Characterization of the Methionine Sulfoxide Reductase Activities of PILB, a Probable Virulence Factor from Neisseria meningitidis
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m112350200
ISSN1083-351X
AutoresAlexandre Olry, Sandrine Boschi‐Müller, Michel Marraud, Sarah Cianférani, Alain van Dorsselear, Guy Branlant,
Tópico(s)Sulfur Compounds in Biology
ResumoPILB 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 obtained by cloning internal fragments of the PILB open reading frame synthesized by PCR (sequences of oligonucleotides not shown) using N. meningitidis Z2491 genomic DNA, kindly provided by Dr. M. K. Taha, into the plasmid pDB125KSNN 2S. Marchal, personal communication. between the NdeI and SacI sites. Site-directed mutageneses were performed using the QuikChange site-directed mutagenesis kit (Stratagene). The E. coli strain used for all Msr productions was HB101 (supE44, hsdS20 (r −, m −), recA13, ara-14, proA2, lacY1, galK2, rpsL20, xyl-5, mtl-1) transformed with the plasmidic construction containing the coding sequence under the lac promoter. For PILB-Msrs purification, cells were harvested by centrifugation, resuspended in a minimal volume of buffer A (50 mmTris-HCl, 2 mm EDTA, pH 8) containing 20 mmdithiothreitol (DTT) and sonicated. The Msrs were then precipitated at 40, 50, and 60% ammonium sulfate ((NH4)2SO4) saturation for PILB-MsrA/MsrB, PILB-MsrA, and PILB-MsrB, respectively. The contaminating proteins were removed by applying the enzymatic solutions onto exclusion size chromatography on ACA 54 resin at pH 8 (buffer A). Purified fractions were then pooled and applied onto a Q-Sepharose column equilibrated with buffer A, followed by a linear gradient of KCl (0–0.4 m) using a fast protein liquid chromatography system (Amersham Biosciences). The PILB-MsrA/MsrB was eluted at 100 mm KCl, whereas the PILB-MsrA and PILB-MsrB passed through. PILB-MsrA and PILB-MsrB were further purified on phenyl-Sepharose (Amersham Biosciences) equilibrated with buffer A, containing 1m (NH4)2SO4. PILB-Msrs were eluted with a linear gradient from 1 to 0 m in buffer A. At this stage, wild-type PILB-MsrA/MsrB and wild-type and mutant PILB-MsrAs and PILB-MsrBs were pure as checked by electrophoresis on 12% SDS-polyacrylamide gel (10.Laemmli U.K. Nature. 1970; 227: 680-687Crossref PubMed Scopus (207522) Google Scholar) followed by Coomassie Brilliant Blue R-250 staining and by electrospray mass spectrometry analyses. Purified enzymes were stored at −20 °C in the presence of 50 mm DTT and 60% (NH4)2SO4. Under these conditions, the enzymes were stable for several weeks. Their molecular concentration was determined spectrophotometrically, using theoretical extinction coefficients at 280 nm deduced from the method of Scopes (11.Scopes R.K. Anal. Biochem. 1974; 59: 277-282Crossref PubMed Scopus (603) Google Scholar) (i.e. 26,200m−1·cm−1 for wild-type and mutant PILB-MsrAs, 17,330m−1·cm−1 for wild-type and mutant PILB-MsrBs, and 42,300m−1·cm−1 for wild-type PILB-MsrA/ MsrB. Thel-Met-S-SO and d-Met-R-SO enantiomers were prepared according to Holland et al. (12.Holland H.L. Andreana P.R. Brown F.M. Tetrahedron: Asymmetry. 1999; 10: 1833-1843Crossref Scopus (33) Google Scholar).l- and d-methionine were treated with phthalic (Pht) anhydride using the method of Bose (13.Bose A.K. Org. Synthesis. 1960; 40: 82-85Crossref Google Scholar) to give Pht-l-Met-OH and Pht-d-Met-OH in 90–95% yields, respectively. Oxidation of Pht-l-Met-OH and Pht-d-Met-OH was carried out in methanol with H2O2 and afforded the sulfoxide in a nearly equimolar (S/R)-composition as revealed by the split CεH3 proton singlet in CDCl3/Me2SO-d64:1 (2.487 ppm for the heterochiral and 2.483 ppm for the homochiral diastereomers). Both Pht-l-Met-S-SO and Pht-d-Met-R-SO enantiomers were obtained by crystallization from methanol. The absolute configuration of the isomer R was confirmed by x-ray diffraction on single crystals. 