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Leslie B. Poole - One of the best experts on this subject based on the ideXlab platform.
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Triphenylphosphonium-Derived Protein Sulfenic Acid Trapping Agents: Synthesis, Reactivity, and Effect on Mitochondrial Function
2019Co-Authors: Tom E. Forshaw, Leslie B. Poole, Reetta J. Holmila, Stephen A. Vance, Cristina M. Furdui, Bruce S. KingAbstract:Redox-mediated protein modifications control numerous processes in both normal and disease metabolism. Protein Sulfenic Acids, formed from the oxidation of protein cysteine residues, play a critical role in thiol-based redox signaling. The reactivity of protein Sulfenic Acids requires their identification through chemical trapping, and this paper describes the use of the triphenylphosphonium (TPP) ion to direct known Sulfenic Acid traps to the mitochondria, a verified source of cellular reactive oxygen species. Coupling of the TPP group with the 2,4-(dioxocyclohexyl)propoxy (DCP) unit and the bicyclo[6.1.0]nonyne (BCN) group produces two new probes, DCP-TPP and BCN-TPP. DCP-TPP and BCN-TPP react with C165A AhpC-SOH, a model protein Sulfenic Acid, to form the expected adducts with second-order rate constants of k = 1.1 M–1 s–1 and k = 5.99 M–1 s–1, respectively, as determined by electrospray ionization time-of-flight mass spectrometry. The TPP group does not alter the rate of DCP-TPP reaction with protein Sulfenic Acid compared to dimedone but slows the rate of BCN-TPP reaction compared to a non-TPP-containing BCN-OH control by 4.6-fold. The hydrophobic TPP group may interact with the protein, preventing an optimal reaction orientation for BCN-TPP. Unlike BCN-OH, BCN-TPP does not react with the protein persulfide, C165A AhpC-SSH. Extracellular flux measurements using A549 cells show that DCP-TPP and BCN-TPP influence mitochondrial energetics, with BCN-TPP producing a drastic decrease in basal respiration, perhaps due to its faster reaction kinetics with sulfenylated proteins. Further control experiments with BCN-OH, TPP-COOH, and dimedone provide strong evidence for mitochondrial localization and accumulation of DCP-TPP and BCN-TPP. These results reveal the compatibility of the TPP group with reactive Sulfenic Acid probes as a mitochondrial director and support the use of the TPP group in the design of Sulfenic Acid traps
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Differential Kinetics of Two-Cysteine Peroxiredoxin Disulfide Formation Reveal a Novel Model for Peroxide Sensing
2018Co-Authors: Stephanie Portillo-ledesma, Derek Parsonage, Leslie B. Poole, Lía M. Randall, Joaquín Dalla Rizza, Andrew P. Karplus, Ana Denicola, Gerardo Ferrer-suetaAbstract:Two-cysteine peroxiredoxins (Prx) have a three-step catalytic cycle consisting of (1) reduction of peroxide and formation of Sulfenic Acid on the enzyme, (2) condensation of the Sulfenic Acid with a thiol to form disulfide, also known as resolution, and (3) reduction of the disulfide by a reductant protein. By following changes in protein fluorescence, we have studied the pH dependence of reaction 2 in human peroxiredoxins 1, 2, and 5 and in Salmonella typhimurium AhpC and obtained rate constants for the reaction and pKa values of the thiol and Sulfenic Acid involved for each system. The observed reaction 2 rate constant spans 2 orders of magnitude, but in all cases, reaction 2 appears to be slow compared to the same reaction in small-molecule systems, making clear the rates are limited by conformational features of the proteins. For each Prx, reaction 2 will become rate-limiting at some critical steady-state concentration of H2O2 producing the accumulation of Prx as Sulfenic Acid. When this happens, an alternative and faster-resolving Prx (or other peroxidase) may take over the antioxidant role. The accumulation of Sulfenic Acid Prx at distinct concentrations of H2O2 is embedded in the kinetic limitations of the catalytic cycle and may constitute the basis of a H2O2-mediated redox signal transduction pathway requiring neither inactivation nor posttranslational modification. The differences in the rate constants of resolution among Prx coexisting in the same compartment may partially explain their complementation in antioxidant function and stepwise sensing of H2O2 concentration
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Strained Cycloalkynes as New Protein Sulfenic Acid Traps
