Structural and Biochemical Characterization of Free Methionine-R-sulfoxide Reductase from Neisseria meningitidis
2010; Elsevier BV; Volume: 285; Issue: 32 Linguagem: Inglês
10.1074/jbc.m110.134528
ISSN1083-351X
AutoresArnaud Gruez, Marouane Libiad, Sandrine Boschi‐Müller, Guy Branlant,
Tópico(s)Sulfur Compounds in Biology
ResumoA new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys118 is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain. A new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys118 is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain. IntroductionMethionine (Met) 4The abbreviations used are: MetmethionineMet-Omethionine sulfoxide (dl-Met-R,S-O)MES2-(N-morpholino)ethanesulfonic acid2-PDS2,2′-dithiodipyridineDTNB5,5′-dithiobis(2-nitro)benzoatefRMsrfree methionine-R-sulfoxide reductaseDTTdithiothreitolMPD2-methylpentane-2,4-diolr.m.s.root mean square. is one of the two amino acids in proteins that are the most susceptible to oxidation by reactive oxygen species, forming Met-O (1Schöneich C. Biochim. Biophys. Acta. 2005; 1703: 111-119Crossref PubMed Scopus (300) Google Scholar). Prior to 2007, two families of methionine-sulfoxide reductase (Msr) enzymes, called MsrA and -B were known to reduce Met-O back into Met (2Boschi-Muller S. Olry A. Antoine M. Branlant G. Biochim. Biophys. Acta. 2005; 1703: 231-238Crossref PubMed Scopus (154) Google Scholar, 3Moskovitz J. Biochim. Biophys. Acta. 2005; 1703: 213-219Crossref PubMed Scopus (249) Google Scholar). The MsrA family reduces the S isomer at the sulfoxide function, whereas MsrB is specific for the R isomer. Both Msrs, which reveal distinct unrelated folds, were shown to reduce more efficiently a Met-O within a polypeptide chain. Therefore, Msrs are repair enzymes that play important roles in the protection of cells against oxidative stress (3Moskovitz J. Biochim. Biophys. Acta. 2005; 1703: 213-219Crossref PubMed Scopus (249) Google Scholar, 4Stadtman E.R. Van Remmen H. Richardson A. Wehr N.B. Levine R.L. Biochim. Biophys. Acta. 2005; 1703: 135-140Crossref PubMed Scopus (318) Google Scholar, 5Weissbach H. Resnick L. Brot N. Biochim. Biophys. Acta. 2005; 1703: 203-212Crossref PubMed Scopus (239) Google Scholar).The catalytic mechanism of MsrA and MsrB is now well documented. For most MsrAs and MsrBs (i.e. those possessing a recycling Cys), the mechanism comprises three steps: a reductase step that leads to formation of a sulfenic acid intermediate on a catalytic Cys (6Boschi-Muller S. Azza S. Sanglier-Cianferani S. Talfournier F. Van Dorsselear A. Branlant G. J. Biol. Chem. 2000; 275: 35908-35913Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), a second step in which a disulfide bond is formed between the catalytic Cys and a recycling Cys, and finally, a step in which the intradisulfide bond is reduced by thioredoxin (Trx) or a Trx-like protein (7Boschi-Muller S. Gand A. Branlant G. Arch. Biochem. Biophys. 2008; 474: 266-273Crossref PubMed Scopus (147) Google Scholar). A catalytic residue (i.e. Glu94 in MsrA and His103 in MsrB) was characterized, one of whose major roles is to protonate the oxygen of the sulfoxide function (8Antoine M. Gand A. Boschi-Muller S. Branlant G. J. Biol. Chem. 2006; 281: 39062-39070Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 9Neiers F. Sonkaria S. Olry A. Boschi-Muller S. Branlant G. J. Biol. Chem. 2007; 282: 32397-32405Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In all of the MsrAs and MsrBs studied to date, the reduction of the disulfide bond is rate-limiting, whereas the formation of the sulfenic acid intermediate is rate-determining in the process leading to intradisulfide bond formation. In other words, in the absence of Trx, only the intradisulfide intermediate accumulates.Recently, a third family of Msr, named fRMsr, was discovered (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar). The fRMsrs exhibit a GAF-type fold. GAF domains are one of the largest and most widespread domains found