Laura Dassama is a chemical biologist who uses principles from chemistry and physics to understand complex biological phenomenal, and to leverage that understanding for the modulation of biological processes. Her current research focuses on deciphering the molecular recognition mechanisms of multidrug transporters implicated in drug resistance, rational engineering and repurposing of natural products, and control of transcription factors relevant to sickle cell disease.
Honors & Awards
Hellman Faculty Scholar, Stanford University (2019-2020)
Alumni Achievement Award, Pennsylvania State University (2019)
Gabilan Junior Faculty Fellowship, Stanford University (2018-2021)
Terman Faculty Fellowship, Stanford University (2018-2021)
Postdoctoral Enrichment Program Grant, Burroughs Wellcome Fund (2015-2018)
Ruth L. Kirschstein National Research Service Award, National Institutes of Health (2014-2017)
Alumni Association Dissertation, Pennsylvania State University (2013)
Carl Storm Underrepresented Minority Fellowship, Gordon Research Conference (2011)
Minority Ph.D. Scholar, Alfred P. Sloan Foundation (2009-2013)
B.S., Temple University, Biochemistry (2007)
Ph.D., Pennsylvania State University, Biochemistry and Molecular Biology (2013)
Postdoctoral fellow, Northwestern University, Molecular Biosciences (2017)
Research Associate, Boston Children's Hospital, Harvard Medical School, and Dana-Farber Cancer Institute, Hematology/Oncology (2018)
- Biochemistry I
CHEM 181, CHEMENG 181, CHEMENG 281 (Aut)
- Biological Chemistry Laboratory
CHEM 184 (Spr)
- Independent Studies (5)
- Prior Year Courses
Doctoral Dissertation Reader (AC)
Augustin Braun, Madeline Chosy, Dory DeWeese, Alex Heyer, Taylor Jones, Rachel Mardjuki, Wilson Sinclair
Postdoctoral Faculty Sponsor
Paul Ludford, Fangfang Shen, Liting Zhai
Doctoral Dissertation Advisor (AC)
Chiu-Chun Chou, Jeandele Elliot, Lisha Ou, Mekedlawit Setegne
The enzymology of oxazolone and thioamide synthesis in methanobactin.
Methods in enzymology
2021; 656: 341-373
Methanobactins are ribosomally synthesized and post-translationally modified peptidic (RiPP) natural products that are known for their ability to chelate copper ions. Crucial for their high copper affinity is a pair of bidentate ligands comprising a nitrogen-containing heterocycle and an adjacent thioamide or enethiol group. The previously uncharacterized proteins MbnB and MbnC were recently shown to synthesize these groups. In this chapter, we describe the methods that were used to determine that MbnB and MbnC are the core biosynthetic enzymes in methanobactin biosynthesis. The two proteins form a heterodimeric complex (MbnBC) which, through a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA), modifies a cysteine residue in order to install the oxazolone and thioamide moieties. This overview covers the heterologous expression and purification of MbnBC, characterization of the iron cluster found in MbnB, and characterization of the modification installed on MbnA. While this chapter is specific to MbnBC, the methods outlined here can be broadly applied to the enzymology of other proteins that install similar groups as well as enzyme pairs related to MbnB and MbnC.
View details for DOI 10.1016/bs.mie.2021.04.008
View details for PubMedID 34325792
Nuclear Resonance Vibrational Spectroscopic Definition of the Facial Triad FeIV═O Intermediate in Taurine Dioxygenase: Evaluation of Structural Contributions to Hydrogen Atom Abstraction.