2015Co-Authors: Thomas H. Poole, Leslie B. Poole, Julie A. Reisz, Weiling Zhao, Cristina M. Furdui, Bruce S. KingAbstract:Protein Sulfenic Acids are formed by the reaction of biologically relevant reactive oxygen species with protein thiols. Sulfenic Acid formation modulates the function of enzymes and transcription factors either directly or through the subsequent formation of protein disulfide bonds. Identifying the site, timing, and conditions of protein Sulfenic Acid formation remains crucial to understanding cellular redox regulation. Current methods for trapping and analyzing Sulfenic Acids involve the use of dimedone and other nucleophilic 1,3-dicarbonyl probes that form covalent adducts with cysteine-derived protein Sulfenic Acids. As a mechanistic alternative, the present study describes highly strained bicyclo[6.1.0]nonyne (BCN) derivatives as concerted traps of Sulfenic Acids. These strained cycloalkynes react efficiently with Sulfenic Acids in proteins and small molecules yielding stable alkenyl sulfoxide products at rates more than 100× greater than 1,3-dicarbonyl reagents enabling kinetic competition with physiological sulfur chemistry. Similar to the 1,3-dicarbonyl reagents, the BCN compounds distinguish the Sulfenic Acid oxoform from the thiol, disulfide, sulfinic Acid, and S-nitrosated forms of cysteine while displaying an acceptable cell toxicity profile. The enhanced rates demonstrated by these strained alkynes identify them as new bioorthogonal probes that should facilitate the discovery of previously unknown Sulfenic Acid sites and their parent proteins
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Dissecting Peroxiredoxin Catalysis: Separating Binding, Peroxidation, and Resolution for a Bacterial AhpC
2015Co-Authors: Derek Parsonage, Cristina M. Furdui, Andrew P. Karplus, Gerardo Ferrer-sueta, Kimberly J. Nelson, Samantha Alley, Leslie B. PooleAbstract:Peroxiredoxins make up a ubiquitous family of cysteine-dependent peroxidases that reduce hydroperoxide or peroxynitrite substrates through formation of a cysteine Sulfenic Acid (R-SOH) at the active site. In the 2-Cys peroxiredoxins, a second (resolving) cysteine reacts with the Sulfenic Acid to form a disulfide bond. For all peroxiredoxins, structural rearrangements in the vicinity of the active site cysteine(s) are necessary to allow disulfide bond formation and subsequent reductive recycling. In this study, we evaluated the rate constants for individual steps in the catalytic cycle of Salmonella typhimurium AhpC. Conserved Trp residues situated close to both peroxidatic and resolving cysteines in AhpC give rise to large changes in fluorescence during the catalytic cycle. For recycling, AhpF very efficiently reduces the AhpC disulfide, with a single discernible step and a rate constant of 2.3 × 107 M–1 s–1. Peroxide reduction was more complex and could be modeled as three steps, beginning with a reversible binding of H2O2 to the enzyme (k1 = 1.36 × 108 M–1 s–1, and k–1 = 53 s–1), followed by rapid Sulfenic Acid generation (620 s–1) and then rate-limiting disulfide bond formation (75 s–1). Using bulkier hydroperoxide substrates with higher Km values, we found that different efficiencies (kcat/Km) for turnover of AhpC with these substrates are primarily caused by their slower rates of binding. Our findings indicate that this bacterial peroxiredoxin exhibits rates for both reducing and oxidizing parts of the catalytic cycle that are among the fastest observed so far for this diverse family of enzymes
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TITLE RUNNING HEAD: Synthesis of New Sulfenic Acid-Reactive Compounds
2013Co-Authors: Leslie B. Poole, Bubing Zeng, Sarah A. Knaggs, Mamudu Yakubu, Bruce S. KingAbstract:1 Abstract: Cysteine Sulfenic Acids in proteins can be identified by their ability to form adducts with dimedone, but this reagent imparts no spectral or affinity tag for subsequent analyses of such tagged proteins. Given its similar reactivity toward cysteine Sulfenic Acids, 1, 3-cyclohexadione was synthetically modified to an alcohol derivative and linked to fluorophores based on isatoic Acid and 7-methoxycoumarin. The resulting compounds retain full reactivity and specificity toward cysteine Sulfenic Acids in proteins, allowing for incorporation of the fluorescent label into the protein and “tagging ” it based on its Sulfenic Acid redox state. Control experiments using dimedone further show the specificity of the reaction of 1, 3-diones with protein Sulfenic Acids in aqueous media. These new compounds provide the basis for an improved method for the detection of protein Sulfenic Acids.