in all kingdoms of life. They are dimeric and are generally arranged in tandem in modular proteins to provide a large variety of regulation functions. However, most of the functions of GAF domains remain to be studied in detail. The fRMsr is the first case of a GAF domain that bears a catalytic activity. The fRMsrs are present in eubacteria and unicellular eukaryotes (11Le D.T. Lee B.C. Marino S.M. Zhang Y. Fomenko D.E. Kaya A. Hacioglu E. Kwak G.H. Koc A. Kim H.Y. Gladyshev V.N. J. Biol. Chem. 2009; 284: 4354-4364Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The family displays a methionine-sulfoxide reductase activity, reducing selectively free Met-O with an R configuration at the sulfoxide (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar). Such a function led Lowther and co-workers (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar) to propose that Met-R-O can represent a signaling molecule in response to oxidative stress.Only the crystal structures of fRMsr from Escherichia coli and Saccharomyces cerevisiae have been solved to date, without substrate (12Badger J. Sauder J.M. Adams J.M. Antonysamy S. Bain K. Bergseid M.G. Buchanan S.G. Buchanan M.D. Batiyenko Y. Christopher J.A. Emtage S. Eroshkina A. Feil I. Furlong E.B. Gajiwala K.S. Gao X. He D. Hendle J. Huber A. Hoda K. Kearins P. Kissinger C. Laubert B. Lewis H.A. Lin J. Loomis K. Lorimer D. Louie G. Maletic M. Marsh C.D. Miller I. Molinari J. Muller-Dieckmann H.J. Newman J.M. Noland B.W. Pagarigan B. Park F. Peat T.S. Post K.W. Radojicic S. Ramos A. Romero R. Rutter M.E. Sanderson W.E. Schwinn K.D. Tresser J. Winhoven J. Wright T.A. Wu L. Xu J. Harris T.J. Proteins. 2005; 60: 787-796Crossref PubMed Scopus (207) Google Scholar, 13Ho Y.S. Burden L.M. Hurley J.H. EMBO J. 2000; 19: 5288-5299Crossref PubMed Scopus (254) Google Scholar). Both structures are described as being composed of six β-strands, four α-helices, and two prominent loops, loop 1 and loop 2, located on the surface of the protein between β2 and β3 and between β4 and β5, respectively. Formation of a disulfide bond between Cys84 of loop 1 and Cys118 of loop 2 is assumed to close off the cavity in which a molecule of MES, which derives from the crystallization buffer, is bound in the E. coli enzyme (Protein Data Bank entry 1vhm). The sulfonic acid moiety is closed to another Cys94, which is located at the N terminus of an α-helix. The rest of the cavity is lined by several invariant residues. From the inspection of the crystal structure of E. coli fRMr in complex with a MES buffer molecule and by carrying out computational docking of Met-R-O into the cavity of the S. cerevisiae enzyme, a reductase mechanism was proposed in which the Cys94 located at one of the sides of the cavity is assumed to be the catalytic Cys (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar). The sulfenic acid postulated to be formed on Cys94 is then attacked by Cys118, forming a disulfide bond. A disulfide bond exchange then occurs with Cys84, which finally leads to the Cys118–Cys84 disulfide intermediate.In the present study, we report the crystal structure of the fRMsr from Neisseria meningitidis in complex with l-Met-R-O, solved at 1.25 Å resolution. By combining genomic analyses, structural information, and biochemical and kinetic data from the wild type and mutants in which Cys was substituted by Ala, Cys118 is shown to be the catalytic residue on which the sulfenic acid is formed. 