIOCHEMISTRY
1997; 36 (7): 1846-1851
Abstract
Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. DEBS is a dimeric protein complex that consists of three large multidomain polypeptide chains, DEBS 1, DEBS 2, and DEBS 3. In turn, each polypeptide includes two modules, where one module is responsible for a single round of condensation and associated reduction reactions. A hybrid protein comprised of the first two modules of DEBS fused to a thioesterase domain (DEBS 1 + TE) was purified to homogeneity in a fully active form (Kcat = 4.8 min-1). Synthesis of the anticipated triketide lactone required the presence of (2RS)-methylmalonyl-CoA and NADPH. When available, propionyl-CoA is the preferred source of primer units. However, in its absence the enzyme can derive primer units via decarboxylation of a methylmalonyl extender. The two subunits of an engineered trimodular derivative of DEBS, DEBS 1 and module 3 of DEBS 2 linked to the TE domain (module 3 + TE), were also individually purified and reconstituted to produce the expected tetraketide lactone in vitro (Kcat = 0.23 min-1). The considerably lower specific activity of this trimodular PKS relative to its bimodular counterpart presumably reflects inefficient association between DEBS 1 and module 3 + TE. As expected, module 3 + TE could be efficiently cross-linked as a homodimer. In contrast, no cross-links were detectable between modules 2 and 3, even though biosynthesis of the tetraketide requires transient interactions to occur between these two modules. Since module 3 only contains the minimal set of active sites required in a module (a ketosynthase, an acyltransferase, and an acyl carrier protein domain) and is the first active unimodular protein to be purified to homogeneity, it represents an attractive target for future biophysical and structural studies.
View details for Web of Science ID A1997WJ05800031
View details for PubMedID 9048569
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Rational design and engineered biosynthesis of a novel 18-carbon aromatic polyketide
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1997; 119 (4): 635-639
View details for Web of Science ID A1997WE82600001
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Harnessing the Biosynthetic Potential of Modular Polyketide Synthases.
Chemical reviews
1997; 97 (7): 2577–90
View details for PubMedID 11851472
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The chemistry and biology of fatty acid, polyketide, and nonribosomal peptide biosynthesis
BIOORGANIC CHEMISTRY DEOXYSUGARS, POLYKETIDES AND RELATED CLASSES: SYNTHESIS, BIOSYNTHESIS, ENZYMES
1997; 188: 85-126
View details for Web of Science ID A1997BH74A00002
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6-deoxyerythronolide B synthase 1 is specifically acylated by a diketide intermediate at the beta-ketoacyl-acyl carrier protein synthase domain of module 2
BIOCHEMISTRY
1996; 35 (48): 15244-15248
Abstract
We have used 6-deoxyerythronolide B synthase (DEBS) as a model system to investigate molecular recognition by a modular polyketide synthase (PKS). DEBS consists of three proteins (DEBS1, -2, and -3) that biosynthesize the polyketide skeleton of the antibiotic erythromycin from propionyl-CoA and methylmalonyl-CoA. Active sites within these multifunctional proteins are organized into biosynthetic "modules", each of which catalyzes a discrete round of polyketide chain elongation and adjusts the appropriate level of beta-ketoacylthioester reduction. Using DEBS1, we demonstrate that there is a substantial degree of molecular recognition in the processing of the natural diketide chain elongation intermediate. Exogenously added (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester is exclusively recognized by its cognate beta-ketoacyl-acyl carrier protein synthase domain in module 2 (KS2). Labeled diketide specifically acylated DEBS1 in crude protein extracts and limited proteolysis localized the binding to module 2. The precise site of acylation in DEBS1 was established by the finding that a Cys2200 Ala mutant of DEBS1, lacking the KS2 active-site cysteine, did not undergo acylation by the diketide. Pretreatment of the wild-type protein with the beta-ketoacyl-ACP synthase inhibitor cerulenin also blocked acylation. These results indicate that in addition to the purely organizational consequences resulting from the order of active-site domains, the programming of polyketide biosynthesis by modular PKSs involves a substantial level of molecular recognition. This conclusion has important implications for the use of PKSs to rationally design novel polyketides.
View details for Web of Science ID A1996VW43900015
View details for PubMedID 8952473
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A new enzyme superfamily - The phosphopantetheinyl transferases
CHEMISTRY & BIOLOGY
1996; 3 (11): 923-936
Abstract
All polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases require posttranslational modification of their constituent acyl carrier protein domain(s) to become catalytically active. The inactive apoproteins are converted to their active holo-forms by posttranslational transfer of the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a conserved serine residue in each acyl carrier protein domain. The first P-pant transferase to be cloned and characterized was the recently reported Escherichia coli enzyme ACPS, responsible for apo to holo conversion of fatty acid synthase. Surprisingly, initial searches of sequence databases did not reveal any proteins with significant peptide sequence similarity with ACPS.Through refinement of sequence alignments that indicated low level similarity with the ACPS peptide sequence, we identified two consensus motifs shared among several potential ACPS homologs. This has led to the identification of a large family of proteins having 12-22 % similarity with ACPS, which are putative P-pant transferases. Three of these proteins, E. coli EntD and o195, and B. subtilis Sfp, have been overproduced, purified and found to have P-pant transferase activity, confirming that the observed low level of sequence homology correctly predicted catalytic function. Three P-pant transferases are now known to be present in E. coli (ACPS, EntD and o195); ACPS and EntD are specific for the activation of fatty acid synthase and enterobactin synthetase, respectively. The apo-protein substrate for o195 has not yet been identified. Sfp is responsible for the activation of the surfactin synthetase.The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains. This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.
