Bio


Christopher Walsh is a consulting professor to the Stanford University Department of Chemistry and an advisor to the Stanford ChEM-H institute. He was the Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology at Harvard Medical School from 1987 to 2013, when he took emeritus status. He has had extensive academic leadership experience, including Chairmanship of the MIT Chemistry Department and of the HMS Biological Chemistry & Molecular Pharmacology Department, as well as serving as President and CEO of the Dana Farber Cancer Institute. At Stanford he has taught short courses including Posttranslational Modifications of Proteins: Expanding Nature’s Inventory (Chem 187/287) and also Antibiotics: Mechanisms and Resistance.

Dr. Walsh’s research has focused on enzymes and enzyme inhibitors, with specialization on antibiotics and biosynthesis of other biologically and medicinally active natural products. He and his group authored 810 research papers, and four books: Enzymatic Reaction Mechanisms (1979); Antibiotics: Origins, Actions, Resistance (2003); Posttranslational Modification of Proteins: Expanding Nature’s Inventory (2005); and Antibiotics: Challenges, Mechanisms, Opportunities (2016).

Dr. Walsh is a member of the U.S. National Academy of Sciences, the U.S. National Academy of Medicine, the American Academy of Arts and Sciences, the American Philosophical Society, and a co-recipient of the 2010 Welch Prize in Chemistry. At Harvard and MIT he taught biochemistry, chemical biology, and pharmacology to medical students and graduate students and organic chemistry to undergraduates.

He has been involved in a variety of venture-based biotechnology companies since 1981, including Genzyme, Immunogen, Leukosite, Millenium, Kosan, Vicuron, Epizyme. Currently he is on the board of directors of Ironwood, and Proteostasis, and the non profits: California Institute for Biomedical Research and Ludwig Institute for Cancer Research. He is a member of the scientific advisory groups at Hua, Abide, Cidara, and Flex Pharma, an advisor to Health Care Ventures and a limited investor in Health Care Ventures, MPM bioventures, Clarus, and the Longwood Venture Funds.

Dr. Walsh is married to Diana Chapman Walsh, who was president of Wellesley College from 1993-2007 and was the founding chair of the board of the Broad Institute of MIT and Harvard. Their daughter Allison Walsh Kurian is an Associate Professor of Medicine at Stanford and co-director of the High Risk Center for women with genetic predisposition to breast and ovarian cancer.

Academic Appointments


Administrative Appointments


  • Emeritus Faculty, Harvard Medical School (2014 - Present)
  • Member, Stanford Chem-H (2013 - Present)
  • Scientific Advisory Board Member, The Stanley Center, Broad Institute of Harvard and MIT (2010 - Present)
  • Senior Associate Member, The Broad Institute of Harvard and MIT (2010 - Present)
  • President, Dana-Farber Cancer Institute (1992 - 1995)
  • Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology, Harvard Medical School (1991 - 2014)
  • Chairman, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School (1987 - 1987)
  • Karl Taylor Compton Professor, Massachusetts Institute of Technology (1985 - 1987)
  • Chairman, Chemistry Department, Massachusetts Institute of Technology (1982 - 1987)
  • Uncas and Helen Whitaker Professor, Massachusetts Institute of Technology (1980 - 1985)
  • Assistant, Associate and Full Professor of Chemistry and Biology, Massachusetts Institute of Technology (1972 - 1987)

