Mechanism Of Methionine Sulfoxide Reductase Research Articles

A Molecular Dynamics and Quantum Mechanics/Molecular Mechanics Study of the Catalytic Reductase Mechanism of Methionine Sulfoxide Reductase A: Formation and Reduction of a Sulfenic Acid

The catalytic mechanism of MsrA in Mycobacterium tuberculosis, in which S-methionine sulfoxide (Met-O) is reduced to methionine (Met), has been investigated using docking, molecular dynamics (MD) simulations, and ONIOM (quantum mechanics/molecular mechanics) methods. In addition, the roles of specific active site residues, including an aspartyl (Asp87) near the recycling cysteine, tyrosyls (Tyr44 and Tyr92), and glutamyl (Glu52), have been examined, as well as the general effects of the protein and active site on the nature and properties of mechanistic intermediates. The mechanism is initiated by the transfer of a proton from the catalytic cysteine's thiol (Cys13SH) via a bridging water to the R group carboxylate of Glu52. The now anionic sulfur of Cys13 nucleophilically attacks the substrate's sulfur with concomitant transfer of a proton from Glu52 to the sulfoxide oxygen, generating a sulfurane. The active site enhances the proton affinity of the sulfurane oxygen, which can readily accept a proton from the phenolic hydroxyls of Tyr44 or Tyr92 to give a sulfonium cation. Subsequently, Asp87 and the recycling cysteine (Cys154) can facilitate nucleophilic attack of a solvent water at the Cys13S center of the sulfonium to give a sulfenic acid (Cys13SOH) and Met. For the subsequent reduction of Cys13SOH with intramolecular disulfide bond formation, Asp87 can help facilitate nucleophilic attack of Cys154S at the sulfur of Cys13SOH by deprotonating its thiol. This reduction is found likely to occur readily upon suitable positioning of the active site hydrogen bond network and the sulfur centers of both Cys13 and Cys154. The calculated rate-limiting barrier is in good agreement with experiment.

Reply to the “Comment on 'A Sulfonium Cation Intermediate in the Mechanism of Methionine Sulfoxide Reductase B: A DFT Study’”

ADVERTISEMENT RETURN TO ISSUEPREVCommentNEXTReply to the “Comment on 'A Sulfonium Cation Intermediate in the Mechanism of Methionine Sulfoxide Reductase B: A DFT Study’”Jesse J. Robinet, Hisham. M. Dokainish, David J. Paterson, and James W. Gauld*View Author Information Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, CanadaE-mail: [email protected]Cite this: J. Phys. Chem. B 2011, 115, 36, 10776–10777Publication Date (Web):July 25, 2011Publication History Received20 July 2011Published online24 August 2011Published inissue 15 September 2011https://doi.org/10.1021/jp2069418Copyright © 2011 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views309Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (632 KB) Get e-AlertsSUBJECTS:Cations,Crystal structure,Monomers,Peptides and proteins,X-rays Get e-Alerts

Comment on “A Sulfonium Cation Intermediate in the Mechanism of Methionine Sulfoxide Reductase B: A DFT Study”

ADVERTISEMENT RETURN TO ISSUEPREVCommentNEXTComment on “A Sulfonium Cation Intermediate in the Mechanism of Methionine Sulfoxide Reductase B: A DFT Study”Fabrice Neiers, Sandrine Boschi-Muller, and Guy Branlant*View Author Information AREMS, UMR CNRS-UHP 7214, Nancy Université, Bâtiment Biopôle, Faculté de Médecine, BP 184, 9 avenue de la Forêt de Haye, 54506 Vandoeuvre-les-Nancy, FranceE-mail: [email protected]Cite this: J. Phys. Chem. B 2011, 115, 36, 10775Publication Date (Web):July 25, 2011Publication History Received8 July 2011Revised22 July 2011Published online24 August 2011Published inissue 15 September 2011https://doi.org/10.1021/jp2064744Copyright © 2011 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views362Altmetric-Citations2LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (613 KB) Get e-AlertsSUBJECTS:Cations,Genetics,Monomers,Peptides and proteins,Recycling Get e-Alerts

A Sulfonium Cation Intermediate in the Mechanism of Methionine Sulfoxide Reductase B: A DFT Study

The hybrid density functional theory method B3LYP in combination with three systematically larger active site models has been used to investigate the substrate binding and catalytic mechanism by which Neisseria gonorrhoeae methionine sulfoxide reductase B (MsrB) reduces methionine-R-sulfoxide (Met-R-SO) to methionine. The first step in the overall mechanism is nucleophilic attack of an active site thiolate at the sulfur of Met-R-SO to form an enzyme-substrate sulfurane. This occurs with concomitant proton transfer from an active site histidine (His480) residue to the substrates oxygen center. The barrier for this step, calculated using our largest most complete active site model, is 17.2 kJ mol(-1). A subsequent conformational rearrangement and intramolecular -OH transfer to form an enzyme-derived sulfenic acid ((Cys495)S-OH) is not enzymatically feasible. Instead, transfer of a second proton from a second histidyl active site residue (His477) to the sulfurane's oxygen center to give water and a sulfonium cation intermediate is found to be greatly preferred, occurring with a quite low barrier of just 1.2 kJ mol(-1). Formation of the final product complex in which an intraprotein disulfide bond is formed with generation of methionine preferably occurs in one step via nucleophilic attack of the sulfur of a second enzyme thiolate ((Cys440)S(-)) at the S(Cys495) center of the sulfonium intermediate with a barrier of 23.8 kJ mol(-1). An alternate pathway for formation of the products via a sulfenic acid intermediate involves enzymatically feasible, but higher energy barriers. The role and impact of hydrogen bonding and active site residues on the properties and stability of substrate and mechanism intermediates and the affects of mutating His477 are also examined and discussed.

Investigation of the Ligand-binding Mechanism of Methionine Sulfoxide Reductase A of E.coli

The rise of free oxygen in the atmosphere over 2.5 billion years ago made it possible for large land-based plants and animals to thrive. But oxygen, and the energy it provides, comes at great cost. Aerobic metabolism generates highly reactive intermediates and by-products in the form of hydroxyl radicals, superoxide anions and hydrogen peroxide. Within the cell, these reactive oxygen species attack biological macromolecules, producing covalent modifications that can affect both function and structure. One amino acid residue in proteins that is particularly sensitive to oxidation is the sulfur-containing side chain of methionine. Fortunately, methionine oxidation can be reversed by the actions of peptide methionine sulfoxide reductase (MsrA), which reduces methionine sulfoxide back to methionine and restores function to damaged proteins. We have used multiple spectroscopic techniques to investigate the mechanism by which MsrA recognizes and binds to a wide range of oxidized substrates in need of repair. Substrates studied include proteins, peptides and the non-steroidal anti-inflammatory drug Sulindac. Competition experiments with the fluorescent reporter ANS suggest the existence of weak, but specific, hydrophobic interactions between MsrA and unstructured and/or hydrophobic ligands.

A Structural Analysis of the Catalytic Mechanism of Methionine Sulfoxide Reductase A from Neisseria meningitidis

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.

Kinetic Characterization of the Chemical Steps Involved in the Catalytic Mechanism of Methionine Sulfoxide Reductase A from Neisseria meningitidis

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.