View details for Web of Science ID A1996VW72300009
View details for PubMedID 8939709
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Evolutionally guided enzyme design
Biochemical Engineering IX - Interdisciplinary Foundations for Creating New Biotechnology
JOHN WILEY & SONS INC. 1996: 122–28
Abstract
A combination of "rational" and "irrational" strategies for the creation of enzymes with novel properties is proving to be a powerful concept in the field of enzyme engineering. Guided by principles of physical organic chemistry, rational design strategies are used to identify suitable target enzymes and to choose appropriate molecular biological methods for engineering purposes. In contrast, irrational (or random) strategies are centered around the biological paradigm of stochastic molecular evolution. As illustrated in this review, such a hybrid approach is particularly useful for the design of new modular enzymes. (c) 1996 John Wiley & Sons, Inc.
View details for Web of Science ID A1996VH41300013
View details for PubMedID 18629858
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A functional chimeric modular polyketide synthase generated via domain replacement
CHEMISTRY & BIOLOGY
1996; 3 (10): 827-831
Abstract
Modular polyketide synthases (PKSs), such as 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzymes that catalyze the biosynthesis of structurally complex and medically important natural products. Active sites within these assemblies are organized into 'modules', such that each module catalyzes the stereospecific addition of a new monomer onto a growing polyketide chain and also sets the reduction level of the beta-carbon atom of the resulting intermediate. The core of each module is made up of a 'reductive segment', which includes all, some, or none of a set of ketoreductase (KR), dehydratase, and enoylreductase domains, in addition to a large interdomain region which lacks overt function but may contribute to structural stability and inter-domain dynamics within modules. The highly conserved organization of reductive segments within modules suggests that they might be able to function in unnatural contexts to generate novel organic molecules.To investigate domain substitution as a method for altering PKS function, a chimeric enzyme was engineered. Using a bimodular derivative of DEBS (DEBS1+TE), the reductive segment of module 2, which includes a functional KR, was replaced with its homolog from module 3 of DEBS, which contains a (naturally occurring) nonfunctional KR. A recombinant strain expressing the chimeric gene produced the predicted ketolactone with a yield (35 %) comparable to that of a control strain in which the KR2 domain was retained but mutationally inactivated.These results demonstrate considerable structural tolerance within an important segment found in virtually every PKS module. The domain boundaries defined here could be exploited for the construction of numerous loss-of-function and possibly even gain-of-function mutants within this remarkable family of multifunctional enzymes.
View details for Web of Science ID A1996VR14500006
View details for PubMedID 8939701
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Specificity and versatility in erythromycin biosynthesis
CHEMICAL SOCIETY REVIEWS
1996; 25 (5): 297-?
View details for Web of Science ID A1996VZ78000001
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Engineered biosynthesis of structurally diverse tetraketides by a trimodular polyketide synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1996; 118 (38): 9184-9185
View details for Web of Science ID A1996VJ48100029
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Evidence for two catalytically independent clusters of active sites in a functional modular polyketide synthase
BIOCHEMISTRY
1996; 35 (38): 12363-12368
Abstract
Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multifunctional enzymes are organized into "modules", where each module contains active sites homologous to those of higher eucaryotic fatty acid synthases (FASs). Like FASs, modular PKSs are known to be dimers. Here we provide functional evidence for the existence of two catalytically independent clusters of active sites within a modular PKS. In three bimodular derivatives of DEBS, the ketosynthase domain of module 1 (KS-1) or module 2 (KS-2) or the acyl carrier protein domain of module 2 (ACP-2) was inactivated via site-directed mutagenesis. As expected, the purified proteins were unable to catalyze polyketide synthesis (although the KS-1 mutant could convert a diketide thioester into the predicted triketide lactone). Remarkably however, the KS-1/KS-2 and the KS-2/ACP-2 mutant pairs could efficiently complement each other and catalyze polyketide formation. In contrast, the KS-1 and ACP-2 mutants did not complement each other. On the basis of these and other results, a model is proposed in which the individual modules of a PKS dimer form head-to-tail homodimers, thereby generating two equivalent and independent clusters of active sites for polyketide biosynthesis. Specifically, each subunit contributes half of the KS and ACP domains in each cluster. A similar complementation approach should also be useful in dissecting the organization of the remaining types of active sites within this family of multienzyme assemblies. Finally, blocked systems, such as the KS-1 mutant described here, present a new strategy for the noncompetitive conversion of unnatural substrates into polyketides by modular PKSs.
View details for Web of Science ID A1996VJ27000018
View details for PubMedID 8823171
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Generation of polyketide libraries via combinatorial biosynthesis
TRENDS IN BIOTECHNOLOGY
1996; 14 (9): 335-341
Abstract
Polyketides are a family of structurally complex natural products that include a number of important pharmaceuticals. Motivated by the value of these natural products, there has been much research focused on developing guidelines for engineering polyketide synthases (PKSs) to generate novel polyketides. Recent studies have provided interesting insights into the enzymatic specificity of the polyketide synthesis pathway, and have demonstrated that various PKSs can be genetically manipulated to synthesize 'unnatural' polyketide natural products. In this article, we discuss the synthesis of polyketides and polyketide libraries by combinatorial biosynthesis.
View details for Web of Science ID A1996VF73800007
View details for PubMedID 8818287
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Erythromycin biosynthesis: Exploiting the catalytic versatility of the modular polyketide synthase
BIOORGANIC & MEDICINAL CHEMISTRY
1996; 4 (7): 995-999
Abstract
DEBS 1 + TE is a recombinant modular polyketide synthase (PKS) in which the first two biosynthetic modules of the 6-deoxyerythronolide B synthase are linked to the thioesterase domain normally found at the C-terminus of DEBS 3. Incubation of DEBS 1 + TE with propionyl-CoA, methylamalonyl-CoA, and NADPH gives the triketide lactone (2R,3S,4S,5R)-2,4-dimethyl-3, 5-dihydroxy-n-heptanoic acid delta-lactone (2), the cyclized form of the normal triketide chain elongation product of DEBS 1. In order to probe the molecular recognition features of the PKS and to explore its synthetic versatility, [2,3-13C2]-(2S,3R)-2-methyl-3-hydroxypentanoyl-NAC thioester (3), an analogue of the normal diketide chain elongation intermediate, and (2RS)-methyl-malonyl-CoA were incubated with DEBS 1 + TE, leading to the formation of the predicted labeled triketide ketolactone [4,5-13C2]-8, as established by 13C NMR analysis and comparison with spectra of synthetic 8. This stereoselective conversion illustrates the potential of using modular PKSs as multifunctional catalysts for the enzymatic synthesis of novel polyketides.