3C. Didierjean, unpublished results. The phthalic protecting group was eliminated with hydrazine hydrate in ethanol, and the l-Met-S-SO and d-Met-R-SO enantiomers were recovered by precipitation from water with acetone. On the other hand, it was not possible by crystallization to obtain Pht-l-Met-R-SO and Pht-d-Met-S-SO enantiomers in a pure form. In both cases, the resulting sulfoxides were found to contain about 25% of the other isomer. Therefore, l-Met-R-SO and d-Met-S-SO were isolated from thel-Met-R,S-SO and d-Met-R,S-SO diastereomers by consuming the other isomer by enzymatic reduction with PILB-MsrA and PILB-MsrB, respectively. The experimental conditions were 100 mm DTT, 500 μm Msr, 50 mm Tris-HCl, 2 mmEDTA, pH 8, and the reaction mixtures were incubated overnight at 25 °C. Each MetSO isomer was then separated from Met on a 25-cm sephasil C18 reverse phase column on an ÄKTA explorer system (Amersham Biosciences) equilibrated with H2O/trifluoroacetic acid 0.1% buffer in the presence of 10% acetonitrile. Met was eluted isocratically after MetSO. The fractions corresponding to each sulfoxide isomer were pooled and concentrated in order to eliminate acetonitrile and trifluoroacetic acid. l-Met-R, S-SO-NHMe and Ac-l-Met-R,S-SO-NHMe were classically prepared (14.Bodanszky M. Bodanszky A. The Practice of Peptide Synthesis. Springer-Verlag, New York1984: 103-112Google Scholar) from N-(tert-butoxycarbonyl)-l-Met-OH via the mixed anhydride with Me2CHCH2OCOCl/NEt(CHMe2)2 and MeNH2. Treatment of the resulting N-(tert-butoxycarbonyl)-l-Met-NHMe with 3 n HCl in EtOAc gave l-Met-NHMe, which was acetylated with AcCl/CHMe2 into Ac-l-Met-NHMe. Oxidation of l-Met-NHMe and Ac-l-Met-NHMe with H2O2 in methanol (12.Holland H.L. Andreana P.R. Brown F.M. Tetrahedron: Asymmetry. 1999; 10: 1833-1843Crossref Scopus (33) Google Scholar) resulted in l-Met-R, S-SO-NHMe and Ac-l-Met-R, S-SO-NHMe. Ac-l-Met-R, S-SO was purchased from Bachem. Cysteine content was determined using DTNB under nondenaturing (buffer A) and denaturing conditions (final concentration of 1% SDS in buffer A), either in the absence or in the presence of 150 mmdl-Met-R,S-SO without the addition of any exogenous reducing system as previously described by Boschi-Muller et al. (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 activities were determined with dl-Met-R,S-SO as a substrate at a concentration of 150 mm. The reaction mixture also contained 10 mm DTT and 5 μmwild-type PILB-MsrA/MsrB or wild-type or mutant PILB-MsrA or PILB-MsrB in buffer A. Initial rate measurements were carried out at 25 °C by following the appearance of free methionine measured by HPLC. To do so, aliquots of the reaction mixture were removed at different times of incubation up to 2.5 min, and the reaction was stopped by the addition of trifluoroacetic acid to a final concentration of 1%. In each aliquot, the quantity of Met formed was measured as previously described by Boschi-Muller et al. (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 ability of wild-type and mutant PILB-MsrAs and PILB-MsrBs and of wild-type PILB-MsrA/MsrB to reduce substrates was assayed in the presence of 1.28 μm E. coli thioredoxin reductase, 0.3 mm NADPH, and various concentrations of E. coli thioredoxin in buffer A. Thioredoxin and thioredoxin reductase from E. coli were prepared following experimental procedures already published (15.Mössner E. Huber-Wunderlich M. Glockshuber R. Protein Sci. 1998; 7: 1233-1244Crossref PubMed Scopus (157) Google Scholar,16.Mulrooney S.B. Protein Expression Purif. 1997; 9: 372-378Crossref PubMed Scopus (40) Google Scholar). Initial rate measurements were carried out at 25 °C on a Kontron Uvikon 933 spectrophotometer by following the decrease of the absorbance at 340 nm. The initial rate data were fitted to the Michaelis-Menten relationship using least squares analysis to determine kcat and Km. All Km values were determined at saturating concentrations of the other substrate. The reaction mixture, containing 150 mmdl-Met-R, S-SO and a 100–500 μm concentration of wild-type or mutant PILB-MsrA or PILB-MsrB or wild-type PILB-MsrA/MsrB, was incubated at 25 °C for 10 min in buffer A. Then the Met formed was quantified as previously described by Boschi-Muller et al. (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 was characterized spectrophotometrically by using thionitrobenzoate (TNB−) under nondenaturing conditions and by mass spectrometry analyses after modification (or no modification) with 5,5-dimethyl-1,3-cyclohexanedione (dimedone). For spectrophotometric characterization, TNB− was prepared by reducing the corresponding disulfide using the procedure of Silver (17.Silver M. Methods Snzymol. 