Honors & Awards


  • Honorary Doctoral Degree, The Scripps Research Institute (2017)
  • Benjamin Franklin Medal in Chemistry, The Franklin Institute (2014)
  • Gordon Hammes Lecture, American Chemical Society Division of Biological Chemistry (2013)
  • Inhoffen Medal for Natural Products Chemistry, Helmholtz Centre for Infection Research and the Technical University of Braunschweig, Germany (2013)
  • Keynote Lecture, Interscience Conference on Antimicrobial Agents and Chemotherapy (2012)
  • Max Tishler Prize Lecture, Harvard University Department of Chemistry and Chemical Biology (2012)
  • Co-recipient, Welch Award in Chemistry, The Welch Foundation (2010)
  • Pauling Medal and Lecture, Stanford University School of Medicine (2010)
  • Ian Scott Medal, Texas A&M University (2009)
  • Whelan Medal, University of Chicago (2008)
  • Murray Goodman Award, American Chemical Society (2007)
  • Fritz Lipmann Medal, American Society for Biochemistry and Molecular Biology (2005)
  • Promega Award, American Society of Microbiology (2004)
  • Bader Award, Bioorganic Chemistry, American Chemical Society (2003)
  • Member, American Philosophical Society (2003)
  • Repligen Award, Chemistry of Life Processes, American Chemical Society (1999)
  • Arthur C. Cope Scholar Award, American Chemical Society (1998)
  • Remsen Award, American Chemical Society, Maryland section (1993)
  • Member, National Academy of Sciences (1989)
  • Member, Institute of Medicine (1989)
  • Member, American Academy of Arts and Sciences (1988)
  • Centenary Medal & Lectureship, Royal Society of Chemistry (1987)
  • Eli Lilly Award in Biochemistry, American Chemical Society Division of Biological Chemistry (1979)
  • Dreyfus Teacher-Scholar Grant, Camille & Henry Dreyfus Foundation (1976)
  • Sloan Fellow, Alfred P. Sloan Foundation (1975)

Boards, Advisory Committees, Professional Organizations


  • Advisory Board Member, MPM Capital, Clarus Ventures, Health Care Ventures
  • Member, Board of Directors, Transform Pharmaceuticals, Kosan Biosciences, Magen Biosciences, Achaogen
  • Member, Board of Directors, Leukosite, Diacrin, Critical Theraputics, Vicuron
  • Scientific Advisory Board Member, Millennium Pharmaceuticals, Dyax, Caliper, LS9, Sirtris, Epizyme, Verastem
  • Scientific Advisory Board Member, Hua, Abide, Flex Pharma, Cidara
  • Scientific Advisory Board Member, Immunogen, Genzyme, Cambridge Neurosciences, Epix Medical
  • Chair and Member, Board of Directors, California Institute of Biomedical Research (Calibr) Scientific Advisory Board (2012 - Present)
  • Member, Board of Scientific Governors, The Scripps Research Institute (2012 - 2014)
  • Member, Scientific Advisory Board, The Ludwig Cancer Institute (2011 - Present)
  • Member, Board of Directors, Proteostasis (2009 - Present)
  • Consultant, Eisai (2008 - 2013)
  • Editorial Board Member, ACS Chemical Biology (2005 - 2015)
  • Member, Board of Directors, Ironwood Pharmaceuticals (2003 - Present)
  • Editorial Board Member, Editorial Board Member (2003 - 2007)
  • Member, Board of Directors, Helen Hay Whitney Foundation (2001 - 2011)
  • Member, Board of Reviewing Editors, Science (2001 - 2006)
  • Editorial Board Member, ChemBioChem (2000 - Present)
  • Scientific Review Group Member, Howard Hughes Medical Institute (2000 - 2013)
  • Member, Board of Directors, Whitehead Institute (1998 - 2004)
  • Member, NIH General Medical Sciences Advisory Council (1996 - 1999)
  • Consultant, Abbott (1996 - 1997)
  • Editorial Board Member, Chemistry and Biology (1993 - Present)
  • Member, Board of Directors, Association of American Cancer Institutes (1993 - 1996)
  • Advisory Committee Member, Cardiovascular Research Center, MGH (1991 - 1994)
  • Editorial Board Member, Journal of the American Chemical Society (1991 - 1994)
  • Associate Editor, Protein Science (1991 - 1992)
  • Scientific Advisory Board Member, Burroughs Welcome Fund in Experimental Therapeutics (1991 - 1992)
  • Associate Editor, Annual Review of Biochemistry (1990 - 1995)
  • Scientific Advisory Committee Member and Chairman, Children’s Hospital (1988 - 1992)
  • Member, Visiting Committee in Biological Sciences, Yale University (1985 - 1988)
  • Member, Visiting Committee in Chemistry, Princeton University (1984 - 1986)
  • Co-chairman, Conference on Methanogenesis (1984 - 1984)
  • Consultant, Roche (1982 - 1995)
  • Chair, NIH Study Section in Biochemistry (1982 - 1982)
  • Panel Member, NIH Study Section in Biochemistry (1978 - 1982)
  • Biology Section Editor, Annual Reports in Medicinal Chemistry (1978 - 1980)
  • Editorial Board Member, Journal of Biological Chemistry (1978 - 1980)
  • Co-chairman, Gordon Research Conference on Enzymes, Coenzymes, and Metabolic Pathways (1978 - 1978)
  • Panel Member, NSF Research Grants Study (1977 - 1979)
  • Consultant, Merck (1975 - 1982)