View details for Web of Science ID A1996UX08200005
View details for PubMedID 8831969
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Efficient synthesis of aromatic polyketides in vitro by the actinorhodin polyketide synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1996; 118 (21): 5158-5159
View details for Web of Science ID A1996UN39600038
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Engineered biosynthesis of novel polyketides: Regiospecific methylation of an unnatural substrate by the tcmO O-methyltransferase
BIOCHEMISTRY
1996; 35 (21): 6527-6532
Abstract
TcmO is an O-methyltransferase that methylates the C-8 hydroxyl to Tcm B3, a four-ring aromatic intermediate in the tetracenomycin biosynthetic pathway of Streptomyces glaucescens. The gene encoding this enzyme was expressed in Streptomyces coelicolor CH999 together with the actinorhodin polyketide synthase (PKS) gene cluster, which is responsible for the biosynthesis of 3,8-dihydroxy-methylanthraquinone carboxylic acid (DMAC) and its decarboxylated analog, aloesaponarin. The resulting recombinant strain produced approximately equal quantities of aloesaponarin and a new product but no DMAC. Spectroscopic analysis revealed that the novel polyketide was the 3-O-methylated analog of DMAC. An in vitro radioisotopic assay was developed for tcmO. The enzyme requires S-adenosylmethionine as a co-substrate. It has a Km of 3 microM and a kcat of 2.7 min-1 for DMAC. A series of monocyclic, bicyclic, and tricyclic aromatic compounds were also tested as candidate substrates in vitro. Remarkably, none was modified by tcmO within detectable limits of the assay. Together, these results highlight the interesting molecular recognition features of this enzyme. On one hand, there appears to be some flexibility in the number and structures of unreactive rings, since both Tcm and B3 and DMAC are good substrates. However, 6-methylsalicylic acid, a monocyclic analog of the reactive ring, is not recognized by the enzyme. Likewise, neither aloesaponarin (which only differs from DMAC in the reactive ring) nor carminic acid (which only differs in the distal nonreactive ring) is modified. Thus, the binding energy for the tcmO-catalyzed methyl transfer appears to involve significant contributions from both the aromaticity and the functionality of polycyclic substrates.
View details for Web of Science ID A1996UN28800002
View details for PubMedID 8639600
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Combinatorial chemistry and biology: An opportunity for engineers
CURRENT OPINION IN BIOTECHNOLOGY
1996; 7 (2): 219-222
Abstract
Within the past two years, the burgeoning field of combinatorial chemistry and biology has witnessed major advances in both technologies and applications. New ideas have emerged, and continue to be sought, with regard to library design, construction, and analysis. The highly multi-disciplinary nature of the field, together with its need for a systems-based view of pertinent challenges and problems, makes it an ideal area for biochemical engineers.
View details for Web of Science ID A1996UE90500016
View details for PubMedID 8791337
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Engineered biosynthesis of novel polyketides: Properties of the whiE aromatase/cyclase
NATURE BIOTECHNOLOGY
1996; 14 (3): 335-338
Abstract
The ORFVI from the cluster of genes, which is responsible for the biosynthesis of the Streptomyces coelicolor spore pigment, the whiE cluster, has been described as a bifunctional aromatase/cyclase. In order to evaluate its potential use for generating novel polyketides, combinations of this gene with those encoding minimal polyketide synthase enzymes with or without a ketoreductase from S. coelicolor A3(2) were constructed and analyzed in vivo. Analysis of the polyketide products generated from these constructs indicates that the whiE-ORFVI enzyme has properties similar to those of TcmN, although the whiE aromatase/cyclase normally acts on a polyketide intermediate that is four carbons longer than the TcmN substrate. The whiE aromatase/cyclase can influence the regiospecificity of the first cyclization of unreduced, but not reduced, backbones and is also responsible for the second ring aromatization. An unusual new polyketide, EM18, was identified which is not seen in equivalent strains expressing the tcmN aromatase/cyclase or the act aromatase genes. The structure of EM18 suggests that the WhiE-ORFVI product might have some unique properties within this family of polyketide synthase subunits, and may therefore be useful in the design of combinatorial biosynthetic strategies.
View details for Web of Science ID A1996UL38000028
View details for PubMedID 9630896
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Deciphering the biosynthetic origin of the aglycone of the aureolic acid group of anti-tumor agents
CHEMISTRY & BIOLOGY
1996; 3 (3): 193-196
Abstract
Mithramycin, chromomycin, and olivomycin belong to the aureolic acid family of clinically important anti-tumor agents. These natural products share a common aromatic aglycone. Although isotope labeling studies have firmly established the polyketide origin of this aglycone, they do not distinguish between alternative biosynthetic models in which the aglycone is derived from one, two or three distinct polyketide moieties. We set out to determine the biosynthetic origin of this moiety using a recombinant approach in which the ketosynthase and chain-length factor proteins from the antibiotic-producer strain, which determine the chain length of a polyketide, are produced in a heterologous bacterial host.The ketosynthase and chain-length factor genes from the polyketide synthase gene cluster from the mithramycin producer, Streptomyces argillaceus ATCC 12956, and the acyl carrier protein and ketoreductase genes from the actinorhodin polyketide synthase were expressed in Streptomyces coelicolor CH999. The recombinant strain produced a 20-carbon polyketide, comprising the complete backbone of the aglycone of mithramycin.The aglycone moieties of mithramycin, chromomycin, and olivomycin are derived from a single polyketide backbone. The nascent polyketide backbone must undergo a series of regiospecific cyclizations to form a tetracenomycin-like tetracyclic intermediate. The final steps in the aglycone biosynthetic pathway presumably involve decarboxylation and oxidative cleavage between C-18 and C-19, followed by additional oxidation, reduction, and methylation reactions.