Shuguang Zhang - One of the best experts on this subject based on the ideXlab platform.
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peptergents peptide detergents that improve stability and functionality of a membrane protein glycerol 3 phosphate dehydrogenase
Biochemistry, 2005Co-Authors: Joanne I Yeh, Antoni Tortajada, Joao Paulo, Shuguang ZhangAbstract:Toward enhancing in vitro membrane protein studies, we have utilized small self-assembling peptides with detergent properties ("peptergents") to extract and stabilize the integral membrane flavoenzyme, glycerol-3-phosphate dehydrogenase (GlpD), and the soluble redox flavoenzyme, NADH peroxidase (Npx). GlpD is a six transmembrane spanning redox enzyme that catalyzes the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate. Although detergents such as n-octyl-beta-D-glucpyranoside can efficiently solubilize the enzyme, GlpD is inactivated within days once reconstituted into detergent micelles. In contrast, peptergents can efficiently extract and solubilize GlpD from native Escherichia coli membrane and maintain its enzymatic activity up to 10 times longer than in traditional detergents. Intriguingly, peptergents also extended the activity of a soluble flavoenzyme, Npx, when used as an additive. Npx is a flavoenzyme that catalyzes the two-electron reduction of hydrogen peroxide to water using a cysteine-Sulfenic Acid as a secondary redox center. The lability of the peroxidase results from oxidation of the Sulfenic Acid to the sulfinic or sulfonic Acid forms. Oxidation of the Sulfenic Acid, the secondary redox center, results in inactivation, and this reaction proceeds in vitro even in the presence of reducing agents. Although the exact mechanism by which peptergents influence solution stability of Npx remains to be determined, the positive effects may be due to antioxidant properties of the peptides. Peptide-based detergents can be beneficial for many applications and may be particularly useful for structural and functional studies of membrane proteins due to their propensity to enhance the formation of ordered supramolecular assemblies.
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peptergents peptide detergents that improve stability and functionality of a membrane protein glycerol 3 phosphate dehydrogenase
Biochemistry, 2005Co-Authors: Shoucheng Du, Antoni Tortajada, Joao Paulo, Shuguang ZhangAbstract:Toward enhancing in vitro membrane protein studies, we have utilized small self-assembling peptides with detergent properties ("peptergents") to extract and stabilize the integral membrane flavoenzyme, glycerol-3-phosphate dehydrogenase (GlpD), and the soluble redox flavoenzyme, NADH peroxidase (Npx). GlpD is a six transmembrane spanning redox enzyme that catalyzes the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate. Although detergents such as n-octyl-‚-D-glucpyra- noside can efficiently solubilize the enzyme, GlpD is inactivated within days once reconstituted into detergent micelles. In contrast, peptergents can efficiently extract and solubilize GlpD from native Escherichia coli membrane and maintain its enzymatic activity up to 10 times longer than in traditional detergents. Intriguingly, peptergents also extended the activity of a soluble flavoenzyme, Npx, when used as an additive. Npx is a flavoenzyme that catalyzes the two-electron reduction of hydrogen peroxide to water using a cysteine-Sulfenic Acid as a secondary redox center. The lability of the peroxidase results from oxidation of the Sulfenic Acid to the sulfinic or sulfonic Acid forms. Oxidation of the Sulfenic Acid, the secondary redox center, results in inactivation, and this reaction proceeds in vitro even in the presence of reducing agents. Although the exact mechanism by which peptergents influence solution stability of Npx remains to be determined, the positive effects may be due to antioxidant properties of the peptides. Peptide-based detergents can be beneficial for many applications and may be particularly useful for structural and functional studies of membrane proteins due to their propensity to enhance the formation of ordered supramolecular assemblies.