Professional Education


  • Postdoc, Brandeis University, Biochemistry (1972)
  • PhD, The Rockefeller University, Life Sciences (1970)
  • BA, Harvard College, Biology (1965)

2017-18 Courses


All Publications


  • Structure-Activity Relationship and Molecular Mechanics Reveal the Importance of Ring Entropy in the Biosynthesis and Activity of a Natural Product. Journal of the American Chemical Society Tran, H. L., Lexa, K. W., Julien, O., Young, T. S., Walsh, C. T., Jacobson, M. P., Wells, J. A. 2017; 139 (7): 2541-2544

    Abstract

    Macrocycles are appealing drug candidates due to their high affinity, specificity, and favorable pharmacological properties. In this study, we explored the effects of chemical modifications to a natural product macrocycle upon its activity, 3D geometry, and conformational entropy. We chose thiocillin as a model system, a thiopeptide in the ribosomally encoded family of natural products that exhibits potent antimicrobial effects against Gram-positive bacteria. Since thiocillin is derived from a genetically encoded peptide scaffold, site-directed mutagenesis allows for rapid generation of analogues. To understand thiocillin's structure-activity relationship, we generated a site-saturation mutagenesis library covering each position along thiocillin's macrocyclic ring. We report the identification of eight unique compounds more potent than wild-type thiocillin, the best having an 8-fold improvement in potency. Computational modeling of thiocillin's macrocyclic structure revealed a striking requirement for a low-entropy macrocycle for activity. The populated ensembles of the active mutants showed a rigid structure with few adoptable conformations while inactive mutants showed a more flexible macrocycle which is unfavorable for binding. This finding highlights the importance of macrocyclization in combination with rigidifying post-translational modifications to achieve high-potency binding.

    View details for DOI 10.1021/jacs.6b10792

    View details for PubMedID 28170244

  • At the Intersection of Chemistry, Biology, and Medicine. Annual review of biochemistry Walsh, C. T. 2017

    Abstract

    After an undergraduate degree in biology at Harvard, I started graduate school at The Rockefeller Institute for Medical Research in New York City in July 1965. I was attracted to the chemical side of biochemistry and joined Fritz Lipmann's large, hierarchical laboratory to study enzyme mechanisms. That work led to postdoctoral research with Robert Abeles at Brandeis, then a center of what, 30 years later, would be called chemical biology. I spent 15 years on the Massachusetts Institute of Technology faculty, in both the Chemistry and Biology Departments, and then 26 years on the Harvard Medical School Faculty. My research interests have been at the intersection of chemistry, biology, and medicine. One unanticipated major focus has been investigating the chemical logic and enzymatic machinery of natural product biosynthesis, including antibiotics and antitumor agents. In this postgenomic era it is now recognized that there may be from 10(5) to 10(6) biosynthetic gene clusters as yet uncharacterized for potential new therapeutic agents. Expected final online publication date for the Annual Review of Biochemistry Volume 86 is June 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

    View details for DOI 10.1146/annurev-biochem-110716-121241

    View details for PubMedID 28125288

  • Insights into the chemical logic and enzymatic machinery of NRPS assembly lines. Natural product reports Walsh, C. T. 2016; 33 (2): 127-135

    Abstract

    Appreciation that some cyclic peptide antibiotics such as gramicidin S and tyrocidine were nonribosomally synthesized has been known for 50 years. The past two decades of research including advances in bacterial genetics, genomics, protein biochemistry and mass spectrometry have codified the principles of assembly line enzymology for hundreds of nonribosomal peptides and in parallel for thousands of polyketides. The advances in understanding the strategies used for chain initiation, elongation and termination from these assembly lines have revitalized natural product biosynthetic communities.