1979; 62: 135-137Crossref PubMed Scopus (18) Google Scholar). Progress curves of TNB− disappearance for wild-type and mutant PILB-MsrAs and PILB-MsrBs were recorded at 412 nm in buffer A. Enzyme concentrations were 7.35 and 14.7 μm, and the TNB− concentration was 60 μm. The amount of TNB− consumed was calculated using an extinction coefficient at 412 nm of 13,600m−1·cm−1. For spectrometric characterization, analyses were performed for wild-type and mutant PILB-MsrAs and PILB-MsrBs, either after modification or not byd,l-Met-R,S-SO and dimedone. All of the modification reactions were performed in buffer A in the presence of 20 μm enzyme.d,l-Met-R,S-SO was added at a concentration of 150 mm, and the mixture was incubated 10 min at 25 °C. Then dimedone at a concentration of 20 mmwas added, and the mixture was incubated overnight in the dark at room temperature. Mass spectrometric measurements were performed on a LCT electrospray time-of-flight mass spectrometer (Micromass, Manchester, UK). For mass analysis in denaturing conditions, Msr samples were diluted to 10 μm in a 1:1 water/acetonitrile mixture (v/v) containing 1% formic acid. Samples were continuously infused into the ion source at a flow rate of 5 μl/min. Spectra were recorded in the positive ion mode in the mass range 400–4000m/z, after calibration of the instrument with a solution of horse heart myoglobin (Sigma) diluted to 2 μmin the 1:1 water/acetonitrile (1% formic acid) mixture. PILB contains 522 amino acids and is composed of three subdomains. The N-subdomain is suggested to encode a disulfide oxidoreductase. The central subdomain is an ortholog to E. coli and Saccharomyces cerevisiae MsrAs, whereas the C-subdomain displays high sequence similarities to E. coli and S. aureus MsrBs (Fig. 1). Sequence comparisons show that at least two MsrAs from S. pneumoniae and Bacillus subtilis (Pedant code gi_14972133 and gi_2634588) and one recently produced by truncation of the N terminus of E. coli MsrA (18.Boschi-Muller S. Azza S. Branlant G. Protein Sci. 2001; 10: 2272-2279Crossref PubMed Scopus (44) Google Scholar) have an N terminus starting from the position corresponding to amino acid 196 of PILB. All 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, 18.Boschi-Muller S. Azza S. Branlant G. Protein Sci. 2001; 10: 2272-2279Crossref PubMed Scopus (44) Google Scholar, 19.Hayes C.S. Illades-Aguiar B. Casillas-Martinez L. Setlow P. J. Bacteriol. 1998; 180: 2694-2700Crossref PubMed Google Scholar). Thus, truncation was done after position 194 of PILB. The PILB-MsrA/MsrB protein (amino acids 195–521) was produced in a soluble form. Two plasmidic constructs were used to produce soluble MsrA and MsrB subdomains. In the first construct, the truncation was done after position 388 of PILB which corresponds to the N terminus of putative MsrB from Klebsiella pneumoniae(Pedant code b_kpn.contig523 orf8). In this case, the PILB-MsrA (amino acids 195–388) was produced in a soluble form, whereas the truncated form corresponding to the MsrB subdomain (amino acids 390–521) was not soluble. Thus, to obtain a soluble form of the MsrB subdomain, another construct was used, where truncation was done after position 375 of PILB, which corresponds to the N terminus of the putative Sinorhizobium meliloti MsrB (NCBI accession numberAE007290.1) 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 this case, PILB-MsrB was produced in a soluble form (amino acids 376–521). Such a result suggests that both Msrs fold independently within PILB. This is also consistent with the fact that the two genes are often located at different positions on chromosomal DNAs. Truncated PILB variants and their mutants were overexpressed in an E. coli strain using