Andrew Claiborne - One of the best experts on this subject based on the ideXlab platform.
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13c nmr analysis of the cysteine Sulfenic Acid redox center of enterococcal nadh peroxidase
Biochemistry, 1997Co-Authors: Edward J Crane, Jacques Vervoort, Andrew ClaiborneAbstract:In order to characterize the native Cys42-Sulfenic Acid redox center of the flavoprotein NADH peroxidase by NMR, an expression protocol has been developed which yields the [3-13C]Cys42-labeled protein in 100 mg quantities. Difference spectra of the labeled minus unlabeled oxidized enzyme (E) give a peak at 41.3 ppm (relative to dioxane) which represents the Cys42-Sulfenic Acid. Reduction of labeled E with 1 equiv of NADH gives the air-stable two-electron reduced (EH2) species, and oxidized minus reduced difference spectra give maxima and minima at 41.3 and 30.8 ppm, respectively, corresponding to the Cys42-Sulfenic Acid and -thiolate species. Peroxide inactivation of E, which has previously been attributed to oxidation of the Cys42-Sulfenic Acid to the Cys42-sulfinic and/or sulfonic Acid states, gives rise to a new maximum in the difference spectrum of Einactive minus E at 57.0 ppm. A similar expression protocol was used to obtain the [ring-2-13C]His-labeled peroxidase HHAA mutant (His10His23Ala87Ala258);...
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an l40c mutation converts the cysteine Sulfenic Acid redox center in enterococcal nadh peroxidase to a disulfide
Biochemistry, 1995Co-Authors: Holly Miller, Derek Parsonage, Sharmila S. Mande, Shlomo Sarfaty, Andrew ClaiborneAbstract:: Multiple sequence alignments including the enterococcal NADH peroxidase and NADH oxidase indicate that residues Ser38 and Cys42 align with the two cysteines of the redox-active disulfides found in glutathione reductase (GR), lipoamide dehydrogenase, mercuric reductase, and trypanothione reductase. In order to evaluate those structural determinants involved in the selection of the cysteine-Sulfenic Acid (Cys-SOH) redox centers found in the two peroxide reductases and the redox-active disulfides present in the GR class of disulfide reductases, NADH peroxidase residues Ser38, Phe39, Leu40, and Ser41 have been individually replaced with Cys. Both the F39C and L40C mutant peroxidases yield active-site disulfides involving the new Cys and the native Cys42; formation of the Cys39-Cys42 disulfide, however, precludes binding of the FAD coenzyme. In contrast, the L40C mutant contains tightly-bound FAD and has been analyzed by both kinetic and spectroscopic approaches. In addition, the L40C and S41C mutant structures have been determined at 2.1 and 2.0 A resolution, respectively, by X-ray crystallography. Formation of the Cys40-Cys42 disulfide bond requires a movement of Cys42-SG to a new position 5.9 A from the flavin-C(4a) position; this is consistent with the inability of the new disulfide to function as a redox center in concert with the flavin. Stereochemical constraints prohibit formation of the Cys41-Cys42 disulfide in the latter mutant.