5The numbering of fRMsr amino acid residues is based on the numbering of the E. coli fRMsr sequence according to sequence alignment presented in Fig. 1. All of the molecular and structural factors involved in the stereoselective binding of the l-Met-R-O stereoisomer are identified from the crystal structure and explain why neither a Met-S-O, a Met-R-O in a peptide or a protein context, nor a DMSO molecule are substrates. Scenarios for both substrate binding and catalysis are proposed for the reductase step.A disulfide bond between Cys84 and Cys118, present in all of the fRMsr crystal structures described so far, including the N. meningitidis fRMsr, stabilized the loop 2 as a flap that is an integral part of the active site. Formation of this disulfide bond seems to be a prerequisite to obtaining crystals. However, it is the free reduced form that is catalytically relevant to the reductase step. Based on the content of 1) the Cys in wild type and Cys-to-Ala mutants before and after Met-O reduction and 2) the Cys titrated under native conditions, a scenario for the Trx-recycling process is proposed in which a disulfide Cys118–Cys84 bond is formed first. All of the results are compared with those obtained for MsrA and MsrB and discussed in terms of the evolution of function of the GAF domain.DISCUSSIONA scenario of the catalytic mechanism, similar to that of MsrA and MsrB, can be proposed for fRMsr based on the genomic, biochemical, kinetic, and structural analyses carried out in the present study. First, a sulfenic acid intermediate is formed on the catalytic Cys118. This conclusion is based on four types of data. First, only the substitution of Cys118 by Ala leads to a complete loss of activity, whereas the mutants in which the other three Cys are individually substituted with Ala, in particular the C84A and C94A mutants, remain active under steady-state conditions. These latter results contradict those described for fRMsr from S. cerevisiae, for which substitution of Cys94 and Cys84 by Ser led to a complete loss of activity (11Le D.T. Lee B.C. Marino S.M. Zhang Y. Fomenko D.E. Kaya A. Hacioglu E. Kwak G.H. Koc A. Kim H.Y. Gladyshev V.N. J. Biol. Chem. 2009; 284: 4354-4364Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Second, the C84A/C94A/C136A mutant is active when tested under steady-state conditions with the Trx-regenerating system. Third, in the absence of Trx, several species with the C84A/C94A/C136A mutant are efficiently formed, including a covalent intermediate, which migrates as a dimer of fRMsr on denaturating SDS-polyacrylamide gel. Its mass corresponds to a dimer linked by a thiosulfinate bond (data not shown). Because formation of a thiosulfinate bond can only proceed via condensation of two sulfenic acids, this result demonstrates that reduction of Met-O passes through the sulfenic acid chemistry. Fourth, inspection of the alignment of the 358 protein sequences of putative fRMsrs deduced from the DNA sequences available to date shows that Cys118 is invariant, whereas Cys84 and Cys94 are 94.5 and 98.6% conserved (see the alignment shown in supplemental Fig. S1). Taken together, these data definitively prove that 1) Cys118 is the catalytic Cys and 2) the catalytic mechanism involves the formation of a sulfenic acid intermediate on Cys118. Therefore, these results exclude Cys94 as the catalytic Cys as postulated from molecular modeling on the S. cerevisiae fRMsr (11Le D.T. Lee B.C. Marino S.M. Zhang Y. Fomenko D.E. Kaya A. Hacioglu E. Kwak G.H. Koc A. Kim H.Y. Gladyshev V.N. J. Biol. Chem. 2009; 284: 4354-4364Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and from inspection of the E. coli fRMsr in complex with a MES molecule trapped from the crystallization buffer (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar).The crystal structure of the N. meningitidis fRMsr in complex with l-Met-R-O illuminates not only the catalysis of the reductase step but also how fRMsr binds Met-O with a strong preference for the l-Met-R-O isomer. As already indicated, the catalysis implies a proton transfer from the catalytic Cys to the oxygen of the sulfoxide. Such a function in fRMsr is probably played by the invariant Asp143, which is located on strand β6, possibly via Wat125. In accord with this assumption, a D143A fRMsr shows a 350-fold decrease in activity (data not shown), similar to that observed for E94A MsrA and H103A MsrB, when determined under similar steady-state conditions (8Antoine M. Gand A. Boschi-Muller S. Branlant