Key points are not available for this paper at this time.
PILB has been described as being involved in the virulence of bacteria of Neisseria genus. The PILB protein is composed of three subdomains. In the present study, the central subdomain (PILB-MsrA), the C terminus subdomain (PILB-MsrB), and the fused subdomain (PILB-MsrA/MsrB) of N. meningitidis were produced as folded entities. The central subdomain shows a methionine sulfoxide reductase A (MsrA) activity, whereas PILB-MsrB displays a methionine sulfoxide reductase B (MsrB) activity. The catalytic mechanism of PILB-MsrB can be divided into two steps: 1) an attack of the Cys-494 on the sulfur atom of the sulfoxide substrate, leading to formation of a sulfenic acid intermediate and release of 1 mol of methionine/mol of enzyme and 2) a regeneration of Cys-494 via formation of an intradisulfide bond with Cys-439 followed by reduction with thioredoxin. The study also shows that 1) MsrA and MsrB display opposite stereoselectivities toward the sulfoxide function; 2) the active sites of both Msrs, particularly MsrB, are rather adapted for binding protein-bound MetSO more efficiently than free MetSO; 3) the carbon Cα is not a determining factor for efficient binding to both Msrs; and 4) the presence of the sulfoxide function is a prerequisite for binding to Msrs. The fact that the two Msrs exhibit opposite stereoselectivities argues for a structure of the active site of MsrBs different from that of MsrAs. This is further supported by the absence of sequence homology between the two Msrs in particular around the cysteine that is involved in formation of the sulfenic acid derivative. The fact that the catalytic mechanism takes place through formation of a sulfenic acid intermediate for both Msrs supports the idea that sulfenic acid chemistry is a general feature in the reduction of sulfoxides by thiols. PILB has been described as being involved in the virulence of bacteria of Neisseria genus. The PILB protein is composed of three subdomains. In the present study, the central subdomain (PILB-MsrA), the C terminus subdomain (PILB-MsrB), and the fused subdomain (PILB-MsrA/MsrB) of N. meningitidis were produced as folded entities. The central subdomain shows a methionine sulfoxide reductase A (MsrA) activity, whereas PILB-MsrB displays a methionine sulfoxide reductase B (MsrB) activity. The catalytic mechanism of PILB-MsrB can be divided into two steps: 1) an attack of the Cys-494 on the sulfur atom of the sulfoxide substrate, leading to formation of a sulfenic acid intermediate and release of 1 mol of methionine/mol of enzyme and 2) a regeneration of Cys-494 via formation of an intradisulfide bond with Cys-439 followed by reduction with thioredoxin. The study also shows that 1) MsrA and MsrB display opposite stereoselectivities toward the sulfoxide function; 2) the active sites of both Msrs, particularly MsrB, are rather adapted for binding protein-bound MetSO more efficiently than free MetSO; 3) the carbon Cα is not a determining factor for efficient binding to both Msrs; and 4) the presence of the sulfoxide function is a prerequisite for binding to Msrs. The fact that the two Msrs exhibit opposite stereoselectivities argues for a structure of the active site of MsrBs different from that of MsrAs. This is further supported by the absence of sequence homology between the two Msrs in particular around the cysteine that is involved in formation of the sulfenic acid derivative. The fact that the catalytic mechanism takes place through formation of a sulfenic acid intermediate for both Msrs supports the idea that sulfenic acid chemistry is a general feature in the reduction of sulfoxides by thiols. Peptide methionine sulfoxide reductase (MsrA) 1The abbreviations used are: MsrMsrA, and MsrB, methionine sulfoxide reductase, methionine sulfoxide reductase A, and methionine sulfoxide reductase B, respectivelydimedone5,5-dimethyl-1,3-cyclohexanedioneDTTdithiothreitolDTNB5,5′-dithiobis(2-nitro)benzoateMetSOmethionine sulfoxidePhtphthalylTNB−thionitrobenzoate (3-carboxy-4-nitrobenzenthiol)HPLChigh pressure liquid chromatography activity is described as being involved in the virulence of the pathogens Escherichia coli, Streptococcus pneumoniae, Erwinia chrysanthemi, Mycoplasma genitalium, and Neisseria gonorrhoeae (1.Wizemann T.M. Moskovitz J. Pearce B.J. Cundell D. Arvidson C.G. So M. Weissbach H. Brot N. Masure H.