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analysis of the kinetic and redox properties of nadh peroxidase c42s and c42a mutants lacking the cysteine Sulfenic Acid redox center
Biochemistry, 1995Co-Authors: Derek Parsonage, Andrew ClaiborneAbstract:: The flavoprotein NADH peroxidase from Enterococcus faecalis 10C1 has been shown to contain, in addition to FAD, an unusual cysteine-Sulfenic Acid (Cys-SOH) redox center. The non-flavin center cycles between reduced (Cys-SH) and oxidized (Cys-SOH) states, and the 2.16 A crystal structure of the non-native cysteine-sulfonic Acid (Cys-SO3H) form of the wild-type peroxidase supports the proposed catalytic role of Cys42. In this study, we have employed a site-directed mutagenesis approach in which Cys42 is replaced with Ser and Ala, neither side chain of which is capable of redox activity. Reductive titrations of both C42S and C42A mutants lead directly to full FAD reduction with 1 equiv of either dithionite or NADH, consistent with elimination of the Cys-SOH center. Direct determinations of the redox potentials for the FAD/FADH2 couples yield values of -219 and -197 mV, respectively, for C42S and C42A peroxidases, indicating that the presence of Cys42-SH in the two-electron-reduced wild-type enzyme lowers the flavin potential by approximately 100 mV. Anaerobic stopped-flow analyses of the reduction of C42S and C42A peroxidases by NADH demonstrate that in both cases flavin reduction is rapid; these results are confirmed by enzyme-monitored, steady-state kinetic analyses which, in addition, give turnover numbers approximately 0.04% that of wild-type enzyme. These results are entirely consistent with the role proposed for Cys42 in the catalytic redox cycle of wild-type NADH peroxidase and indirectly support its function as a peroxidatic center in the homologous NADH oxidase.
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Protein-Sulfenic Acid stabilization and function in enzyme catalysis and gene regulation.
The FASEB Journal, 1993Co-Authors: Andrew Claiborne, Derek Parsonage, Holly Miller, R.p. RossAbstract:Sulfenic Acids (R-SOH) result from the stoichiometric oxidations of thiols with mild oxidants such as H2O2; in solution, however, these derivatives accumulate only transiently due to rapid self-condensation reactions, further oxidations to the sulfinic and/or sulfonic Acids, and reactions with nucleophiles such as R-SH. In contrast, oxidations of cysteinyl side chains in proteins, where disulfide bond formation can be prevented and where the reactivity of the nascent cysteine-Sulfenic Acid (Cys-SOH) can be controlled, have previously been shown to yield stable active-site Cys-SOH derivatives of papain and glyceraldehyde-3-phosphate dehydrogenase. More recently, however, functional Cys-SOH residues have been identified in the native oxidized forms of the FAD-containing NADH peroxidase and NADH oxidase from Streptococcus faecalis; these two proteins constitute a new class within the flavoprotein disulfide reductase family. In addition, Cys-SOH derivatives have been suggested to play important roles in redox regulation of the DNA-binding activities of transcription factors such as Fos and Jun, OxyR, and bovine papillomavirus type 1 E2 protein. Structural inferences for the stabilization of protein-Sulfenic Acids, drawn from the refined 2.16-A structure of the streptococcal NADH peroxidase, provide a molecular basis for understanding the proposed redox functions of these novel cofactors in both enzyme catalysis and transcriptional regulation.
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nadh binding site and catalysis of nadh peroxidase
FEBS Journal, 1993Co-Authors: Thilo Stehle, Andrew Claiborne, Georg E SchulzAbstract:The structure of the complex between cofactor NADH and the enzyme NADH peroxidase from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been determined by crystal soaking, X-ray data collection, model building of NADH and refinement at 0.24-nm resolution based on the known enzyme structure [Stehle, T., Ahmed, S. A., Claiborne, A. & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325–1344]. Apart from NADH, the catalytic center of the enzyme contains FAD and a cysteine that shuttles between thiolate and Sulfenic Acid states. Unfortunately, this cysteine was irreversibly oxidized to a cysteine sulfonic Acid in the established enzyme structure. Based on the geometry of the catalytic center, we discuss the stabilization of the oxidation-sensitive Sulfenic Acid and propose a reaction mechanism.