    View details for DOI 10.1039/c5np00035a

    View details for PubMedID 26175103

  • A chemocentric view of the natural product inventory NATURE CHEMICAL BIOLOGY Walsh, C. T. 2015; 11 (9): 620-624

    View details for Web of Science ID 000359954700002

    View details for PubMedID 26284660

  • Nature loves nitrogen heterocycles TETRAHEDRON LETTERS Walsh, C. T. 2015; 56 (23): 3075-3081
  • In Vitro Reconstitution of Metabolic Pathways: Insights into Nature's Chemical Logic SYNLETT Lowry, B., Walsh, C. T., Khosla, C. 2015; 26 (8): 1008-1025
  • Biological matching of chemical reactivity: pairing indole nucleophilicity with electrophilic isoprenoids. ACS chemical biology Walsh, C. T. 2014; 9 (12): 2718-2728

    Abstract

    The indole side chain of tryptophan has latent nucleophilic reactivity at both N1 and all six (nonbridgehead) carbons, which is not generally manifested in post-translational reactions of proteins. On the other hand, all seven positions can be prenylated by the primary metabolite Δ(2)-isopentenyl diphosphate by dimethyallyl transferase (DMATs) family members as initial steps in biosynthetic pathways to bioactive fungal alkaloids including ergots and tremorgens. These are formulated as regioselective capture of isopentenyl allylic cationic transition states by the indole side chain as a nucleophile. The balance of regiospecificity and promiscuity among these indole prenyltransferases continues to raise questions about possible Cope and azaCope rearrangements of nascent products. In addition to these two electron reaction manifolds, there is evidence for one electron reaction manifolds in indole ring biosynthetic functionalization.

    View details for DOI 10.1021/cb500695k

    View details for PubMedID 25303280

  • Blurring the Lines between Ribosomal and Nonribosomal Peptide Scaffolds ACS CHEMICAL BIOLOGY Walsh, C. T. 2014; 9 (8): 1653-1661

    Abstract

    Two of the canons of protein science have been (1) that there are 20-22 amino acids that are proteinogenic and (2), with the exception of achiral glycine, that the other residues are L-amino acids. By contrast, the presence of nonproteinogenic amino acid residues and D-enantiomers has been regarded as hallmarks of nonribosomal peptides. The recent discoveries that bottromycins and polytheonamides, containing β-methyl and D-amino acid residues, are of ribosomal origin blur the distinctions between peptide structures derivable by ribosomal and nonribosomal assembly lines and reveal new chemistry for posttranslational maturation of proteins.

    View details for DOI 10.1021/cb5003587

    View details for Web of Science ID 000340517500004

    View details for PubMedID 24883916

  • Assembly line polyketide synthases: mechanistic insights and unsolved problems. Biochemistry Khosla, C., Herschlag, D., Cane, D. E., Walsh, C. T. 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

  • Prospects for new antibiotics: a molecule-centered perspective JOURNAL OF ANTIBIOTICS Walsh, C. T., Wencewicz, T. A. 2014; 67 (1): 7-22

    Abstract

    There is a continuous need for iterative cycles of antibiotic discovery and development to deal with the selection of resistant pathogens that emerge as therapeutic application of an antibiotic becomes widespread. A short golden age of antibiotic discovery from nature followed by a subsequent golden half century of medicinal chemistry optimization of existing molecular scaffolds emphasizes the need for new antibiotic molecular frameworks. We bring a molecule-centered perspective to the questions of where will new scaffolds come from, when will chemogenetic approaches yield useful new antibiotics and what existing bacterial targets merit contemporary re-examination.

    View details for DOI 10.1038/ja.2013.49

    View details for Web of Science ID 000330222300003

    View details for PubMedID 23756684

  • Nonproteinogenic Amino Acid Building Blocks for Nonribosomal Peptide and Hybrid Polyketide Scaffolds ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Walsh, C. T., Brien, R. V., Khosla, C. 2013; 52 (28): 7098-7124
  • Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds. Angewandte Chemie (International ed. in English) Walsh, C. T., O'Brien, R. V., Khosla, C. 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 PubMedID 23729217

  • Short Pathways to Complexity Generation: Fungal Peptidyl Alkaloid Multicyclic Scaffolds from Anthranilate Building Blocks ACS CHEMICAL BIOLOGY Walsh, C. T., Haynes, S. W., Ames, B. D., Gao, X., Tang, Y. 2013; 8 (7): 1366-1382