G. J. Biol. Chem. 2006; 281: 39062-39070Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 9Neiers F. Sonkaria S. Olry A. Boschi-Muller S. Branlant G. J. Biol. Chem. 2007; 282: 32397-32405Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The crystal structure also shows that the oxygen of the sulfoxide is located in a hydrophilic cavity, where it strongly interacts not only with Asp143 but also with Wat99, which is itself hydrogen-bonded with the main chain nitrogen of Ser119. Wat99 recalls the water molecule observed in MsrB that is stabilized through a hydrogen-bonding network with the side chains of Thr26, His100, and Asn119 (21Ranaivoson F.M. Neiers F. Kauffmann B. Boschi-Muller S. Branlant G. Favier F. J. Mol. Biol. 2009; 394: 83-93Crossref PubMed Scopus (35) Google Scholar). In the scenario, a sulfurane intermediate/transition state is formed that rearranges into a sulfenic acid intermediate (see Scheme 1A). Therefore, Asp143 and Wat99 should interact not only with the oxygen of the sulfoxide and its polarized form but probably also with the OH of the sulfurane transition state and directly or indirectly with the OH of the sulfenic acid, as observed in the crystal structure of the MsrA sulfenic acid intermediate (22Ranaivoson F.M. Antoine M. Kauffmann B. Boschi-Muller S. Aubry A. Branlant G. Favier F. J. Mol. Biol. 2008; 377: 268-280Crossref PubMed Scopus (30) Google Scholar). To form a sulfurane, the Cys118 has to be under the thiolate form to attack the sulfur of the sulfoxide, the geometry of which is tetrahedral. Clearly, because the pKapp of Cys118 in the free enzyme is 8.5, binding of the substrate should contribute to the decrease of the pKapp of Cys118. This situation is similar to that observed in MsrA and MsrB. As shown by the crystal structure, the sulfur is closer to Cys118 than to Cys84, and consequently it is in a better position to form a covalent bond with Cys118. Moreover, if Cys118 is reduced, the thiolate can adopt a favorable orientation that permits formation of the sulfurane with the SH and the OH groups in apical positions.As shown previously (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar), fRMsrs only reduce the R isomer at the sulfoxide function. This stereochemical preference is confirmed by the structure of N. meningitidis fRMsr in complex with l-Met-R-O. The methyl group of the sulfoxide points perpendicularly toward the indole ring of the invariant Trp62, whose conformation is strongly stabilized within the active site. Therefore, the indole ring is the major factor involved in the binding and stabilization of the ɛ-methyl group, as already observed in MsrA and MsrB, where at minimum a Trp residue is involved. The β- and δ-methylene groups are stabilized by van der Waals interactions with the side chains of the invariant Ile87, Ile116, and Tyr66. Together, these structural data explain why only the R isomer at the sulfoxide function is recognized by the fRMsrs.As shown in the present study from kinetic studies, fRMsr is highly selective for the l-isomer at the Cα carbon. Moreover, as already described, it is the free form of Met-O that is active. Inspection of the crystal structure of fRMsr shows the following. 1) The negatively charge of the carboxylate function, which is completely delocalized, is buried and points toward the positive helix dipole at the N-terminal end of the helix α3. In addition, the backbone amides of Val93, Cys94, and Ile116 are within hydrogen distance and form an oxyanion hole. A similar situation is observed with the negative charge of the sulfonic acid function of the MES molecule in the E. coli fRMsr (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar), the negatively charged 3′,5′-cyclic phosphate of cGMP and cAMP in the phosphodiesterase 5A (23Heikaus C.C. Stout J.R. Sekharan M.R. Eakin C.M. Rajagopal P. Brzovic P.S. Beavo J.A. Klevit R.E. J. Biol. Chem. 2008; 283: 22749-22759Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and adenylate cyclase from Anabaena (24Martinez S.E. Bruder S. Schultz A. Zheng N. Schultz J.E. Beavo J.A. Linder J.U. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 3082-3087Crossref PubMed Scopus (75) Google Scholar), respectively. 2) The NH3+ group is strongly stabilized by a hydrogen-bonding network involving (i) the side chain of the invariant Glu125, which itself is stabilized by interactions with the invariant Asp141 and Asp143, and (ii) the main chain oxygen of Ile116. All of these stabilizing interactions 1) favor binding of the l-Met-O epimer, 2) prevent the binding of a Met-R-O within a peptide or protein context due to steric hindrance, and 3) are responsible for the absence of activity with DMSO because of the absence of COO− and NH3+ groups in DMSO.As clearly revealed by the crystal structure, the l-Met-R-O is strongly bound in a small cavity that is capped by the flap (residues 107–124, including Cys118), which thus protects the active site from the solvent. In the structure, the Cys84–Cys118 disulfide bond should restrict the conformational freedom of the flap, which is situated between strands β4 and β5, respectively. Because Cys118 has to be free to play its catalytic role, the disulfide-bonded Cys118–Cys84 form is not catalytically relevant for the reductase step. Nevertheless, the positioning of Cys118 is compatible with a nucleophilic attack of the sulfoxide function of the substrate. The disulfide bond Cys118–Cys84 is, however, essential to obtaining crystals because the addition of DTT dissolves them (data not shown) and thus prevents obtention of crystals of the reduced form.Formation of a sulfenic acid intermediate on Cys118 implies a recycling process in the wild type that can involve formation of an intradisulfide bond, which is then reduced by Trx. Two Cys, Cys94 or Cys84, could be implicated. All of the fRMsr mutants in which either Cys84 or Cys94 was substituted by Ala were active. In agreement with these results is the fact that 5.6 and 1.4% of the putative sequences of fRMsrs only contain the signatures Cys118/Cys94 and Cys118/Cys84, respectively (see the alignment shown in supplemental Fig. S1). When Cys118 and Cys94 were individually mutated to Ala in the wild type from N. meningitidis, a stoichiometry of 1 mol of Met/subunit was observed in the absence of Trx with formation of a disulfide bond between Cys118 and Cys94 or between Cys118 and Cys84, depending on the mutant considered. Such results are based on the difference in thiol content observed before and after the addition of Met-O. For the wild type and the C136A fRMsr, a stoichiometry of 1 mol of Met/subunit was also observed with formation of a disulfide bond, the nature of which remains to be determined. Because 1) the disulfide bond in the crystal is between Cys118 and Cys84 when Met-O is bound, 2) Cys94 is located at the N terminus of helix α3 on one of the two sides of the active site far away from Cys118 and Cys84 (i.e. ∼8 Å), and 3) Cys118 is located within the flap, it is probable that the first disulfide formed for recycling the activity of the fRMsr from N. meningitidis is Cys118–Cys84 (see Scheme 1B). This is in accord with the DTNB titration results obtained under native conditions, which showed that Cys118 and Cys84 are titrated by DTNB and formed a disulfide bond in the wild type and C136A/C94A mutant. In fact, one of the two Cys residues, probably Cys118 that is located on the flexible flap forms an adduct with the TNB− moiety, which, by an intramolecular attack of Cys84, leads to formation of the Cys84–Cys118 disulfide bond. When one of these two Cys residues, either Cys84 or Cys118, is substituted by Ala, Cys94 is now titrated together with either Cys118 or Cys84, respectively, and thus is accessible. But no disulfide bond between Cys94 and either Cys118 or Cys84 is formed, in contrast to that observed between Cys118 and Cys84. Clearly, formation of the Cys84–Cys118 disulfide bond in the wild type prevents accessibility of the DTNB