Journal of the American Chemical Society
The alpha-ketoglutarate (alphaKG)-dependent oxygenases catalyze a diverse range of chemical reactions using a common high-spin FeIV═O intermediate that, in most reactions, abstract a hydrogen atom from the substrate. Previously, the FeIV═O intermediate in the alphaKG-dependent halogenase SyrB2 was characterized by nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations, which demonstrated that it has a trigonal-pyramidal geometry with the scissile C-H bond of the substrate calculated to be perpendicular to the Fe-O bond. Here, we have used NRVS and DFT calculations to show that the FeIV═O complex in taurine dioxygenase (TauD), the alphaKG-dependent hydroxylase in which this intermediate was first characterized, also has a trigonal bipyramidal geometry but with an aspartate residue replacing the equatorial halide of the SyrB2 intermediate. Computational analysis of hydrogen atom abstraction by square pyramidal, trigonal bipyramidal, and six-coordinate FeIV═O complexes in two different substrate orientations (one more along [sigma channel] and another more perpendicular [pi channel] to the Fe-O bond) reveals similar activation barriers. Thus, both substrate approaches to all three geometries are competent in hydrogen atom abstraction. The equivalence in reactivity between the two substrate orientations arises from compensation of the promotion energy (electronic excitation within the d manifold) required to access the pi channel by the significantly larger oxyl character present in the ppi orbital oriented toward the substrate, which leads to an earlier transition state along the C-H coordinate.
View details for DOI 10.1021/jacs.0c08903
View details for PubMedID 33103886
Rational targeting of a NuRD subcomplex guided by comprehensive in situ mutagenesis.
Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for therapeutic intervention of β-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in γ-globin repression. We used pooled CRISPR screening to disrupt NuRD protein coding sequences comprehensively in human adult erythroid precursors. Essential for fetal hemoglobin (HbF) control is a non-redundant subcomplex of NuRD protein family paralogs, whose composition we corroborated by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identified key protein interfaces where in-frame alleles resulted in loss-of-function due to destabilization or altered function of subunits. We ascertained mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrated that sequestering CHD4 from NuRD phenocopied these mutations. These results indicate a generalizable approach to discover protein complex features amenable to rational biochemical targeting.
View details for DOI 10.1038/s41588-019-0453-4
View details for PubMedID 31253978
MbnH is a diheme MauG-like protein associated with microbial copper homeostasis.
The Journal of biological chemistry
Methanobactins (Mbns) are ribosomally-produced, post-translationally-modified peptidic copper-binding natural products produced under conditions of copper limitation. Genes encoding Mbn biosynthetic and transport proteins have been identified in a wide variety of bacteria, indicating a broader role for Mbns in bacterial metal homeostasis. Many of the genes in the Mbn operons have been assigned functions, but two genes usually present, mbnP and mbnH, encode uncharacterized proteins predicted to reside in the periplasm. MbnH belongs to the bacterial diheme cytochrome c peroxidase (bCcP)/MauG protein family, and MbnP contains no domains of known function. Here, we performed a detailed bioinformatic analysis of both proteins and have biochemically characterized MbnH from Methylosinus (Ms.) trichosporium OB3b. We note that the mbnH and mbnP genes typically co-occur and are located proximal to genes associated with microbial copper homeostasis. Our bioinformatics analysis also revealed that the bCcP/MauG family is significantly more diverse than originally appreciated, and that MbnH is most closely related to the MauG subfamily. A 2.6 Å resolution structure of (Ms.) trichosporium OB3b MbnH combined with spectroscopic data and peroxidase activity assays provided evidence that MbnH indeed more closely resembles MauG than bCcPs, although its redox properties are significantly different from those of MauG. The overall similarity of MbnH to MauG suggested that MbnH could post-translationally modify a macromolecule, such as internalized CuMbn or its uncharacterized partner protein, MbnP. Our results indicate that MbnH is a MauG-like diheme protein that is likely involved in microbial copper homeostasis and represents a new family within the bCcP/MauG superfamily.
View details for DOI 10.1074/jbc.RA119.010202
View details for PubMedID 31511324
The biosynthesis of methanobactin
2018; 359 (6382): 1411-+
Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.