    Abstract

    Complexity generation in naturally occurring peptide scaffolds can occur either by posttranslational modifications of nascent ribosomal proteins or through post assembly line tailoring of nonribosomal peptides. Short enzymatic pathways utilizing bimodular and trimodular nonribosomal peptide synthetase (NRPS) assembly lines, followed by tailoring oxygenases and/or prenyltransferases, efficiently construct complex fungal peptidyl alkaloid scaffolds in Aspergilli, Neosartorya, and Penicillium species. Use of the nonproteinogenic amino acid anthranilate as chain-initiating building block and chain-terminating intramolecular nucleophile leads efficiently to peptidyl alkaloid scaffolds with two to seven fused rings.

    View details for DOI 10.1021/cb4001684

    View details for Web of Science ID 000322210100002

    View details for PubMedID 23659680

  • The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Malcolmson, S. J., Young, T. S., Ruby, J. G., Skewes-Cox, P., Walsh, C. T. 2013; 110 (21): 8483-8488

    Abstract

    Berninamycin is a member of the pyridine-containing thiopeptide class of antibiotics that undergoes massive posttranslational modifications from ribosomally generated preproteins. Berninamycin has a 2-oxazolyl-3-thiazolyl-pyridine core embedded in a 35-atom macrocycle rather than typical trithiazolylpyridine cores embedded in 26-atom and 29-atom peptide macrocycles. We describe the cloning of an 11-gene berninamycin cluster from Streptomyces bernensis UC 5144, its heterologous expression in Streptomyces lividans TK24 and Streptomyces venezuelae ATCC 10712, and detection of variant and incompletely processed scaffolds. Posttranslational maturation in S. lividans of both the wild-type berninamycin prepeptide (BerA) and also a T3A mutant generates macrocyclic compounds as well as linear variants, which have failed to form the pyridine and the macrocycle. Expression of the gene cluster in S. venezuelae generates a variant of the 35-atom skeleton of berninamycin, containing a methyloxazoline in the place of a methyloxazole within the macrocyclic framework.

    View details for DOI 10.1073/pnas.1307111110

    View details for Web of Science ID 000320328700046

    View details for PubMedID 23650400

  • Flavoenzymes: versatile catalysts in biosynthetic pathways. Natural product reports Walsh, C. T., Wencewicz, T. A. 2013; 30 (1): 175-200

    Abstract

    Riboflavin-based coenzymes, tightly bound to enzymes catalyzing substrate oxidations and reductions, enable an enormous range of chemical transformations in biosynthetic pathways. Flavoenzymes catalyze substrate oxidations involving amine and alcohol oxidations and desaturations to olefins, the latter setting up Diels-Alder cyclizations in lovastatin and solanapyrone biosyntheses. Both C(4a) and N(5) of the flavin coenzymes are sites for covalent adduct formation. For example, the reactivity of dihydroflavins with molecular oxygen leads to flavin-4a-OOH adducts which then carry out a diverse range of oxygen transfers, including Baeyer-Villiger type ring expansions, olefin epoxidations, halogenations via transient HOCl generation, and an oxidative Favorskii rerrangement during enterocin assembly.

    View details for DOI 10.1039/c2np20069d

    View details for PubMedID 23051833

  • Identification and Characterization of the Echinocandin B Biosynthetic Gene Cluster from Emericella rugulosa NRRL 11440 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Cacho, R. A., Jiang, W., Chooi, Y., Walsh, C. T., Tang, Y. 2012; 134 (40): 16781-16790