View details for Web of Science ID A1996UH69100007
View details for PubMedID 8807845
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Erythromycin biosynthesis: Kinetic studies on a fully active modular polyketide synthase using natural and unnatural substrates
BIOCHEMISTRY
1996; 35 (7): 2054-2060
Abstract
6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) that catalyzes the biosynthesis of the parent macrolide of erythromycin. On the basis of a recently developed cell-free assay (Pieper et al., 1995a) we report the results of steady-state kinetic studies on a modular PKS. A truncated form of DEBS (DEBS 1+TE), in which DEBS 1 is fused to the thioesterase domain from the C-terminal end of DEBS 3, was used for most of these studies. The overall k(cat) for (2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid delta-lactone (C9-lactone) synthesis is 3.4 min(-1), indicating that the enzyme is at least as active in vitro as in vivo. The apparent K(m) for (2S)-methylmalonyl-CoA consumption by DEBS 1+TE is 24 microM. The catalytic activity of DEBS 1+TE is strongly dependent on the phosphate concentration in the reaction buffer in the range 0-250 mM, suggesting that hydrophobic interactions may be crucial to the assembly of DEBS monomers into a functional complex. Although DEBS 1+TE can convert acetyl-, propionyl-, or butyryl-CoA into the corresponding C8-, C9-, and C10-lactones (Pieper et al., 1995b), it has a 32-fold preference for a propionate primer over an acetate primer and a 7.5-fold preference for a propionate primer over a butyrate primer. In the absence of any added primer unit, synthesis can be primed via decarboxylation of methylmalonyl-CoA; under these conditions the overall k(cat) for polyketide synthesis remains unchanged. Decarboxylation of methylmalonyl-CoA is negligible in the presence of saturating concentrations of propionyl-CoA but competes with the priming of the enzyme by acetyl-CoA or butyryl-CoA. The k(cat) for 6-deoxyerythronolide B synthesis by the complete DEBS is 0.5 min(-1). Under these assay conditions, the C9-lactone is also produced as an abortive chain elongation product with a k(cat) of 0.23 min(-1), presumably due to inefficient assembly of the multimeric protein complex involving DEBS 1, 2, and 3. Together, these results provide the first comprehensive kinetic insights into a fully active modular PKS.
View details for Web of Science ID A1996TW34500002
View details for PubMedID 8652546
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Antibiotic activity of polyketide products derived from combinatorial biosynthesis: Implications for directed evolution
MOLECULAR DIVERSITY
1996; 1 (2): 121-124
Abstract
A library of over 100 polyketides, generated via combinatorial cloning of genes encoding subunits of aromatic polyketide synthases, was screened for molecules capable of inhibiting the growth of gram-positive bacteria. A total of 26 polyketides, with varying levels of antibiotic activity in filter-disk assays, were purified. Most bioactive polyketides were produced as relatively minor compounds (< 1 mg/l), although two major anthraquinones, with yields in the range of 10-100 mg/l, were also identified and structurally characterized. When tested against Bacillus subtilis 168 beta, they were found to cause a 50% reduction in colony-forming units at concentrations of 20 and 300 micrograms/ml, respectively. We speculate that many of the minor (and possibly more potent) bioactive polyketides are synthesized via nonspecific enzymatic modifications of shunt products derived from engineered polyketide synthase pathways. If so, then these 'fortuitous' pathways should be amenable to further rationally guided manipulation. Our results support the notion that combinatorial biosynthesis can be used to generate novel, biologically active molecules. They also point to the feasibility of designing mutagenesis selection experiments aimed at the directed evolution of organic molecules with desirable pharmaceutical properties.
View details for Web of Science ID A1996VV71400006
View details for PubMedID 9237201
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Engineering of novel polyketides - Progress and prospects
13th International Enzyme Engineering Conference
NEW YORK ACAD SCIENCES. 1996: 32–45
View details for Web of Science ID 000070949000006
View details for PubMedID 8958070
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CELL-FREE SYNTHESIS OF POLYKETIDES BY RECOMBINANT ERYTHROMYCIN POLYKETIDE SYNTHASES
NATURE
1995; 378 (6554): 263-266
Abstract
Modular polyketide synthases (PKSs) are complex multi-enzyme proteins that catalyse the bacterial biosynthesis of many pharmaceutically useful polyketides. The PKSs are organized into a series of modules, each containing the active catalytic sites required for one step in the synthesis process. Here we report a method for cell-free enzymatic synthesis of 6-deoxyerythronolide B (6-dEB), the parent molecule of the antibiotic erythromycin A, using recombinant 6-deoxyerythronolide B synthase (DEBS), a modular PKS with at least 28 distinct active sites. We have also synthesized in vitro a triketide lactone by using a truncated mutant of DEBS. The availability of such cell-free synthetic routes will allow direct investigation of the structural and mechanistic basis for the unusual combination of high substrate specificity and tolerance to genetic reprogramming found in this enzyme family.
View details for Web of Science ID A1995TE85800043
View details for PubMedID 7477343
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MANIPULATION OF MACROLIDE RING SIZE BY DIRECTED MUTAGENESIS OF A MODULAR POLYKETIDE SYNTHASE
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1995; 117 (35): 9105-9106
View details for Web of Science ID A1995RT72400043
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EXPRESSION OF A FUNCTIONAL FUNGAL POLYKETIDE SYNTHASE IN THE BACTERIUM STREPTOMYCES-COELICOLOR A3(2)
JOURNAL OF BACTERIOLOGY
1995; 177 (15): 4544-4548
Abstract
The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor. Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy. These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined.