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7985-7990Crossref PubMed Scopus (117) Google Scholar, 2.El Hassouni M. Chambost J.P. Expert D. Van Gijsegem F. Barras F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 887-892Crossref PubMed Scopus (149) Google Scholar, 3.Dhandayuthapani S. Blaylock M.W. Bebear C.M. Rasmussen W.G. Baseman J.B. J. Bacteriol. 2001; 183: 5645-5650Crossref PubMed Scopus (93) Google Scholar, 4.Taha M.K. Dupuis B. Saurin W. So M. Marchal C. Mol. Microbiol. 1991; 5: 137-148Crossref PubMed Scopus (37) Google Scholar). Inspection of the alignment of the corresponding protein sequences shows that all possess in common a sequence that displays an MsrA activity. This MsrA activity has now been well characterized at the structural level (5.Tête-Favier F. Cobessi D. Boschi-Muller S. Azza S. Branlant G. Aubry A. Structure. 2000; 8: 1167-1178Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 6.Lowther W.T. Brot N. Weissbach H. Matthews B.W. Biochemistry. 2000; 39: 13307-13312Crossref PubMed Scopus (125) Google Scholar) and the enzymatic level (7.Boschi-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 (164) Google Scholar). In particular, a sulfenic acid intermediate has been shown to be formed on Cys-51 of E. coli MsrA during the reduction of the sulfoxide function of methionine sulfoxide (MetSO). The active site can be represented as an open basin in which Cys-51, located at the N terminus of an α-helix, is accessible. Compared with the E. coli MsrA, the MsrAs from S. pneumoniae and from N. meningitidis or N. gonorrhoeae (called PILB) contain, in addition, an extension at the C terminus and at the C and N termini, respectively. This raised the question of the role of these extensions, in particular of the C-terminal extension. Sequence comparisons of the C-extension of PILB show amino acid identities with open reading frames of which no function has been assigned until recently. These sequences are detected in all kingdoms. Recently, the functions of the E. coli ortholog YeaA and an open reading frame downstream from the msrA gene from Staphylococcus aureus, which both have at least 50% amino acid identities with the C-subdomain of PILB, has been determined and shown to display a new Msr activity, called MsrB (8.Grimaud R. Ezraty B. Mitchell J.K. Lafitte D. Briand C. Derrick P.J. Barras F. J. Biol. Chem. 2001; 276: 48915-48920Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 9.Singh V.K. Moskovitz J. Wilkinson B.J. Jayaswal R.K. Microbiology. 2001; 147: 3037-3045Crossref PubMed Scopus (51) Google Scholar). MsrA, and MsrB, methionine sulfoxide reductase, methionine sulfoxide reductase A, and methionine sulfoxide reductase B, respectively 5,5-dimethyl-1,3-cyclohexanedione dithiothreitol 5,5′-dithiobis(2-nitro)benzoate methionine sulfoxide phthalyl thionitrobenzoate (3-carboxy-4-nitrobenzenthiol) high pressure liquid chromatography The fact that the MsrB activity of YeaA is thioredoxin-dependent (8.Grimaud R. Ezraty B. Mitchell J.K. Lafitte D. Briand C. Derrick P.J. Barras F. J. Biol. Chem. 2001; 276: 48915-48920Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) indicates that at least a Cys residue is involved in the catalytic mechanism. Inspection of the amino acid sequences shows that two Cys are often conserved in putative MsrBs (see Fig. 1). One Cys, Cys-439, which is located in a CGWP(S/A)F motif is at least 50% conserved. The second one, Cys-494, which is included in an RYC(I/V/M)N motif is almost conserved. In the present study, we show that in addition to an MsrA activity that is displayed by the central subdomain, called PILB-MsrA, the C terminus of PILB, called PILB-MsrB, possesses a thioredoxin-dependent MsrB activity. The catalytic mechanism of PILB-MsrB is shown to proceed via the sulfenic acid chemistry. The role of Cys-439 and Cys-494 has been demonstrated. The stereoselectivity in the reduction of the sulfoxide function and the catalytic