View details for DOI 10.1126/science.aap9437
View details for Web of Science ID 000428043600048
View details for PubMedID 29567715
View details for PubMedCentralID PMC5944852
Copper transport in methanotrophic bacteria
AMER CHEMICAL SOC. 2017
View details for Web of Science ID 000430569103238
Bacterial copper acquisition
FEDERATION AMER SOC EXP BIOL. 2017
View details for Web of Science ID 000405461402700
O-H Activation by an Unexpected Ferryl Intermediate during Catalysis by 2-Hydroxyethylphosphonate Dioxygenase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2017; 139 (5): 2045–52
Activation of O-H bonds by inorganic metal-oxo complexes has been documented, but no cognate enzymatic process is known. Our mechanistic analysis of 2-hydroxyethylphosphonate dioxygenase (HEPD), which cleaves the C1-C2 bond of its substrate to afford hydroxymethylphosphonate on the biosynthetic pathway to the commercial herbicide phosphinothricin, uncovered an example of such an O-H-bond-cleavage event. Stopped-flow UV-visible absorption and freeze-quench Mössbauer experiments identified a transient iron(IV)-oxo (ferryl) complex. Maximal accumulation of the intermediate required both the presence of deuterium in the substrate and, importantly, the use of 2H2O as solvent. The ferryl complex forms and decays rapidly enough to be on the catalytic pathway. To account for these unanticipated results, a new mechanism that involves activation of an O-H bond by the ferryl complex is proposed. This mechanism accommodates all available data on the HEPD reaction.
View details for DOI 10.1021/jacs.6b12147
View details for Web of Science ID 000393848400055
View details for PubMedID 28092705
View details for PubMedCentralID PMC5302023
Methanobactins: from genome to function
2017; 9 (1): 7–20
Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptide (RiPP) natural products that bind copper with high affinity using nitrogen-containing heterocycles and thioamide groups. In some methanotrophic bacteria, Mbns are secreted under conditions of copper starvation and then re-internalized as a copper source for the enzyme particulate methane monooxygenase (pMMO). Genome mining studies have led to the identification and classification of operons encoding the Mbn precursor peptide (MbnA) as well as a number of putative transport, regulatory, and biosynthetic proteins. These Mbn operons are present in non-methanotrophic bacteria as well, suggesting a broader role in and perhaps beyond copper acquisition. Genetic and biochemical studies indicate that specific operon-encoded proteins are involved in Mbn transport and provide insight into copper-responsive gene regulation in methanotrophs. Mbn biosynthesis is not yet understood, but combined analysis of Mbn structures, MbnA sequences, and operon content represents a powerful approach to elucidating the roles of specific biosynthetic enzymes. Future work will likely lead to the discovery of unique pathways for natural product biosynthesis and new mechanisms of microbial metal homeostasis.
View details for DOI 10.1039/c6mt00208k
View details for Web of Science ID 000394948300001
View details for PubMedID 27905614
View details for PubMedCentralID PMC5269455
Methanobactin transport machinery
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (46): 13027–32
Methanotrophic bacteria use methane, a potent greenhouse gas, as their primary source of carbon and energy. The first step in methane metabolism is its oxidation to methanol. In almost all methanotrophs, this chemically challenging reaction is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent integral membrane enzyme. Methanotrophs acquire copper (Cu) for pMMO by secreting a small ribosomally produced, posttranslationally modified natural product called methanobactin (Mbn). Mbn chelates Cu with high affinity, and the Cu-loaded form (CuMbn) is reinternalized into the cell via an active transport process. Bioinformatic and gene regulation studies suggest that two proteins might play a role in CuMbn handling: the TonB-dependent transporter MbnT and the periplasmic binding protein MbnE. Disruption of the gene that encodes MbnT abolishes CuMbn uptake, as reported previously, and expression of MbnT in Escherichia coli confers the ability to take up CuMbn. Biophysical studies of MbnT and MbnE reveal specific interactions with CuMbn, and a crystal structure of apo MbnE is consistent with MbnE's proposed role as a periplasmic CuMbn transporter. Notably, MbnT and MbnE exhibit different levels of discrimination between cognate and noncognate CuMbns. These findings provide evidence for CuMbn-protein interactions and begin to elucidate the molecular mechanisms of its recognition and transport.