to Cys94, whereas in the absence of the Cys84–Cys118 disulfide bond, as is the case for the C118A or C84A mutant, Cys94 becomes accessible to DTNB.The fact that only one Met molecule was formed in the wild type and in the C136A mutant requires explanation. Either Cys118 remains engaged in a disulfide bond with Cys84, it participates in a new disulfide bond with Cys94, or it is free. The last hypothesis, although less probable, implies formation of a Cys84–Cys94 disulfide bond and an active site that is no longer catalytically competent to reduce another Met-O molecule. In other words, formation of the Cys84–Cys94 disulfide bond would perturb the conformation of the active site, which in return would prevent efficient binding of l-Met-R-O. In any case, the behavior of the wild-type fRMsr is clearly different from that of the MsrA from E. coli (6Boschi-Muller S. Azza S. Sanglier-Cianferani S. Talfournier F. Van Dorsselear A. Branlant G. J. Biol. Chem. 2000; 275: 35908-35913Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Indeed, the E. coli MsrA, which also possesses three Cys residues, forms two successive disulfide bonds, first between the catalytic Cys51 and the recycling Cys198 and then between Cys198 and Cys206. In that case, formation of the second disulfide bond renders the catalytic Cys51 free and renders the active site competent to reduce a second molecule of Met-O.The fact that oxidized disulfide forms of fRMsr are formed (i.e. containing a Cys118–Cys84 or Cys118–Cys94 bond) or would be formed in the absence of reductant (i.e. Cys84–Cys94) does not mean that all of these oxidized forms are catalytically competent under steady-state conditions (i.e. in the presence of Trx). For instance, in the 5.6% of putative fRMsrs where only Cys94 is present, a disulfide bond probably forms between Cys118 and Cys94 in the absence of reductant, as shown for the C84A/C136A fRMsr from N. meningitidis. However, inspection of the crystal structure shows, as already pointed out, that Cys94 is situated at one of the sides of the active site far away from Cys118. Thus, Cys94 is not in a good position to form a Cys118–Cys94 bond efficiently unless the enzyme is highly flexible. At present, we do not know the rate at which this disulfide bond is formed in the C84A mutant and whether it is this form that is recognized and reduced by Trx.Like all GAF domains, fRMsr is dimeric. An obvious question is whether not only both subunits of fRMsr are active as shown in the absence of Trx but also express their activity with the same catalytic efficiency. Another question concerns the nature of the rate-limiting step. This has to be determined in order to validate the amino acids that play a role in the catalysis and in the substrate specificity in the reductase step.The fRMsr represents a unique case in which a GAF fold behaves as an enzyme and moreover as an independent folded unit not included in a modular larger protein. Like MsrA and MsrB, it catalyzes the reduction of Met-O and therefore constitutes the third protein fold to exhibit a Met-O reductase activity that involves the sulfenic acid chemistry. From a structural point of view, the active site of fRMsr is clearly different from those of MsrA and MsrB. All of the amino acids within the active site that underlie the l-Met-R-O reductase activity are not present in the other GAF domains. From an evolutionary point of view, this observation raises the question of how a GAF fold has evolved to acquire a broad range of different functions by binding various molecules, including cyclic nucleotides, heme, amino acids, or Met-O. IntroductionMethionine (Met) 4The abbreviations used are: MetmethionineMet-Omethionine sulfoxide (dl-Met-R,S-O)MES2-(N-morpholino)ethanesulfonic acid2-PDS2,2′-dithiodipyridineDTNB5,5′-dithiobis(2-nitro)benzoatefRMsrfree methionine-R-sulfoxide reductaseDTTdithiothreitolMPD2-methylpentane-2,4-diolr.m.s.root mean square. is one of the two amino acids in proteins that are the