Guy Branlant - One of the best experts on this subject based on the ideXlab platform.
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methionine sulfoxide reductase chemistry substrate binding recycling process and oxidase activity
Bioorganic Chemistry, 2014Co-Authors: Sandrine Boschimuller, Guy BranlantAbstract:Three classes of methionine sulfoxide reductases are known: MsrA and MsrB which are implicated stereo-selectively in the repair of protein oxidized on their methionine residues; and fRMsr, discovered more recently, which binds and reduces selectively free L-Met-R-O. It is now well established that the chemical mechanism of the reductase step passes through formation of a Sulfenic Acid intermediate. The oxidized catalytic cysteine can then be recycled by either Trx when a recycling cysteine is operative or a reductant like glutathione in the absence of recycling cysteine which is the case for 30% of the MsrBs. Recently, it was shown that a subclass of MsrAs with two recycling cysteines displays an oxidase activity. This reverse activity needs the accumulation of the Sulfenic Acid intermediate. The present review focuses on recent insights into the catalytic mechanism of action of the Msrs based on kinetic studies, theoretical chemistry investigations and new structural data. Major attention is placed on how the Sulfenic Acid intermediate can be formed and the oxidized catalytic cysteine returns back to its reduced form.
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the role of methionine sulfoxide reductase in redox signaling
Journal of Biological Chemistry, 2012Co-Authors: Guy BranlantAbstract:This letter concerns the article published recently by Lim et al. (1). The mechanism of methionine sulfoxide reductases begins with formation of a Sulfenic Acid on the catalytic Cys. Most of the methionine sulfoxide reductases (Msr) then form only one intradisulfide bond via the attack of a recycling Cys. The disulfide bond is then reduced by thioredoxin to regenerate the Msr. Kinetic experiments clearly demonstrated that the rate-limiting step is associated with the Trx-recycling process, whereas the rate of formation of the intradisulfide bond is limited by that of the Sulfenic Acid intermediate (2). Therefore, the Sulfenic Acid never accumulates in vivo whatever the subclass of MsrA/B. Recently, Levine and coworkers determined a pKapp of 7.2 for the catalytic Cys of the mouse MsrA (1). In fact, the pKapp can vary from 9.5 in Neisseria meningitidis (3), a value determined using 2-PDS as a probe, to 7.2 in mouse MsrA, indicating a catalytic Cys environment depending on the MsrA. The reaction of H2O2 can give two isomers of the Sulfenic Acid function, e.g. one with the OH oriented within the active site and strongly stabilized by a network of hydrogen bond interactions that will favor formation of disulfide bond (4) and the other one oriented in the opposite direction within the solvent, not stabilized, and thus whose population will shift to the first one. Therefore, a role of Msr, present in the three kingdoms, in redox signaling is excluded because the Sulfenic Acid never accumulates and its formation rate of 32 m−1 s−1 is low compared with 105–107 m−1 s−1 in peroxiredoxins (5).
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e coli methionine sulfoxide reductase with a truncated n terminus or c terminus or both retains the ability to reduce methionine sulfoxide
Protein Science, 2008Co-Authors: Sandrine Boschimuller, Said Azza, Guy BranlantAbstract:The monomeric peptide methionine sulfoxide reductase (MsrA) catalyzes the irreversible thioredoxin-dependent reduction of methionine sulfoxide. The crystal structure of MsrAs from Escherichia coli and Bos taurus can be described as a central core of about 140 amino Acids that contains the active site. The core is wrapped by two long N- and C-terminal extended chains. The catalytic mechanism of the E. coli enzyme has been recently postulated to take place through formation of a Sulfenic Acid intermediate, followed by reduction of the intermediate via intrathiol-disulfide exchanges and thioredoxin oxidation. In the present work, truncated MsrAs at the N- or C-terminal end or at both were produced as folded entities. All forms are able to reduce methionine sulfoxide in the presence of dithiothreitol. However, only the N-terminal truncated form, which possesses the two cysteines located at the C-terminus, reduces the Sulfenic Acid intermediate in a thioredoxin-dependent manner. The wild type displays a ping-pong mechanism with either thioredoxin or dithiothreitol as reductant. Kinetic saturation is only observed with thioredoxin with a low KM value of 10 μM. Thus, thioredoxin is likely the reductant in vivo. Truncations do not significantly modify the kinetic properties, except for the double truncated form, which displays a 17-fold decrease in kcat/KMetSO. Alternative mechanisms for Sulfenic Acid reduction are also presented based on analysis of available MsrA sequences.