View details for DOI 10.1073/pnas.1603578113
View details for Web of Science ID 000388970100054
View details for PubMedID 27807137
View details for PubMedCentralID PMC5135309
Direct Measurement of the Radical Translocation Distance in the Class I Ribonucleotide Reductase from Chlamydia trachomatis
JOURNAL OF PHYSICAL CHEMISTRY B
2015; 119 (43): 13777–84
Ribonucleotide reductases (RNRs) catalyze conversion of ribonucleotides to deoxyribonucleotides in all organisms via a free-radical mechanism that is essentially conserved. In class I RNRs, the reaction is initiated and terminated by radical translocation (RT) between the α and β subunits. In the class Ic RNR from Chlamydia trachomatis (Ct RNR), the initiating event converts the active S = 1 Mn(IV)/Fe(III) cofactor to the S = 1/2 Mn(III)/Fe(III) "RT-product" form in the β subunit and generates a cysteinyl radical in the α active site. The radical can be trapped via the well-described decomposition reaction of the mechanism-based inactivator, 2'-azido-2'-deoxyuridine-5'-diphosphate, resulting in the generation of a long-lived, nitrogen-centered radical (N(•)) in α. In this work, we have determined the distance between the Mn(III)/Fe(III) cofactor in β and N(•) in α to be 43 ± 1 Å by using double electron-electron resonance experiments. This study provides the first structural data on the Ct RNR holoenzyme complex and the first direct experimental measurement of the inter-subunit RT distance in any class I RNR.
View details for DOI 10.1021/acs.jpcb.5b04057
View details for Web of Science ID 000363994000035
View details for PubMedID 26087051
View details for PubMedCentralID PMC5840866
Geometric and electronic structure of the Mn(IV)Fe(III) cofactor in class Ic ribonucleotide reductase: correlation to the class Ia binuclear non-heme iron enzyme.
Journal of the American Chemical Society
2013; 135 (46): 17573-17584
The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) utilizes a Mn/Fe heterobinuclear cofactor, rather than the Fe/Fe cofactor found in the β (R2) subunit of the class Ia enzymes, to react with O2. This reaction produces a stable Mn(IV)Fe(III) cofactor that initiates a radical, which transfers to the adjacent α (R1) subunit and reacts with the substrate. We have studied the Mn(IV)Fe(III) cofactor using nuclear resonance vibrational spectroscopy (NRVS) and absorption (Abs)/circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD spectroscopies to obtain detailed insight into its geometric/electronic structure and to correlate structure with reactivity; NRVS focuses on the Fe(III), whereas MCD reflects the spin-allowed transitions mostly on the Mn(IV). We have evaluated 18 systematically varied structures. Comparison of the simulated NRVS spectra to the experimental data shows that the cofactor has one carboxylate bridge, with Mn(IV) at the site proximal to Phe127. Abs/CD/MCD/VTVH MCD data exhibit 12 transitions that are assigned as d-d and oxo and OH(-) to metal charge-transfer (CT) transitions. Assignments are based on MCD/Abs intensity ratios, transition energies, polarizations, and derivative-shaped pseudo-A term CT transitions. Correlating these results with TD-DFT calculations defines the Mn(IV)Fe(III) cofactor as having a μ-oxo, μ-hydroxo core and a terminal hydroxo ligand on the Mn(IV). From DFT calculations, the Mn(IV) at site 1 is necessary to tune the redox potential to a value similar to that of the tyrosine radical in class Ia RNR, and the OH(-) terminal ligand on this Mn(IV) provides a high proton affinity that could gate radical translocation to the α (R1) subunit.
View details for DOI 10.1021/ja409510d
View details for PubMedID 24131208
Geometric and Electronic Structure of the Mn(IV)Fe(III) Cofactor in Class Ic Ribonucleotide Reductase: Correlation to the Class Ia Binuclear Non-Heme Iron Enzyme.
Journal of the American Chemical Society
2013; 135 (46): 17573-17584
View details for DOI 10.1021/ja409510d
View details for PubMedID 24131208
A 2.8 angstrom Fe-Fe Separation in the Fe-2(III/IV) Intermediate, X, from Escherichia coli Ribonucleotide Reductase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2013; 135 (45): 16758–61
A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe2(III/III)/tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ~35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coli RNR-β cofactor, reaction of the protein's Fe2(II/II) complex with O2 results in accumulation of an Fe2(III/IV) cluster, termed X, which oxidizes the adjacent tyrosine (Y122) to the radical (Y122(•)) as the cluster is converted to the μ-oxo-Fe2(III/III) product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe-Fe separation (d(Fe-Fe)) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O(2-), HO(-), and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have d(Fe-Fe) ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O2 in situ from chlorite using the enzyme chlorite dismutase to prepare X at ~2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured d(Fe-Fe) = 2.78 Å is fully consistent with computational models containing a (μ-oxo)2-Fe2(III/IV) core. Correction of the d(Fe-Fe) brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y122(•).