parameters of the two subdomains have also been determined. The results are in favor of a structure of the active site of MsrBs different from that of the MsrAs. Plasmids pSKPILBMsrA, pSKPILBMsrB, and pSKPILBMsrAMsrB, designed for PILB-MsrA, PILB-MsrB, and PILB-MsrA/MsrB production, respectively, were by of the PILB open reading frame by of not N. meningitidis by M. into the between the and were the The E. coli used for all Msr with the the sequence the were by in a of A and The Msrs were at and for PILB-MsrA, and PILB-MsrB, respectively. The were by the enzymatic chromatography on at were and a with A, followed by a of a protein liquid chromatography The PILB-MsrA/MsrB at whereas the and PILB-MsrB and PILB-MsrB were further on with A, were with a from 1 to in A. PILB-MsrA/MsrB and and and were as by on PubMed Scopus Google Scholar) followed by and by were at in the presence of and these the were for determined at from the of R.K. PubMed Scopus Google Scholar) for and for and and for and were to 1999; Scopus Google and were with the of Google Scholar) to and in respectively. of and in with and the sulfoxide in a as by the in for the and for the and were by from The of the by on The with in and the and were by from with the not by to and in a In both the sulfoxides were to of the and were from and by the by enzymatic reduction with and PILB-MsrB, respectively. The were and the were at MetSO from on a on an with acid in the presence of The corresponding to sulfoxide were and in to and and were M. A. The of Peptide Scholar) from via the with and of the with in which with into of and with in 1999; Scopus Google Scholar) in and from determined and of in in the absence or in the presence of the addition of as described by Boschi-Muller (7.Boschi-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 (164) Google Scholar). Msrs were determined with as a at a of The also and PILB-MsrA/MsrB or or or PILB-MsrB in A. were at by the of free methionine by of the were at different of to and the by the addition of acid to a of In the of formed as described by Boschi-Muller (7.Boschi-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 (164) Google Scholar). The of and and and of PILB-MsrA/MsrB to in the presence of E. coli reductase, and of E. coli in A. and reductase from E. coli were E. M. R. Sci. PubMed Scopus Google PubMed Scopus Google Scholar). were at on a by the of the at The were to the least to and were determined at of the The and a of or or PILB-MsrB or at for in A. the formed as described by Boschi-Muller (7.Boschi-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 (164) Google Scholar). The sulfenic acid intermediate characterized by thionitrobenzoate and by no with 5,5-dimethyl-1,3-cyclohexanedione by the corresponding the of M. PubMed Scopus Google Scholar). of for and and were at in A. were and and the The of an at of were for and and or not and of the were in A in the presence of at a of and the at at a of and the in the at were on a in Msr were to in a were into the at a of were in the in the of the with a of to the PILB amino and is composed of three subdomains. The is to a The central subdomain is an ortholog to E. coli and whereas the C-subdomain displays high sequence to E. coli and S. MsrBs 1). Sequence comparisons show that at least two MsrAs from S. pneumoniae and and and produced by of the N terminus of E. coli MsrA S. Azza S. Branlant G. Sci. 2001; PubMed Scopus Google Scholar) have an N terminus from the corresponding to amino acid of three MsrAs were shown to be active (1.Wizemann T.M. Moskovitz J. Pearce B.J. Cundell D. Arvidson C.G. So M. Weissbach H. Brot N. Masure H.