View details for Web of Science ID A1995RL82800050
View details for PubMedID 7635840
View details for PubMedCentralID PMC177212
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ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - ANALYSIS OF TCMN FUNCTION IN TETRACENOMYCIN BIOSYNTHESIS
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1995; 117 (26): 6805-6810
View details for Web of Science ID A1995RG97600001
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ERYTHROMYCIN BIOSYNTHESIS - HIGHLY EFFICIENT INCORPORATION OF POLYKETIDE CHAIN ELONGATION INTERMEDIATES INTO 6-DEOXYERYTHRONOLIDE-B IN AN ENGINEERED STREPTOMYCES HOST
JOURNAL OF ANTIBIOTICS
1995; 48 (7): 647-651
Abstract
Feeding of (2S,3R)-[2,3-13C2]-2-methyl-3-hydroxypentanoyl NAC thioester (1a) to the recombinant organism Streptomyces coelicolor CH999/pCK7 harboring the complete set of eryA genes from Saccharopolyspora erythraea encoding the 6-deoxyerythronolide B synthase (DEBS) resulted in the formation of 6-deoxyerythronolide B (2a) labeled with 13C at C-12 and C-13, as evidenced by the appearance of a pair of enhanced and coupled doublets in the 13C NMR spectrum. The level of 13C enrichment was 15-20 atom% 13C, as much as 100 times higher than the usually observed efficiency of incorporation of NAC thioesters into polyketide metabolites. Similar incorporation of (2S,3R)-[3-2H,3-13C]-2-methyl-3-hydroxypentanoyl NAC thioester (1b) gave 6-deoxyerythronolide B (2b) labeled with both 13C and deuterium at C-13. The intact incorporation of both precursors confirms the normal functioning of the recombinant DEBS proteins in the heterologous host.
View details for Web of Science ID A1995RL59200016
View details for PubMedID 7649863
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RATIONAL DESIGN OF AROMATIC POLYKETIDE NATURAL-PRODUCTS BY RECOMBINANT ASSEMBLY OF ENZYMATIC SUBUNITS
NATURE
1995; 375 (6532): 549-554
Abstract
Recent advances in understanding of bacterial aromatic polyketide biosynthesis allow the development of a set of design rules for the rational manipulation of chain synthesis, reduction of keto groups and early cyclization steps by genetic engineering. The concept of rational design is illustrated by the preparation of Streptomyces strains that produce two new polyketides by expression of combinations of appropriate enzymatic subunits from naturally occurring polyketide synthases. The potential for generating molecular diversity within this class of molecules by genetic engineering is enormous.
View details for Web of Science ID A1995RD28700041
View details for PubMedID 7791871
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COMBINATORIAL BIOSYNTHESIS OF UNNATURAL NATURAL-PRODUCTS - THE POLYKETIDE EXAMPLE
CHEMISTRY & BIOLOGY
1995; 2 (6): 355-362
Abstract
Multi-enzyme systems, such as those involved in the biosynthesis of polyketides, typically catalyze several distinct reactions that are combined in different ways to generate diverse natural products. The variability available in such systems has not been fully harnessed from nature. It may therefore be possible to create 'unnatural' natural products, which may be as structurally diverse and medicinally valuable as existing natural products, using combinatorial biosynthesis.
View details for Web of Science ID A1995RH72200003
View details for PubMedID 9383437
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ENGINEERED BIOSYNTHESIS OF A TRIKETIDE LACTONE FROM AN INCOMPLETE MODULAR POLYKETIDE SYNTHASE
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1994; 116 (25): 11612-11613
View details for Web of Science ID A1994PX38600069
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Engineered biosynthesis of novel polyketides: evidence for temporal, but not regiospecific, control of cyclization of an aromatic polyketide precursor.
Chemistry & biology
1994; 1 (4): 205-210
Abstract
Aromatic polyketide synthases (PKSs) catalyze the formation and cyclization of polyketide chains of variable lengths, generating a family of compounds of proven medical significance. Initial control over the regiospecificity of cyclization is believed to be exercised by the minimal PKS, composed of the three essential components for polyketide biosynthesis, which catalyzes an intramolecular aldol condensation towards the middle of the chain. Subsequent cyclization reactions are either catalyzed by additional components of the PKS, or occur in the absence of specific catalysts.Structural and biosynthetic studies on SEK4b, a novel octaketide product of a minimal PKS, revealed an unusual cyclization pattern. The first cyclization (an aldol condensation) occurs at the methyl end of the unreduced polyketide backbone precursor. This is followed by hemiketal formation and lactonization. The overall structure of SEK4b is similar to that of SEK4, a previously-identified product of the same genetically-engineered strain, differing only in the positions of a methyl and a pyrone group around a common fused-ring system. The biosynthetic pathways of the two molecules are quite different, however. The yield of SEK4b relative to SEK4 is much higher in the absence of PKS components (aromatases and cyclases) acting later in the pathway.In this cyclization pathway, the regiospecificity of cyclization is not directly controlled by the minimal PKS. Instead, we propose that the enzyme influences cyclization by controlling the timing of chain release. Chain release and cyclization may be concurrent with synthesis. Other PKS subunits appear to stabilize the complex of the PKS with the nascent chain, preventing premature release.