the corresponding DNA sequences under the lac promoter. All forms of PILB variants were obtained pure as judged by SDS-PAGE gels and mass spectrometry analyses. DTNB reagent revealed four Cys for PILB-MsrA/MsrB and two Cys for both PILB-MsrA and PILB-MsrB under denaturating conditions (Table I). These results are in agreement with the PILB DNA sequence that indicates four Cys at positions 206 and 348 in PILB-MsrA and at positions 439 and 494 in PILB-MsrB. Under native conditions, all of the Cys were also reactive regardless of the subdomains. This shows that 1) both Cys of the MsrA subdomain are easily accessible, similar to what is described for E. coli MsrA; 2) both Cys of the MsrB subdomain are also accessible; and 3) the accessibility in PILB-MsrA/MsrB of each couple of Cys within each domain is not significantly altered by the presence of the second subdomain. This again supports an independent folding of each subdomain.Table IFree sulfhydryl content of wild-type and Cys to Ser mutants of N. meningitidis PILB-MsrA and PILB-MsrB and PILB-MsrA/MsrB wild typeEnzymeNo. of CysNo. of Cys measuredWithout dl-Met-R, S-SOWithdl-Met- R, S-SODecrease in free thiolsaThe difference in the number of free cysteine thiols upon treatment withdl-Met-R, S-SO versus no treatment.CalculatedTheoreticalmol Cys/mol enzymePILB-MsrA21.90.11.82C206S PILB-MsrA11.01.000C348S PILB-MsrA10.900.91PILB-MsrB22.00.11.92C439S PILB-MsrB11.00.10.91C494S PILB-MsrB11.01.000PILB-MsrA/MsrB44.00.43.64The values indicated represent the average of two independent measurements of at least two enzyme concentrations (S.D. range 10%). Cysteine content was determined spectrophotometrically using DTNB under nondenaturing and denaturing conditions (10% SDS), in the absence or in the presence of 150 mmdl-Met-R, S-SO without any regenerating system (see "Experimental Procedures"). Data presented in the table are those obtained under nondenaturing conditions. Data obtained under denaturing conditions are identical but are not presented for reasons of clarity of the table.a The difference in the number of free cysteine thiols upon treatment withdl-Met-R, S-SO versus no treatment. Open table in a new tab The values indicated represent the average of two independent measurements of at least two enzyme concentrations (S.D. range 10%). Cysteine content was determined spectrophotometrically using DTNB under nondenaturing and denaturing conditions (10% SDS), in the absence or in the presence of 150 mmdl-Met-R, S-SO without any regenerating system (see "Experimental Procedures"). Data presented in the table are those obtained under nondenaturing conditions. Data obtained under denaturing conditions are identical but are not presented for reasons of clarity of the table. Stoichiometry of methionine formation was determined in the absence of reductant (Table II). One mol of methionine was formed with a loss of two thiols, which is in agreement with formation of a disulfide bond between Cys-206 and Cys-348. One mol of methionine was also formed with mutant C348S with a loss of one thiol, whereas no methionine was formed with mutant C206S. In the presence of thioredoxin, a recycling activity was observed with PILB-MsrA wild type but not with the mutants (Table II). Together, these results are in agreement with formation of a sulfenic acid on Cys-206 and regeneration of Cys-206 via formation of an internal disulfide bond with Cys-348 followed by its reduction by thioredoxin. This is a situation similar to that described for E. coli MsrA except that the Cys-206–Cys-348 bond (equivalent to the Cys-51–Cys-198 bond in E. coli MsrA) is reducible by thioredoxin in PILB-MsrA, whereas only the Cys-198–Cys-206 bond is reducible in E. coli MsrA. Such a difference remains to be explained. Further evidence of the formation of a sulfenic acid comes from the use of TNB− and dimedone, which are specific reagents for sulfenic acid. In the case of mutant C348S, in which no disulfide bond can be formed, a decrease of the absorbance at 410 nm by 1 eq of TNB−/mol was observed when the mutant was first incubated with MetSO and then treated with