    Abstract

    Echinocandins are a family of fungal lipidated cyclic hexapeptide natural products. Due to their effectiveness as antifungal agents, three semisynthetic derivatives have been developed and approved for treatment of human invasive candidiasis. All six of the amino acid residues are hydroxylated, including 4R,5R-dihydroxy-L-ornithine, 4R-hydroxyl-L-proline, 3S,4S-dihydroxy-L-homotyrosine, and 3S-hydroxyl-4S-methyl-L-proline. We report here the biosynthetic gene cluster of echinocandin B 1 from Emericella rugulosa NRRL 11440 containing genes encoding for a six-module nonribosomal peptide synthetase EcdA, an acyl-AMP ligase EcdI, and oxygenases EcdG, EcdH, and EcdK. We showed EcdI activates linoleate as linoleyl-AMP and installs it on the first thiolation domain of EcdA. We have also established through ATP-PP(i) exchange assay that EcdA loads L-ornithine in the first module. A separate hty gene cluster encodes four enzymes for de novo generation of L-homotyrosine from acetyl-CoA and 4-hydroxyphenyl-pyruvate is found from the sequenced genome. Deletions in the ecdA, and htyA genes validate their essential roles in echinocandin B production. Five predicted iron-centered oxygenase genes, ecdG, ecdH, ecdK, htyE, and htyF, in the two separate ecd and hty clusters are likely to be the tailoring oxygenases for maturation of the nascent NRPS lipohexapeptidolactam product.

    View details for DOI 10.1021/ja307220z

    View details for Web of Science ID 000309566400053

    View details for PubMedID 22998630

  • CD and MCD of CytC3 and taurine dioxygenase: Role of the facial triad in alpha-KG-dependent oxygenases JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Neidig, M. L., Brown, C. D., Light, K. M., Fujimori, D. G., Nolan, E. M., Price, J. C., Barr, E. W., Bollinger, J. M., Krebs, C., Walsh, C. T., Solomon, E. I. 2007; 129 (46): 14224-14231

    Abstract

    The alpha-ketoglutarate (alpha-KG)-dependent oxygenases are a large and diverse class of mononuclear non-heme iron enzymes that require FeII, alpha-KG, and dioxygen for catalysis with the alpha-KG cosubstrate supplying the additional reducing equivalents for oxygen activation. While these systems exhibit a diverse array of reactivities (i.e., hydroxylation, desaturation, ring closure, etc.), they all share a common structural motif at the FeII active site, termed the 2-His-1-carboxylate facial triad. Recently, a new subclass of alpha-KG-dependent oxygenases has been identified that exhibits novel reactivity, the oxidative halogenation of unactivated carbon centers. These enzymes are also structurally unique in that they do not contain the standard facial triad, as a Cl- ligand is coordinated in place of the carboxylate. An FeII methodology involving CD, MCD, and VTVH MCD spectroscopies was applied to CytC3 to elucidate the active-site structural effects of this perturbation of the coordination sphere. A significant decrease in the affinity of FeII for apo-CytC3 was observed, supporting the necessity of the facial triad for iron coordination to form the resting site. In addition, interesting differences observed in the FeII/alpha-KG complex relative to the cognate complex in other alpha-KG-dependent oxygenases indicate the presence of a distorted 6C site with a weak water ligand. Combined with parallel studies of taurine dioxygenase and past studies of clavaminate synthase, these results define a role of the carboxylate ligand of the facial triad in stabilizing water coordination via a H-bonding interaction between the noncoordinating oxygen of the carboxylate and the coordinated water. These studies provide initial insight into the active-site features that favor chlorination by CytC3 over the hydroxylation reactions occurring in related enzymes.

    View details for DOI 10.1021/ja074557r

    View details for Web of Science ID 000251182000047

    View details for PubMedID 17967013

  • Probing intra- versus interchain kinetic preferences of L-Thr acylation on dimeric VibF with mass spectrometry BIOPHYSICAL JOURNAL Hicks, L. M., Balibar, C. J., Walsh, C. T., Kelleher, N. L., Hillson, N. J. 2006; 91 (7): 2609-2619

    Abstract

    We present a method to probe intra- and interchain activities within dimeric nonribosomal peptide synthetases. Utilizing domain inactivation and analytical mass mutants in conjunction with rapid-quench, mass spectrometry, and a probabilistic kinetic model, we have elucidated the pre-steady-state intra- and interchain rates and the corresponding flux of the acylation of L-Thr onto VibF. Although the intra rate is significantly faster than the inter rate, the data are most consistent with an even flux of covalent substrate loading where neither pathway dominates. These pre-steady-state results confirm previous steady-state in vitro mutant complementation studies of VibF. Extension of this methodology to other dimeric nonribosomal peptide synthetases, and to the related fatty acid and polyketide synthases, will further our biophysical understanding of their acyl-intermediate-processing pathways.