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a structural analysis of the catalytic mechanism of methionine sulfoxide reductase a from neisseria meningitidis
Journal of Molecular Biology, 2008Co-Authors: Fanomezana M Ranaivoson, Sandrine Boschimuller, Guy Branlant, Mathias Antoine, Brice Kauffmann, Andre Aubry, Frederique FavierAbstract:The methionine sulfoxide reductases (Msrs) are thioredoxin-dependent oxidoreductases that catalyse the reduction of the sulfoxide function of the oxidized methionine residues. These enzymes have been shown to regulate the life span of a wide range of microbial and animal species and to play the role of physiological virulence determinant of some bacterial pathogens. Two structurally unrelated classes of Msrs exist, MsrA and MsrB, with opposite stereoselectivity towards the R and S isomers of the sulfoxide function, respectively. Both Msrs share a similar three-step chemical mechanism including (1) the formation of a Sulfenic Acid intermediate on the catalytic Cys with the concomitant release of the product-methionine, (2) the formation of an intramonomeric disulfide bridge between the catalytic and the regenerating Cys and (3) the reduction of the disulfide bridge by thioredoxin or its homologues. In this study, four structures of the MsrA domain of the PilB protein from Neisseria meningitidis, representative of four catalytic intermediates of the MsrA catalytic cycle, were determined by X-ray crystallography: the free reduced form, the Michaelis-like complex, the Sulfenic Acid intermediate and the disulfide oxidized forms. They reveal a conserved overall structure up to the formation of the Sulfenic Acid intermediate, while a large conformational switch is observed in the oxidized form. The results are discussed in relation to those proposed from enzymatic, NMR and theoretical chemistry studies. In particular, the substrate specificity and binding, the catalytic scenario of the reductase step and the relevance and role of the large conformational change observed in the oxidized form are discussed.
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kinetic characterization of the chemical steps involved in the catalytic mechanism of methionine sulfoxide reductase a from neisseria meningitidis
Journal of Biological Chemistry, 2003Co-Authors: Mathias Antoine, Sandrine Boschimuller, Guy BranlantAbstract:Abstract Oxidation of methionine into methionine sulfoxide is associated with many pathologies and is described to exert regulatory effects on protein functions. Two classes of methionine sulfoxide reductases, called MsrA and MsrB, have been described to reduce the S and the R isomers of the sulfoxide of methionine sulfoxide back to methionine, respectively. Although MsrAs and MsrBs display quite different x-ray structures, they share a similar, new catalytic mechanism that proceeds via the Sulfenic Acid chemistry and that includes at least three chemical steps with 1) the formation of a Sulfenic Acid intermediate and the concomitant release of methionine; 2) the formation of an intra-disulfide bond; and 3) the reduction of the disulfide bond by thioredoxin. In the present study, it is shown that for the Neisseria meningitidis MsrA, 1) the rate-limiting step is associated with the reduction of the Cys-51/Cys-198 disulfide MsrA bond by thioredoxin; 2) the formation of the Sulfenic Acid intermediate is very efficient, thus suggesting catalytic assistance via amino Acids of the active site; 3) the rate-determining step in the formation of the Cys-51/Cys-198 disulfide bond is that leading to the formation of the Sulfenic intermediate on Cys-51; and 4) the apparent affinity constant for methionine sulfoxide in the methionine sulfoxide reductase step is 80-fold higher than the Km value determined under steady-state conditions.
Fillmore Freeman - One of the best experts on this subject based on the ideXlab platform.