most susceptible to oxidation by reactive oxygen species, forming Met-O (1Schöneich C. Biochim. Biophys. Acta. 2005; 1703: 111-119Crossref PubMed Scopus (300) Google Scholar). Prior to 2007, two families of methionine-sulfoxide reductase (Msr) enzymes, called MsrA and -B were known to reduce Met-O back into Met (2Boschi-Muller S. Olry A. Antoine M. Branlant G. Biochim. Biophys. Acta. 2005; 1703: 231-238Crossref PubMed Scopus (154) Google Scholar, 3Moskovitz J. Biochim. Biophys. Acta. 2005; 1703: 213-219Crossref PubMed Scopus (249) Google Scholar). The MsrA family reduces the S isomer at the sulfoxide function, whereas MsrB is specific for the R isomer. Both Msrs, which reveal distinct unrelated folds, were shown to reduce more efficiently a Met-O within a polypeptide chain. Therefore, Msrs are repair enzymes that play important roles in the protection of cells against oxidative stress (3Moskovitz J. Biochim. Biophys. Acta. 2005; 1703: 213-219Crossref PubMed Scopus (249) Google Scholar, 4Stadtman E.R. Van Remmen H. Richardson A. Wehr N.B. Levine R.L. Biochim. Biophys. Acta. 2005; 1703: 135-140Crossref PubMed Scopus (318) Google Scholar, 5Weissbach H. Resnick L. Brot N. Biochim. Biophys. Acta. 2005; 1703: 203-212Crossref PubMed Scopus (239) Google Scholar).The catalytic mechanism of MsrA and MsrB is now well documented. For most MsrAs and MsrBs (i.e. those possessing a recycling Cys), the mechanism comprises three steps: a reductase step that leads to formation of a sulfenic acid intermediate on a catalytic Cys (6Boschi-Muller S. Azza S. Sanglier-Cianferani S. Talfournier F. Van Dorsselear A. Branlant G. J. Biol. Chem. 2000; 275: 35908-35913Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), a second step in which a disulfide bond is formed between the catalytic Cys and a recycling Cys, and finally, a step in which the intradisulfide bond is reduced by thioredoxin (Trx) or a Trx-like protein (7Boschi-Muller S. Gand A. Branlant G. Arch. Biochem. Biophys. 2008; 474: 266-273Crossref PubMed Scopus (147) Google Scholar). A catalytic residue (i.e. Glu94 in MsrA and His103 in MsrB) was characterized, one of whose major roles is to protonate the oxygen of the sulfoxide function (8Antoine M. Gand A. Boschi-Muller S. Branlant G. J. Biol. Chem. 2006; 281: 39062-39070Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 9Neiers F. Sonkaria S. Olry A. Boschi-Muller S. Branlant G. J. Biol. Chem. 2007; 282: 32397-32405Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In all of the MsrAs and MsrBs studied to date, the reduction of the disulfide bond is rate-limiting, whereas the formation of the sulfenic acid intermediate is rate-determining in the process leading to intradisulfide bond formation. In other words, in the absence of Trx, only the intradisulfide intermediate accumulates.Recently, a third family of Msr, named fRMsr, was discovered (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar). The fRMsrs exhibit a GAF-type fold. GAF domains are one of the largest and most widespread domains found in all kingdoms of life. They are dimeric and are generally arranged in tandem in modular proteins to provide a large variety of regulation functions. However, most of the functions of GAF domains remain to be studied in detail. The fRMsr is the first case of a GAF domain that bears a catalytic activity. The fRMsrs are present in eubacteria and unicellular eukaryotes (11Le D.T. Lee B.C. Marino S.M. Zhang Y. Fomenko D.E. Kaya A. Hacioglu E. Kwak G.H. Koc A. Kim H.Y. Gladyshev V.N. J. Biol. Chem. 2009; 284: 4354-4364Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The family displays a methionine-sulfoxide reductase activity, reducing selectively free Met-O with an R configuration at the sulfoxide (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar). Such a function led Lowther and co-workers (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar) to propose that Met-R-O can represent a signaling molecule in response to oxidative