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7985-7990Crossref PubMed Scopus (117) Google Scholar, S. Azza S. Branlant G. Sci. 2001; PubMed Scopus Google Scholar, B. J. Bacteriol. PubMed Google Scholar). of The PILB-MsrA/MsrB protein produced in a were used to MsrA and MsrB subdomains. In the the of PILB which to the N terminus of putative MsrB from In the produced in a whereas the corresponding to the MsrB subdomain not to a of the MsrB subdomain, of PILB, which to the N terminus of the putative MsrB and of the E. coli MsrB (8.Grimaud R. Ezraty B. Mitchell J.K. Lafitte D. Briand C. Derrick P.J. Barras F. J. Biol. Chem. 2001; 276: 48915-48920Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). In PILB-MsrB produced in a a that both Msrs This is also with the fact that the two are often located at different on PILB and were in an E. coli the corresponding sequences the of PILB were as by and Cys for PILB-MsrA/MsrB and two Cys for both and PILB-MsrB These results are in with the PILB sequence that indicates Cys at and in and at and in all of the Cys were also of the subdomains. This shows that 1) both Cys of the MsrA subdomain are to is described for E. coli 2) both Cys of the MsrB subdomain are also and 3) the in PILB-MsrA/MsrB of of Cys is not by the presence of the second This supports an of of and Cys to of N. meningitidis and PILB-MsrB and PILB-MsrA/MsrB of of Cys in free in the of free cysteine no the of two of at least two enzyme determined and in the absence or in the presence of (see in the are are are not for of of the The in the of free cysteine no in a new The the of two of at least two enzyme determined and in the absence or in the presence of (see in the are are are not for of of the of methionine formation determined in the absence of One mol of methionine formed with a of two which is in with formation of a bond between and One mol of methionine also formed with with a of whereas no methionine formed with In the presence of a activity with not with the these results are in with formation of a sulfenic acid on and regeneration of via formation of an bond with followed by reduction by thioredoxin. This is a to that described for E. coli MsrA that the bond to the bond in E. coli is by in PILB-MsrA, whereas the bond is in E. coli a to be of the formation of a sulfenic acid from the of and which are for sulfenic In the of in which no bond can be a of the at by 1 of the with MetSO and with an of of with MetSO and a addition of to an of This is that a is formed with of formed in the absence of and enzymatic activity with or as with with the of three of at least two enzyme The of formed for determined by with as described The of and were with and enzyme or with reductase, and enzyme as described no activity in a new of the sulfenic acid by and with with MetSO and MetSO by to the between the of the protein and second 50% of the enzyme and with a of 1 This to a of with This can to a of sulfenic acid that be into acid the for with the of two of at least two enzyme acid determined with in the absence of as described (see of and and PILB-MsrB were determined by or with as described and in are in by to the between the of the protein and A second 50% of the enzyme and with a of 1 This to a of with This can to a of sulfenic acid that be into acid the in a new The the of three of at least two enzyme The of formed for determined by with as described The of and were with and enzyme or with reductase, and enzyme as described no activity The for with the of two of at least two enzyme acid determined with in the absence of as described (see of and and PILB-MsrB were determined by or with as described and in are in One mol of methionine mol of PILB-MsrB formed in the absence of the of in with formation of a bond between Cys-439 and Cys-494 the role of Cys-439 and Cys-494, of these into were for Cys-494 activity, whereas the a reductase activity with a of 1 mol of methionine in the absence of the with MetSO in the absence of and with a of the at to that of 1 mol of the the and with a of of the of This of to be A is the the that is formed on MsrB can be into sulfoxide In the presence of a activity with PILB-MsrB not with the these results show that 1) the C-terminal subdomain of PILB displays an Msr 2) the mechanism formation of a sulfenic acid 3) the Cys involved in reduction of MetSO and in formation of the sulfenic acid is and 4) the regeneration of Cys-494 is via formation of a bond with Cys-439 followed by reduction by thioredoxin. This mechanism is of that described for MsrA from E. coli that intradisulfide bond is formed in MsrB of PILB for the active The fact that no in indicates to MsrA and more to all a sulfenic acid is formed for the