View details for PubMedID 9383392
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ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - ACT(VII) AND ACT(IV) GENES ENCODE AROMATASE AND CYCLASE ENZYMES, RESPECTIVELY
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1994; 116 (24): 10855-10859
View details for Web of Science ID A1994PX30900001
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ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - INFLUENCE OF A DOWNSTREAM ENZYME ON THE CATALYTIC SPECIFICITY OF A MINIMAL AROMATIC POLYKETIDE SYNTHASE
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1994; 91 (24): 11542-11546
Abstract
To identify the minimum set of polyketide synthase (PKS) components required for in vivo biosynthesis of aromatic polyketides, combinations of genes encoding subunits of three different aromatic PKSs--act from Streptomyces coelicolor A3(2) (an actinorhodin producer), fren from Streptomyces roseofulvus (a frenolicin and nanaomycin producer), and tcm from Streptomyces glaucescens (a tetracenomycin producer)--were expressed in a recently developed Streptomyces host-vector system. The "minimal" components (ketosynthase/putative acyltransferase, chain length-determining factor, and acyl carrier protein) were produced with and without a functional polyketide ketoreductase and/or cyclase, and the polyketide products of these recombinant strains were structurally characterized. Several previously identified polyketides were isolated in addition to two previously unidentified polyketides, dehydromutactin and SEK 15b, described here. The results proved that the act cyclase is not required for the biosynthesis of several aberrantly cyclized products that have been previously reported. They are also consistent with earlier conclusions that the minimal PKS controls chain length as well as the regiospecificity of the first cyclization and that it can do so in the absence of both a ketoreductase and a cyclase. However, the ability of the minimal tcm PKS to synthesize two different singly cyclized intermediates suggests that it is unable to accurately control the course of this reaction by itself. In the presence of a downstream enzyme, the flux through one branch of the cyclization pathway increases relative to the other. We propose that these alternative specificities may be due to the ability of downstream enzymes to associate with the minimal PKS and to selectively inhibit a particular branch of the cyclization pathway.
View details for Web of Science ID A1994PU28500053
View details for PubMedID 7972098
View details for PubMedCentralID PMC45267
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ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - STEREOCHEMICAL COURSE OF 2 REACTIONS CATALYZED BY A POLYKETIDE SYNTHASE
BIOCHEMISTRY
1994; 33 (31): 9321-9326
Abstract
A genetically engineered strain expressing the essential components of the tetracenomycin polyketide synthase (tcm PKS) along with the actinorhodin ketoreductase (act KR) was found to produce two new (diastereomeric) aromatic polyketides, designated RM20b and RM20c, in addition to RM20, whose structure was reported earlier [McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) Science 262, 1546-1550]. Spectroscopic and in vivo isotopic labeling analysis of RM20b and RM20c revealed that their polyketide backbones were identical to that of RM20 with respect to chain length, regiospecificity of ketoreduction, and regiospecificity of the first intramolecular aldol condensation. This is consistent with earlier predictions that the essential components of the PKS--a bifunctional ketosynthase/acyltransferase, a chain length determining factor, and an acyl carrier protein--are responsible for controlling each of these features of the polyketide backbone [McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) Science 262, 1546-1550; McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) J. Am. Chem. Soc. 115, 11671-11675; Fu, H., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1994) J. Am. Chem. Soc. 116, 4166-4170]. In addition, however, RM20b and RM20c possess two unusual features. In both molecules the hydroxyls on sp3 C-9 and C-7 of the first six-membered ring, which arise as a result of ketoreduction and aldol condensation, respectively, are intact, rather than being lost via dehydration. Furthermore, the relative yield of RM20b (in which these hydroxyls are syn) is 7-fold greater than that of RM20c (in which they are anti).(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1994PB76300036
View details for PubMedID 8049233
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ENGINEERED BIOSYNTHESIS OF A COMPLETE MACROLACTONE IN A HETEROLOGOUS HOST
SCIENCE
1994; 265 (5171): 509-512
Abstract
Macrocyclic polyketides have been subjects of great interest in synthetic and biosynthetic chemistry because of their structural complexity and medicinal activities. With expression of the entire 6-deoxyerythronolide B synthase (DEBS) (10,283 amino acids) in a heterologous host, substantial quantities of 6-deoxyerythronolide B (6dEB), the aglycone of the macrolide antibiotic erythromycin, and 8,8a-deoxyoleandolide, a 14-membered lactone ring identical to 6dEB except for a methyl group side chain in place of an ethyl unit, were synthesized in Streptomyces coelicolor. The biosynthetic strategy utilizes a genetic approach that facilitates rapid structural manipulation of DEBS or other modular polyketide synthases (PKSs), including those found in actinomycetes with poorly developed genetic methods. From a technological viewpoint, this approach should allow the rational design of biosynthetic products and may eventually lead to the generation of diverse polyketide libraries by means of combinatorial cloning of naturally occurring and mutant PKS modules.
View details for Web of Science ID A1994NY21600025
View details for PubMedID 8036492
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RELAXED SPECIFICITY OF THE OXYTETRACYCLINE POLYKETIDE SYNTHASE FOR AN ACETATE PRIMER IN THE ABSENCE OF A MALONAMYL PRIMER
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1994; 116 (14): 6443-6444
View details for DOI 10.1021/ja00093a058
View details for Web of Science ID A1994NX54800058
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ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - DISSECTION OF THE CATALYTIC SPECIFICITY OF THE ACT KETOREDUCTASE
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1994; 116 (10): 4166-4170
View details for Web of Science ID A1994NM98800003
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EFFICIENT SAMPLING OF PROTEIN-SEQUENCE SPACE FOR MULTIPLE MUTANTS
BIO-TECHNOLOGY
1994; 12 (5): 517-520
Abstract
We describe here a method capable of generating a very large population of multiple mutants, the size of which is primarily limited by volume constraints. This method, referred to as recombination-enhanced mutagenesis, combines the power of in vitro mutagenesis with the high frequencies of in vivo recombination that can be achieved using single-stranded transduction systems. The recombination frequency between two mutations separated by as little as 19 amino acids is 0.02; this frequency approaches a value of 0.1 for mutations separated by more than 38 amino acids. Up to 10(8) independent recombinants were generated in 1 ml of an E. coli culture, and this number scales linearly (or better) with increasing volume. To prove the method's effectiveness, we applied it to the problem of reverting multiple mutants of mouse dihydrofolate reductase, which could not be reverted using mutagenesis alone. Thus, given an appropriate screen or selection scheme, recombination-enhanced mutagenesis is well-suited for addressing a range of combinatorially complex problems, such as antigen recognition, enzyme catalysis, protein folding, and transport/transduction across biomembranes.