View details for DOI 10.1021/ja407438p
View details for Web of Science ID 000327103600004
View details for PubMedID 24094084
View details for PubMedCentralID PMC4209742
Structural Basis for Assembly of the Mn-IV/Fe-III Cofactor in the Class Ic Ribonucleotide Reductase from Chlamydia trachomatis
2013; 52 (37): 6424–36
The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) employs a Mn(IV)/Fe(III) cofactor in each monomer of its β2 subunit to initiate nucleotide reduction. The cofactor forms by reaction of Mn(II)/Fe(II)-β2 with O2. Previously, in vitro cofactor assembly from apo β2 and divalent metal ions produced a mixture of two forms, with Mn at site 1 (Mn(IV)/Fe(III)) or site 2 (Fe(III)/Mn(IV)), of which the more active Mn(IV)/Fe(III) product predominates. Here we have addressed the basis for metal site selectivity by determining X-ray crystal structures of apo, Mn(II), and Mn(II)/Fe(II) complexes of Ct β2. A structure obtained anaerobically with equimolar Mn(II), Fe(II), and apoprotein reveals exclusive incorporation of Mn(II) at site 1 and Fe(II) at site 2, in contrast to the more modest site selectivity achieved previously. Site specificity is controlled thermodynamically by the apoprotein structure, as only minor adjustments of ligands occur upon metal binding. Additional structures imply that, by itself, Mn(II) binds in either site. Together, the structures are consistent with a model for in vitro cofactor assembly in which Fe(II) specificity for site 2 drives assembly of the appropriately configured heterobimetallic center, provided that Fe(II) is substoichiometric. This model suggests that use of a Mn(IV)/Fe(III) cofactor in vivo could be an adaptation to Fe(II) limitation. A 1.8 Å resolution model of the Mn(II)/Fe(II)-β2 complex reveals additional structural determinants for activation of the cofactor, including a proposed site for side-on (η(2)) addition of O2 to Fe(II) and a short (3.2 Å) Mn(II)-Fe(II) interionic distance, promoting formation of the Mn(IV)/Fe(IV) activation intermediate.
View details for DOI 10.1021/bi400819x
View details for Web of Science ID 000330099600013
View details for PubMedID 23924396
View details for PubMedCentralID PMC3821933
Novel approaches for the accumulation of oxygenated intermediates to multi-millimolar concentrations
COORDINATION CHEMISTRY REVIEWS
2013; 257 (1): 234–43
Metalloenzymes that utilize molecular oxygen as a co-substrate catalyze a wide variety of chemically difficult oxidation reactions. Significant insight into the reaction mechanisms of these enzymes can be obtained by the application of a combination of rapid kinetic and spectroscopic methods to the direct structural characterization of intermediate states. A key limitation of this approach is the low aqueous solubility (< 2 mM) of the co-substrate, O2, which undergoes further dilution (typically by one-third or one-half) upon initiation of reactions by rapid-mixing. This situation imposes a practical upper limit on [O2] (and therefore on the concentration of reactive intermediate(s) that can be rapidly accumulated) of ∼1-1.3 mM in such experiments as they are routinely carried out. However, many spectroscopic methods benefit from or require significantly greater concentrations of the species to be studied. To overcome this problem, we have recently developed two new approaches for the preparation of samples of oxygenated intermediates: (1) direct oxygenation of reduced metalloenzymes using gaseous O2 and (2) the in situ generation of O2 from chlorite catalyzed by the enzyme chlorite dismutase (Cld). Whereas the former method is applicable only to intermediates with half lives of several minutes, owing to the sluggishness of transport of O2 across the gas-liquid interface, the latter approach has been successfully applied to trap several intermediates at high concentration and purity by the freeze-quench method. The in situ approach permits generation of a pulse of at least 5 mM O2 within ∼ 1 ms and accumulation of O2 to effective concentrations of up to ∼ 11 mM (i.e. ∼ 10-fold greater than by the conventional approach). The use of these new techniques for studies of oxygenases and oxidases is discussed.