stress.Only the crystal structures of fRMsr from Escherichia coli and Saccharomyces cerevisiae have been solved to date, without substrate (12Badger J. Sauder J.M. Adams J.M. Antonysamy S. Bain K. Bergseid M.G. Buchanan S.G. Buchanan M.D. Batiyenko Y. Christopher J.A. Emtage S. Eroshkina A. Feil I. Furlong E.B. Gajiwala K.S. Gao X. He D. Hendle J. Huber A. Hoda K. Kearins P. Kissinger C. Laubert B. Lewis H.A. Lin J. Loomis K. Lorimer D. Louie G. Maletic M. Marsh C.D. Miller I. Molinari J. Muller-Dieckmann H.J. Newman J.M. Noland B.W. Pagarigan B. Park F. Peat T.S. Post K.W. Radojicic S. Ramos A. Romero R. Rutter M.E. Sanderson W.E. Schwinn K.D. Tresser J. Winhoven J. Wright T.A. Wu L. Xu J. Harris T.J. Proteins. 2005; 60: 787-796Crossref PubMed Scopus (207) Google Scholar, 13Ho Y.S. Burden L.M. Hurley J.H. EMBO J. 2000; 19: 5288-5299Crossref PubMed Scopus (254) Google Scholar). Both structures are described as being composed of six β-strands, four α-helices, and two prominent loops, loop 1 and loop 2, located on the surface of the protein between β2 and β3 and between β4 and β5, respectively. Formation of a disulfide bond between Cys84 of loop 1 and Cys118 of loop 2 is assumed to close off the cavity in which a molecule of MES, which derives from the crystallization buffer, is bound in the E. coli enzyme (Protein Data Bank entry 1vhm). The sulfonic acid moiety is closed to another Cys94, which is located at the N terminus of an α-helix. The rest of the cavity is lined by several invariant residues. From the inspection of the crystal structure of E. coli fRMr in complex with a MES buffer molecule and by carrying out computational docking of Met-R-O into the cavity of the S. cerevisiae enzyme, a reductase mechanism was proposed in which the Cys94 located at one of the sides of the cavity is assumed to be the catalytic Cys (10Lin Z. Johnson L.C. Weissbach H. Brot N. Lively M.O. Lowther W.T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9597-9602Crossref PubMed Scopus (112) Google Scholar). The sulfenic acid postulated to be formed on Cys94 is then attacked by Cys118, forming a disulfide bond. A disulfide bond exchange then occurs with Cys84, which finally leads to the Cys118–Cys84 disulfide intermediate.In the present study, we report the crystal structure of the fRMsr from Neisseria meningitidis in complex with l-Met-R-O, solved at 1.25 Å resolution. By combining genomic analyses, structural information, and biochemical and kinetic data from the wild type and mutants in which Cys was substituted by Ala, Cys118 is shown to be the catalytic residue on which the sulfenic acid is formed. 5The numbering of fRMsr amino acid residues is based on the numbering of the E. coli fRMsr sequence according to sequence alignment presented in Fig. 1. All of the molecular and structural factors involved in the stereoselective binding of the l-Met-R-O stereoisomer are identified from the crystal structure and explain why neither a Met-S-O, a Met-R-O in a peptide or a protein context, nor a DMSO molecule are substrates. Scenarios for both substrate binding and catalysis are proposed for the reductase step.A disulfide bond between Cys84 and Cys118, present in all of the fRMsr crystal structures described so far, including the N. meningitidis fRMsr, stabilized the loop 2 as a flap that is an integral part of the active site. Formation of this disulfide bond seems to be a prerequisite to obtaining crystals. However, it is the free reduced form that is catalytically relevant to the reductase step. Based on the content of 1) the Cys in wild type and Cys-to-Ala mutants before and after Met-O reduction and 2) the Cys titrated under native conditions, a scenario for the Trx-recycling process is proposed in which a disulfide Cys118–Cys84 bond is formed first. All of the results are compared with those obtained for MsrA and MsrB and discussed in terms of the evolution of function of the GAF domain.
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