View details for Web of Science ID A1994NJ86300029
View details for PubMedID 7764711
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ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES - MANIPULATION AND ANALYSIS OF AN AROMATIC POLYKETIDE SYNTHASE WITH UNPROVED CATALYTIC SPECIFICITIES
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
1993; 115 (25): 11671-11675
View details for Web of Science ID A1993MM51400002
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ENGINEERED BIOSYNTHESIS OF NOVEL POLYKETIDES
SCIENCE
1993; 262 (5139): 1546-1550
Abstract
Polyketide synthases (PKSs) are multifunctional enzymes that catalyze the biosynthesis of a huge variety of carbon chains differing in their length and patterns of functionality and cyclization. Many polyketides are valuable therapeutic agents. A Streptomyces host-vector system has been developed for efficient construction and expression of recombinant PKSs. Using this expression system, several novel compounds have been synthesized in vivo in significant quantities. Characterization of these metabolites has provided new insights into key features of actinomycete aromatic PKS specificity. Thus, carbon chain length is dictated, at least in part, by a protein that appears to be distinctive to this family of PKSs, whereas the acyl carrier proteins of different PKSs can be interchanged without affecting product structure. A given ketoreductase can recognize and reduce polyketide chains of different length; this ketoreduction always occurs at the C-9 position. The regiospecificity of the first cyclization of the nascent polyketide chain is either determined by the ketoreductase, or the chain-extending enzymes themselves. However, the regiospecificity of the second cyclization is determined by a distinct cyclase, which can discriminate between substrates of different chain lengths.
View details for Web of Science ID A1993MK32900033
View details for PubMedID 8248802
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GENETIC CONSTRUCTION AND FUNCTIONAL-ANALYSIS OF HYBRID POLYKETIDE SYNTHASES CONTAINING HETEROLOGOUS ACYL CARRIER PROTEINS
JOURNAL OF BACTERIOLOGY
1993; 175 (8): 2197-2204
Abstract
The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases.
View details for Web of Science ID A1993KX33600005
View details for PubMedID 8468280
View details for PubMedCentralID PMC204504
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TARGETED GENE REPLACEMENTS IN A STREPTOMYCES POLYKETIDE SYNTHASE GENE-CLUSTER - ROLE FOR THE ACYL CARRIER PROTEIN
MOLECULAR MICROBIOLOGY
1992; 6 (21): 3237-3249
Abstract
A methodology was developed to construct any desired chromosomal mutation in the gene cluster that encodes the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2). A positive selection marker (resistance gene) is first introduced by double crossing-over into the chromosomal site of interest by use of an unstable delivery plasmid. This marker is subsequently replaced by the desired mutant allele via a second high-frequency double recombination event. The technology has been used to: (i) explore the significance of translational coupling between two adjacent PKS genes; (ii) prove that the acyl carrier protein (ACP) encoded by a gene in the cluster is necessary for the function of the actinorhodin PKS; (iii) provide genetic evidence supporting the hypothesis that serine 42 is the site of phosphopantetheinylation in the ACP of the actinorhodin PKS; and (iv) demonstrate that this ACP can be replaced by a Saccharopolyspora fatty acid synthase ACP to generate an active hybrid PKS.
View details for Web of Science ID A1992JW88800014
View details for PubMedID 1453961
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EXPRESSION OF INTRACELLULAR HEMOGLOBIN IMPROVES PROTEIN-SYNTHESIS IN OXYGEN-LIMITED ESCHERICHIA-COLI
BIO-TECHNOLOGY
1990; 8 (9): 849-853
Abstract
We have previously cloned the Vitreoscilla hemoglobin gene (VHb) and expressed the protein in Escherichia coli in its active form. Under oxygen-limited conditions the presence of VHb improves protein synthesis as indicated by both total protein content and the activity of an enzyme expressed from a cloned gene present on a multicopy plasmid. Measurements of nitrogen utilization rates corroborate the observation of enhanced protein synthesis; however, the rates of carbon consumption and acid synthesis remain unchanged. This suggests that the net effect of VHb in E. coli is to improve the efficiency, rather than the kinetics, of oxygen-limited aerobic metabolism. We propose two possible models for the mechanism of action of VHb: the facilitated diffusion hypothesis and the intracellular redox effector hypothesis. These suggest other systems in which cloned VHb may enhance bioprocess productivity.
View details for Web of Science ID A1990DW07500018
View details for PubMedID 1366796
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EXPRESSION OF RECOMBINANT PROTEINS IN ESCHERICHIA-COLI USING AN OXYGEN-RESPONSIVE PROMOTER
BIO-TECHNOLOGY
1990; 8 (6): 554-558
Abstract
The oxygen-dependent promoter of the Vitreoscilla hemoglobin (VHb) gene has been shown to be functional in E. coli. Earlier studies established that the promoter is maximally induced under microaerobic conditions and that its activity is also influenced by the cAMP-CAP complex. We demonstrate here that the promoter can be used for regulated, high-level expression of recombinant proteins in two-stage fed-batch fermentations. The promoter is maximally induced at dissolved oxygen levels lower than 5% air saturation. Despite the influence of catabolite repression, glucose and glycerol-containing media give comparable product levels under carbon-limited conditions such as those encountered in typical fed-batch fermentations. The possibility of a third level of control of promoter activity is also indicated. This mode of induction can be repressed by addition of a complex nitrogen source such as yeast extract to the medium. The observed promoter activity can be modulated at least 30-fold over the course of high-cell density fermentations producing either cloned beta-galactosidase or cloned chloramphenicol acetyltransferase (CAT). Densitometer scanning of SDS-polyacrylamide gels revealed that beta-galactosidase was expressed to a level of approximately 10% of total cellular protein.