an excess of TNB− (Table III). Incubation of mutant C348S with MetSO and a subsequent addition of dimedone led to an increased mass of 138 Da. This increase is that expected if a covalent adduct is formed with dimedone.Table IIStoichiometry of Met formed in the absence of regenerating system and enzymatic activity with DTT or thioredoxin as reductantEnzymeStoichiometryActivity with DTTActivity with thioredoxinmol Met formed/mol enzymeμmol Met formed/min/μmol enzymeμmol Met formed/min/μmol enzymePILB-MsrA0.93220C206S PILB-MsrA0NANAC348S PILB-MsrA0.937NAPILB-MsrB1.0312C439S PILB-MsrB1.036NAC494S PILB-MsrB0NANAPILB-MsrA/MsrB1.84.2170The values indicated represent the average of three independent measurements of at least two enzyme concentrations (S.D. range 10%). The quantity of Met formed for stoichiometry determination was determined by HPLC, after reaction with 150 mmdl-Met-R, S-SO without any regenerating system as described under "Experimental Procedures." The activities of wild-type and mutant enzymes were assayed with 150 mmdl-Met-R, S-SO, 10 mm DTT, and 5 μm enzyme or with 150 mmdl-Met-R, S-SO, 200 μm thioredoxin, 1.28 μm thioredoxin reductase, and 0.5–50 μm enzyme as described under "Experimental Procedures." NA, no activity (<6 · 10−2 μmol/min/μmol). Open table in a new tab Table IIICharacterization of the sulfenic acid intermediates by electrospray mass spectrometry analyses and spectrophotometric titration with TNB−EnzymeTNB−Electrospray mass spectrometry analysesBefore modificationAfter modification with MetSO and dimedoneNativeAfter MetSO treatmentTheoretical massMeasured massMeasured massMass differenceaMass differences measured by electrospray ionization-mass spectrometry correspond to the differences between the mass of the protein before and after treatment.mol TNB−/mol enzymeWild-type PILB-MsrA0021,89621,898 ± 121,897 ± 1−1 ± 2C206S PILB-MsrA0021,88021,881 ± 121,880 ± 1−1 ± 2C348S PILB-MsrA0121,88021,880 ± 122,018 ± 1+138 ± 2Wild-type PILB-MsrB0016,37416,374 ± 116,373 ± 1−1 ± 2C439S PILB-MsrB0116,35816,358 ± 116,512 ± 1bA second peak, representing about 50% of the enzyme population, and with a mass of 16,390 ± 1 Da was observed. This corresponds to a mass increase of 32 Da compared with native C439S PILB-MsrB. This peak can correspond to a population of sulfenic acid derivative that would be oxidized into sulfonic acid under the experimental conditions used.+154 ± 2C494S PILB-MsrB0016,35816,358 ± 116,358 ± 1+0 ± 2The values indicated for spectrophotometric titration with TNB− represent the average of two independent measurements of at least two enzyme concentrations (S.D. range 10%). Sulfenic acid content was determined spectrophotometrically using TNB− under denaturing conditions (0.1% SDS), after reaction with 150 mmdl-Met-R, S-SO in the absence of any regenerating system as previously described (see "Experimental Procedures"). Molecular masses of wild-type and mutant PILB-MsrA and PILB-MsrB were determined by electrospray mass analysis without any modification or after reaction with 150 mmdl-Met-R, S-SO and/or 20 mm dimedone as described under "Experimental Procedures." Molecular mass and difference in mass are expressed in daltons.a Mass differences measured by electrospray ionization-mass spectrometry correspond to the differences between the mass of the protein before and after treatment.b A second peak, representing about 50% of the enzyme population, and with a mass of 16,390 ± 1 Da was observed. This corresponds to a mass increase of 32 Da compared with native C439S PILB-MsrB. This peak can correspond to a population of sulfenic acid derivative that would be oxidized into sulfonic acid under the experimental conditions used. Open table in a new tab The values indicated represent the average of three independent measurements of at least two enzyme concentrations (S.D. range 10%). The quantity of Met formed for stoichiometry determination was determined by HPLC, after reaction with 150 mmdl-Met-R, S-SO without any regenerating system as described under "Experimental Procedures." The activ
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