    View details for DOI 10.1529/biophysj.106.084848

    View details for Web of Science ID 000240368700022

    View details for PubMedID 16815901

  • Reconstitution and characterization of a new desosaminyl transferase, EryCIII, from the erythromycin biosynthetic pathway JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lee, H. Y., Chung, H. S., Hang, C., Khosla, C., Walsh, C. T., Kahne, D., Walker, S. 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

  • 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 Admiraal, S. J., Khosla, C., Walsh, C. T. 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

  • Biosynthesis of yersiniabactin, a complex polyketide-nonribosomal peptide, using Escherichia coli as a heterologous host APPLIED AND ENVIRONMENTAL MICROBIOLOGY Pfeifer, B. A., Wang, C. C., Walsh, C. T., Khosla, C. 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

  • Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Watanabe, K., Rude, M. A., Walsh, C. T., Khosla, C. 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

  • Epothilone C macrolactonization and hydrolysis are catalyzed by the isolated thioesterase domain of epothilone polyketide synthase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Boddy, C. N., Schneider, T. L., Hotta, K., Walsh, C. T., Khosla, C. 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

  • The loading and initial elongation modules of rifamycin synthetase collaborate to produce mixed aryl ketide products-1 BIOCHEMISTRY Admiraal, S. J., Khosla, C., Walsh, C. T. 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

  • Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chiu, H. T., Hubbard, B. K., Shah, A. N., Eide, J., Fredenburg, R. A., Walsh, C. T., Khosla, C. 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

  • The loading module of rifamycin synthetase is an adenylation-thiolation didomain with substrate tolerance for substituted benzoates BIOCHEMISTRY Admiraal, S. J., Walsh, C. T., Khosla, C. 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

  • Predicting microbial biodegradation pathways ASM NEWS Wackett, L. P., Ellis, L. B., Speedie, S. M., Hershberger, C. D., Knackmuss, H. J., Spormann, A. M., Walsh, C. T., Forney, L. J., Punch, W. F., Kazic, T., Kanehisa, M., Berndt, D. J. 1999; 65 (2): 87-93
  • Biochemistry - Harnessing the biosynthetic code: Combinations, permutations, and mutations SCIENCE Cane, D. E., Walsh, C. T., Khosla, C. 1998; 282 (5386): 63-68
  • Utilization of enzymatically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase BIOCHEMISTRY Carreras, C. W., Gehring, A. M., Walsh, C. T., Khosla, C. 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

  • Ability of Streptomyces spp acyl carrier proteins and coenzyme A analogs to serve as substrates in vitro for E-coli holo-ACP synthase CHEMISTRY & BIOLOGY Gehring, A. M., Lambalot, R. H., Vogel, K. W., Drueckhammer, D. G., Walsh, C. T. 1997; 4 (1): 17-24

    Abstract

    The polyketide natural products are assembled by a series of decarboxylation/condensation reactions of simple carboxylic acids catalyzed by polyketide synthase (PKS) complexes. The growing chain is assembled on acyl carrier protein (ACP), an essential component of the PKS. ACP requires posttranslational modification on a conserved serine residue by covalent attachment of a 4'-phosphopantetheine (P-pant) cofactor to yield active holo-ACP. When ACPs of Streptomyces type II aromatic PKS are overproduced in E. coli, however, typically little or no active holo-ACP is produced, and the ACP remains in the inactive apo-form.We demonstrate that E. coli holo-ACP synthase (ACPS), a fatty acid biosynthesis enzyme, can catalyze P-pant transfer in vitro to the Streptomyces PKS ACPs required for the biosynthesis of the polyketide antibiotics granaticin, frenolicin, oxytetracycline and tetracenomycin. The catalytic efficiency of this P-pant transfer reaction correlates with the overall negative charge of the ACP substrate. Several coenzyme A analogs, modified in the P-pant portion of the molecule, are likewise able to serve as substrates in vitro for ACPS.E coli ACPS can serve as a useful reagent for the preparation of holo-forms of Streptomyces ACPs as well as holo-ACPs with altered phosphopantetheine moieties. Such modified ACPs should prove useful for studying the role of particular ACPs and the phosphopantetheine cofactor in the subsequent reactions of polyketide and fatty acid biosynthesis.

    View details for Web of Science ID A1997WH80400005

    View details for PubMedID 9070424