View details for Web of Science ID A1990DF72500018
View details for PubMedID 1367436
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STRATEGIES AND CHALLENGES IN METABOLIC ENGINEERING
6TH CONF ON BIOCHEMICAL ENGINEERING
NEW YORK ACAD SCIENCES. 1990: 1–15
View details for Web of Science ID A1990EX28200002
View details for PubMedID 2192652
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EVIDENCE FOR PARTIAL EXPORT OF VITREOSCILLA HEMOGLOBIN INTO THE PERIPLASMIC SPACE IN ESCHERICHIA-COLI - IMPLICATIONS FOR PROTEIN FUNCTION
JOURNAL OF MOLECULAR BIOLOGY
1989; 210 (1): 79-89
Abstract
The Vitreoscilla hemoglobin protein has been implicated in earlier studies to serve a globin-like function under oxygen-limited growth conditions. Evidence is presented using fractionation as well as proteinase K accessibility techniques to prove that a considerable amount of this protein is localized in the periplasmic space of the cell. Genetic evidence points towards the existence of information within the N-terminal domain of the protein that plays a role in the process of protein export. However, this sequence is not cleaved in the process of translocation. Analysis of the primary structure of this region reveals several unusual features. Instead of positively charged residues at its amino terminus, it has a negative charge. The overall hydrophobicity of the central region of this sequence is significantly lower than in typical leader peptides due to the presence of a charged residue. In keeping with the likelihood that such an export signal may not be very efficient, a substantial fraction of the total cellular hemoglobin can also be detected in the cytoplasm. Heme is incorporated in both cytoplasmic and periplasmic globin as indicated by the ability of protein from both fractions to bind carbon monoxide. The secretion of this protein into the periplasm raises questions concerning the physiological significance of its localization. Dimensional analysis of a model based on the facilitated diffusion hypothesis, which was initially proposed to account for the effects of eukaryotic globins on oxygen transport, suggests that periplasmic globin can support an additional oxygen flux to the respiratory apparatus that may be physiologically significant.
View details for Web of Science ID A1989AY73300007
View details for PubMedID 2685332
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CHARACTERIZATION OF THE OXYGEN-DEPENDENT PROMOTER OF THE VITREOSCILLA HEMOGLOBIN GENE IN ESCHERICHIA-COLI
JOURNAL OF BACTERIOLOGY
1989; 171 (11): 5995-6004
Abstract
The gene coding for the Vitreoscilla hemoglobin (VHb) molecule has been cloned and functionally expressed in Escherichia coli. By using a plasmid-encoded gene as well as single-copy integrants, the oxygen-dependent VHb gene (VHb) promoter was shown to be functional in E. coli. The promoter was maximally induced under microaerobic conditions (dissolved oxygen levels of less than 2% air saturation). Direct analysis of mRNA levels as well as the use of gene fusions with lacZ showed that oxygen-dependent regulation occurred at the level of transcription. Transcriptional activity decreased substantially under anaerobic conditions, suggesting the presence of a regulatory mechanism that is maximally induced under hypoxic but not completely anaerobic conditions in E. coli. Primer extension analysis was used to identify the existence of two overlapping promoters within a 150-base-pair region upstream of the structural VHb gene. The oxygen-dependent activity of both promoters was qualitatively similar, suggesting the existence of a common mechanism by which available oxygen concentrations influence expression from the two promoters. Analysis of promoter activity in crp and cya mutants showed that both cyclic AMP and catabolite activator protein were required for full activity of the promoter. The VHb promoter contained a region of significant homology to the catabolite activator protein-binding site near the E. coli lac promoter.
View details for Web of Science ID A1989AX60400031
View details for PubMedID 2681149
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A NEW OXYGEN-REGULATED PROMOTER FOR THE EXPRESSION OF PROTEINS IN ESCHERICHIA-COLI
BIOTECHNIQUES
1989; 7 (9): 1026-1028
Abstract
Several properties of a new oxygen-regulated promoter, OXYPRO, were tested in small-scale Escherichia coli cultures. Using OXYPRO, maximal activity of a reporter gene encoding chloramphenicol acetyltransferase (CAT) occurred in cultures that were tightly capped immediately after inoculation. This is probably a result of the reduced oxygen concentration attained in capped cultures, a condition known to be required for OXYPRO induction. CAT levels were significantly higher when the cells were grown in a glycerol-based medium. Similar levels of CAT expression were obtained when OXYPRO was compared to the trp-lac (tac) promoter. In addition, regulated expression of CAT occurred in a wild type strain of E. coli, suggesting that OXYPRO will be useful in most E. coli strains. Thus, OXYPRO provides a simple, inexpensive, and unobtrusive method to achieve high levels of cloned protein expression in most strains of E. coli. OXYPRO is available in a high copy plasmid with a convenient multiple cloning site for the insertion of genes for direct expression in E. coli.
View details for Web of Science ID A1989CK50700017
View details for PubMedID 2698665
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HETEROLOGOUS EXPRESSION OF A BACTERIAL HEMOGLOBIN IMPROVES THE GROWTH-PROPERTIES OF RECOMBINANT ESCHERICHIA-COLI
NATURE
1988; 331 (6157): 633-635
Abstract
Rational design of novel as well as improved cellular biocatalysts by genetic manipulation of cellular metabolism has recently attracted considerable interest. A wide range of bacteria have been genetically modified by integrating new enzymatic functions into their metabolic network. A central problem in the aerobic growth of any cell culture is the maintenance of dissolved oxygen (DO) concentrations above growth-limiting levels especially in high cell-density fermentations which are usually of a fed-batch type. The optimal rate of nutrient addition (and consequently the productivity) is ultimately limited by the rate at which cells can aerobically catabolize the carbon source without generating growth-inhibitory metabolites such as lactate and acetate. All approaches thus far have concentrated on improving the oxygen mass transfer rates by manipulating various environmental parameters. We have isolated the gene for a haemoglobin-like molecule, expressed by the aerobic bacterium Vitreoscilla in poorly-oxygenated environments, and expressed it in Escherichia coli. The recombinant cells contain enhanced haem as well as active haemoglobin, and they grow faster and to considerably greater cell densities than comparable plasmid-containing cells which do not express haemoglobin. This haemoglobin increases the rate of oxygen use, especially when dissolved oxygen is less than 5% of air saturation.
View details for Web of Science ID A1988M118000063
View details for PubMedID 3277067