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mechanism of the cysteine Sulfenic Acid o sulfenylation of 1 3 cyclohexanedione
Chemical Communications, 2014Co-Authors: Fillmore FreemanAbstract:The density functionals B3LYP, B3PW91, M062X, and CAM-B3LYP with the 6-311+G(d,p) basis set predict the cysteine Sulfenic Acid O-sulfenylation of the s-cis-ketoenol tautomer of 1,3-cyclohexanedione proceeds through a cyclic 14-membered transition state structure containing three water molecules.
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conformers of cysteine and cysteine Sulfenic Acid and mechanisms of the reaction of cysteine Sulfenic Acid with 5 5 dimethyl 1 3 cyclohexanedione dimedone
Journal of Physical Chemistry B, 2013Co-Authors: Fillmore Freeman, Ifeoluwa Taiwo Adesina, Julie Le La, Amelia Ann PoplawskiAbstract:Equilibrium and molecular structures, relative energies of conformers of gaseous cysteine (Cys, C, Cys-SH) and gaseous cysteine Sulfenic Acid (Cys-SOH), and the mechanisms of the reaction of Cys-SOH with 3-hydroxy-5,5-dimethyl-2-cyclohexen-1-one, the enol tautomer of 5,5-dimethyl-1,3-cyclohexadione (dimedone), have been studied using BD(T), CCSD(T), and QCISD(T) with the cc-pVTZ basis set and using MP2 and the density functionals B3LYP, B3PW91, PBE1PBE, PBEh1PBE, M062X, CAM-B3LYP, and WB97XD with the 6-311+G(d,p) basis set. The structures of the six lowest energy conformers of gaseous Cys-SOH are compared with the six lowest energy conformers of gaseous cysteine (Cys-SH). The relative stability of the six lowest energy conformers of Cys-SH and Cys-SOH are influenced by the interplay among many factors including dispersive effects, electronic effects, electrostatic interactions, hydrogen bonds, inductive effects, and noncovalent interactions. The mechanism of the addition of the lowest energy conformer of ...
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Conformers of Cysteine and Cysteine Sulfenic Acid and Mechanisms of the Reaction of Cysteine Sulfenic Acid with 5,5-Dimethyl-1,3-cyclohexanedione (Dimedone)
2013Co-Authors: Fillmore Freeman, Ifeoluwa Taiwo Adesina, Joseph Yonghun Lee, Amelia Ann PoplawskiAbstract:Equilibrium and molecular structures, relative energies of conformers of gaseous cysteine (Cys, C, Cys-SH) and gaseous cysteine Sulfenic Acid (Cys-SOH), and the mechanisms of the reaction of Cys-SOH with 3-hydroxy-5,5-dimethyl-2-cyclohexen-1-one, the enol tautomer of 5,5-dimethyl-1,3-cyclohexadione (dimedone), have been studied using BD(T), CCSD(T), and QCISD(T) with the cc-pVTZ basis set and using MP2 and the density functionals B3LYP, B3PW91, PBE1PBE, PBEh1PBE, M062X, CAM-B3LYP, and WB97XD with the 6-311+G(d,p) basis set. The structures of the six lowest energy conformers of gaseous Cys-SOH are compared with the six lowest energy conformers of gaseous cysteine (Cys-SH). The relative stability of the six lowest energy conformers of Cys-SH and Cys-SOH are influenced by the interplay among many factors including dispersive effects, electronic effects, electrostatic interactions, hydrogen bonds, inductive effects, and noncovalent interactions. The mechanism of the addition of the lowest energy conformer of cysteine Sulfenic Acid (Cys-SOH) to dimedone may proceed through a six-membered ring transition state structure and through cyclic hydrogen-bonded transition state structures with one water molecule (8-membered ring), with two water molecules (10-membered ring), and with three water molecules (12-membered ring). Inclusion of one and two water molecules in the transition state structures lowers the activation barrier, whereas inclusion of a third water molecule raises the activation barrier