protein and W. M.K. J. Biol. Chem. 1999; 275: Full Text Full Text PDF Scopus Google Scholar, A. N. G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google the sulfenic acid intermediate formed in MsrB be via a mechanism formation of a between Cys-494 and of followed by formation of a bond between and of and release of The fact that an efficient activity on with as a indicates that can attack the sulfenic acid intermediate on Cys-494 in to thioredoxin. supports a of the sulfenic acid the active site of PILB-MsrB to thioredoxin. In of the structure of MsrB the sulfenic acid intermediate be PILB-MsrA/MsrB in the absence of mol of methionine mol of subdomain were formed with a of and These results that both Msrs are active PILB-MsrA/MsrB and that the presence of subdomain not the reductase activity of the The catalytic of and PILB-MsrB were determined the of as a were not for MetSO and and of and PILB-MsrB were and and and respectively. The catalytic of are in the of determined for E. coli MsrA S. Azza S. Branlant G. Sci. 2001; PubMed Scopus Google the and for MetSO are different from described by (8.Grimaud R. Ezraty B. Mitchell J.K. Lafitte D. Briand C. Derrick P.J. Barras F. J. Biol. Chem. 2001; 276: 48915-48920Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) for E. coli MsrA and E. coli MsrB for MetSO are and and are and This is the of used in not In the of MsrB, the be to structural between MsrBs from N. meningitidis and E. In a on E. coli MsrB that a of is also at not In is to that of E. coli MsrB and of E. coli MsrA for E. coli are than for not This that is in of N. meningitidis MsrA and MsrB for N. meningitidis are than for E. coli parameters for the methionine sulfoxide reductase activity of PILB-MsrA, PILB-MsrB, and in the presence of to in the presence of to and be divided and by respectively, the fact that is a and that the second is not an and be divided and by respectively, the fact that is a and that the second is not an and be divided and by respectively, the fact that is a and that the second is not an and be divided and by respectively, the fact that is a and that the second is not an and be divided and by respectively, the fact that is a and that the second is not an and be divided and by respectively, the fact that is a and that the second is not an in the presence of to in the presence of to and be divided and by respectively, the fact that is a and that the second is not an and be divided and by respectively, the fact that is a and that the second is not an and be divided and by respectively, the fact that is a and that the second is not an and be divided and by respectively, the fact that is a and that the second is not an and be divided and by respectively, the fact that is a and that the second is not an and be divided and by respectively, the fact that is a and that the second is not an were in at as described for and and for were by the to the no activity not in the presence of to in the presence of to and be divided and by respectively, the fact that is a and that the second is not an in the presence of to in a new were in at as described for and and for were by the to the no activity not determined. shown to be by In 50% of the of of MetSO by or and were by PILB-MsrA, whereas and were by These results the by (8.Grimaud R. Ezraty B. Mitchell J.K. Lafitte D. Briand C. Derrick P.J. Barras F. J. Biol. Chem. 2001; 276: 48915-48920Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) on that MsrB is for the of the sulfoxide of as has been MsrA is for the C. 1999; PubMed Scopus Google J. R. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The catalytic were determined for the that are The of is than whereas the of PILB-MsrB for is with that for This that the at the carbon Cα is not a determining factor for efficient binding to both Msrs. Compared with the of for with the catalytic of PILB-MsrB is at least The catalytic of PILB-MsrB raised the question of protein-bound MetSO is a than free the catalytic were determined for In the amino and of MetSO are in and no is shown in the of MsrB for by a factor of with that of into the fact that is a an (see the is in fact A with MsrB from E. S. and G. PILB-MsrA, by a factor of with that for also into the fact that is not a the the of PILB-MsrB for to that for whereas a in the to PILB-MsrB that a in the for the of both Msrs are for that the active sites of both Msrs are adapted for binding protein-bound more efficiently than is to that no of the two in the presence of methionine and of the which is not a substrate, at a as high as with as a results in study for a structure of the active site of PILB-MsrB different from that of PILB-MsrA, which can be to all MsrBs and MsrAs. In particular, the two Msrs exhibit opposite stereoselectivities toward the sulfoxide and the sulfoxide which is not a substrate, not no sequence is In particular, the conserved sequence of MsrA around Cys-51 which is located at the N terminus of an is different from the conserved around Cys-494 of PILB-MsrB the common are by MsrA and 1) are with sulfenic acid This supports the idea that sulfenic acid chemistry is a general feature in the reduction of sulfoxides by and mechanism by and J. Chem. Scopus Google Scholar). 2) The is on in 3) The presence of the sulfoxide function is a prerequisite for binding to Msrs as supported by the absence of in the presence of a of The fact that MsrA and MsrB subdomains are fused in N. meningitidis PILB and also in S. pneumoniae (1.Wizemann T.M. Moskovitz J. Pearce B.J. Cundell D. Arvidson C.G. So M. Weissbach H. Brot N. Masure H.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7985-7990Crossref PubMed Scopus (117) Google Scholar) in which the subdomain is not present also raised the question of the of MsrA and MsrB subdomains an an or a on the of MetSO the catalytic were also determined on shown in the catalytic determined with in the of that of This that the presence of PILB-MsrB not the catalytic of This is by the fact that and of PILB-MsrA/MsrB were to of The fact that the catalytic of PILB-MsrA/MsrB to that of PILB-MsrB also that the presence of not the catalytic of and of PILB-MsrB were and respectively. These to be Sequence comparisons of putative MsrBs show that Cys to Cys-494 of PILB-MsrB is by a in A. D. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and ortholog into the fact that is more than a Cys, can that for Cys favor the of the reduction of the sulfoxide function at least of the attack on the sulfoxide Inspection of the structure of shows that the Cys to Cys-439 of PILB-MsrB located in the CGWP(S/A)F is by a whereas Cys at to and which are conserved in at least of the MsrB putative The fact that these Cys have been shown to be involved in binding of in E. coli in the reductase activity. are and are that an than that is or Cys at a role as Cys-439 of In be to the activity of the putative MsrBs that have no Cys at virulence on the and of the bacteria to to also on the of the bacteria to by or In that PILB from N. gonorrhoeae to the or the of the of by not (1.Wizemann T.M. Moskovitz J. Pearce B.J. Cundell D. Arvidson C.G. So M. Weissbach H. Brot N. Masure H.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7985-7990Crossref PubMed Scopus (117) Google Scholar). that MsrA is for virulence of the E. Hassouni M. Chambost J.P. Expert D. Van Gijsegem F. Barras F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 887-892Crossref PubMed Scopus (149) Google Scholar) and of the M. S. Blaylock M.W. Bebear C.M. Rasmussen W.G. Baseman J.B. J. Bacteriol. 2001; 183: 5645-5650Crossref PubMed Scopus (93) Google Scholar). The role of the be at least to the protein of in particular from or through The fact that MsrA is for the of MetSO MsrB is and that PILB both Msr that MsrB is also involved in have shown that the MsrB subdomain of PILB MetSO via a catalytic mechanism sulfenic acid chemistry. The to reduction of MetSO into with a formation of a sulfenic acid on the catalytic The second in the regeneration of Cys-494 via formation of a between Cys-494 and Cys-439 followed by reduction of the by thioredoxin. Cys-494 is almost whereas Cys-439 is conserved in 50% of the MsrB putative This argues for the of the sulfenic acid mechanism described in the present study at the the question of in Cys-494 regeneration are
Olry et al. (Mon,) studied this question.