View details for DOI 10.1016/j.ccr.2012.06.020
View details for Web of Science ID 000312972700019
View details for PubMedID 24368870
View details for PubMedCentralID PMC3870000
Radical-Translocation Intermediates and Hurdling of Pathway Defects in "Super-oxidized" (Mn-IV/Fe-IV) Chlamydia trachomatis Ribonucleotide Reductase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2012; 134 (50): 20498-20506
A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y(•)) or a Mn(IV)/Fe(III) cluster in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit, generating a thiyl radical that abstracts hydrogen (H(•)) from the substrate. With either oxidant, the inter-subunit "hole-transfer" or "radical-translocation" (RT) process is thought to occur by a "hopping" mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type (wt) RNR. The Mn(IV)/Fe(III) cofactor of Chlamydia trachomatis RNR assembles via a Mn(IV)/Fe(IV) intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when α is present, accumulating radicals with EPR spectra characteristic of Y(•)'s. The dependence of Y(•) accumulation on the presence of substrate suggests that RT within this "super-oxidized" enzyme form is gated by the protein, and the failure of a β variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y(•)(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant β proteins having pathway substitutions rendering them inactive in their Mn(IV)/Fe(III) states can generate the pathway Y(•)'s in their Mn(IV)/Fe(IV) states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the "hurdling" of engineered defects in the RT pathway.
View details for DOI 10.1021/ja309468s
View details for Web of Science ID 000312430700049
View details for PubMedID 23157728
O-2-Evolving Chlorite Dismutase as a Tool for Studying O-2-Utilizing Enzymes
2012; 51 (8): 1607–16
The direct interrogation of fleeting intermediates by rapid-mixing kinetic methods has significantly advanced our understanding of enzymes that utilize dioxygen. The gas's modest aqueous solubility (<2 mM at 1 atm) presents a technical challenge to this approach, because it limits the rate of formation and extent of accumulation of intermediates. This challenge can be overcome by use of the heme enzyme chlorite dismutase (Cld) for the rapid, in situ generation of O(2) at concentrations far exceeding 2 mM. This method was used to define the O(2) concentration dependence of the reaction of the class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis, in which the enzyme's Mn(IV)/Fe(III) cofactor forms from a Mn(II)/Fe(II) complex and O(2) via a Mn(IV)/Fe(IV) intermediate, at effective O(2) concentrations as high as ~10 mM. With a more soluble receptor, myoglobin, an O(2) adduct accumulated to a concentration of >6 mM in <15 ms. Finally, the C-H-bond-cleaving Fe(IV)-oxo complex, J, in taurine:α-ketoglutarate dioxygenase and superoxo-Fe(2)(III/III) complex, G, in myo-inositol oxygenase, and the tyrosyl-radical-generating Fe(2)(III/IV) intermediate, X, in Escherichia coli RNR, were all accumulated to yields more than twice those previously attained. This means of in situ O(2) evolution permits a >5 mM "pulse" of O(2) to be generated in <1 ms at the easily accessible Cld concentration of 50 μM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O(2) addition steps.
View details for DOI 10.1021/bi201906x
View details for Web of Science ID 000300757100007
View details for PubMedID 22304240
View details for PubMedCentralID PMC3297476
Evidence That the beta Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2012; 134 (5): 2520–23
The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the β subunit oxidizes a cysteine residue ~35 Å away in the α subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn(IV) ion of a Mn(IV)/Fe(III) cluster, which assembles in a reaction between O(2) and the Mn(II)/Fe(II) complex of β. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn(IV) ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn(IV) ion most likely resides in this site (i.e., (1)Mn(IV)/(2)Fe(III)), but a subsequent computational study favored its occupation of site 2 ((1)Fe(III)/(2)Mn(IV)). In this work, we have sought to resolve the location of the Mn(IV) ion in Ct RNR-β by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn(IV)/Fe(III) clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both (1)Mn(II)/(2)Fe(II) and (1)Fe(II)/(2)Mn(II) complexes are competent to react with O(2) to produce the corresponding oxidized states. However, with diminished Mn(II) loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these "low-Mn" samples on a per-Mn basis implies that the (1)Mn(IV)/(2)Fe(III)-β is at least the more active of the two oxidized forms and may be the only active form.
View details for DOI 10.1021/ja211314p
View details for Web of Science ID 000300460600019
View details for PubMedID 22242660
View details for PubMedCentralID PMC3297472