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• W2081097951 abstract "Among theChromatiaceae, the glutathione derivative γ-l-glutamyl-l-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species. The gene (garB) encoding the central enzyme in glutathione amide cycling, glutathione amide reductase (GAR), has been isolated from Chromatium gracile, and its genomic organization has been examined. The garB gene is immediately preceded by an open reading frame encoding a novel 27.5-kDa chimeric enzyme composed of one N-terminal peroxiredoxin-like domain followed by a glutaredoxin-like C terminus. The 27.5-kDa enzyme was established in vitro to be a glutathione amide-dependent peroxidase, being the first example of a prokaryotic low molecular mass thiol-dependent peroxidase. Amino acid sequence alignment of GAR with the functionally homologous glutathione and trypanothione reductases emphasizes the conservation of the catalytically important redox-active disulfide and of regions involved in binding the FAD prosthetic group and the substrates glutathione amide disulfide and NADH. By establishing Michaelis constants of 97 and 13.2 μm for glutathione amide disulfide and NADH, respectively (in contrast toK m values of 6.9 mm for glutathione disulfide and 1.98 mm for NADPH), the exclusive substrate specificities of GAR have been documented. Specificity for the amidated disulfide cofactor partly can be explained by the substitution of Arg-37, shown by x-ray crystallographic data of the human glutathione reductase to hydrogen-bond one of the glutathione glycyl carboxylates, by the negatively charged Glu-21. On the other hand, the preference for the unusual electron donor, to some extent, has to rely on the substitution of the basic residues Arg-218, His-219, and Arg-224, which have been shown to interact in the human enzyme with the NADPH 2′-phosphate group, by Leu-197, Glu-198, and Phe-203. We suggest GAR to be the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases. Among theChromatiaceae, the glutathione derivative γ-l-glutamyl-l-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species. The gene (garB) encoding the central enzyme in glutathione amide cycling, glutathione amide reductase (GAR), has been isolated from Chromatium gracile, and its genomic organization has been examined. The garB gene is immediately preceded by an open reading frame encoding a novel 27.5-kDa chimeric enzyme composed of one N-terminal peroxiredoxin-like domain followed by a glutaredoxin-like C terminus. The 27.5-kDa enzyme was established in vitro to be a glutathione amide-dependent peroxidase, being the first example of a prokaryotic low molecular mass thiol-dependent peroxidase. Amino acid sequence alignment of GAR with the functionally homologous glutathione and trypanothione reductases emphasizes the conservation of the catalytically important redox-active disulfide and of regions involved in binding the FAD prosthetic group and the substrates glutathione amide disulfide and NADH. By establishing Michaelis constants of 97 and 13.2 μm for glutathione amide disulfide and NADH, respectively (in contrast toK m values of 6.9 mm for glutathione disulfide and 1.98 mm for NADPH), the exclusive substrate specificities of GAR have been documented. Specificity for the amidated disulfide cofactor partly can be explained by the substitution of Arg-37, shown by x-ray crystallographic data of the human glutathione reductase to hydrogen-bond one of the glutathione glycyl carboxylates, by the negatively charged Glu-21. On the other hand, the preference for the unusual electron donor, to some extent, has to rely on the substitution of the basic residues Arg-218, His-219, and Arg-224, which have been shown to interact in the human enzyme with the NADPH 2′-phosphate group, by Leu-197, Glu-198, and Phe-203. We suggest GAR to be the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases. glutathione reductase glutathione glutaredoxin peroxiredoxin glutathione amide glutathione amide disulfide glutathione amide reductase chimeric enzyme composed of one Prx and one Grx homologous domain polyacrylamide gel electrophoresis polymerase chain reaction N-(9-fluorenyl)methoxycarbonyl pre-activated pentafluorophenyl esters high pressure liquid chromatography column volumes open reading frame Several different oxidoreductase processes shared by eukaryotes and certain prokaryotes such as the detoxification of environmental chemicals (1Kerklaan P.R. Zoetemelk C.E. Mohn G.R. Biochem. Pharmacol. 1985; 34: 2151-2156Crossref PubMed Scopus (36) Google Scholar, 2Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3229) Google Scholar), formaldehyde dissimilation (3Duine J.A. Biofactors. 1999; 10: 201-206Crossref PubMed Scopus (18) Google Scholar, 4Dolferus R. Osterman J.C. Peacock W.J. Dennis E.S. Genetics. 1997; 146: 1131-1141Crossref PubMed Google Scholar), and the reduction of ribonucleotides (5Aslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (163) Google Scholar) depend to a certain extent on the glutathione reductase (GR1)-dependent glutathione (GSH) redox cycle. Moreover, in eukaryotes, some reactive oxygen intermediates are detoxified directly by the action of glutathione peroxidases (6Ursini F. Maiorino M. Brigelius-Flohe R. Aumann K.D. Roveri A. Schomburg D. Flohe L. Methods Enzymol. 1995; 252: 38-53Crossref PubMed Scopus (663) Google Scholar) and, to a lesser extent, of glutathioneS-transferases (7Ketterer B. Free Radic. Res. 1998; 28: 647-658Crossref PubMed Scopus (129) Google Scholar). Members of another enzyme family of GSH-dependent thiol-disulfide oxidoreductases, designated thioltransferases or glutaredoxins (Grx), are believed to act as one of the primary defenders against mixed disulfides formed after oxidative damage to proteins (8Luikenhuis S. Perrone G. Dawes I.W. Grant C.M. Mol. Biol. Cell. 1998; 9: 1081-1091Crossref PubMed Scopus (196) Google Scholar). Besides these direct and indirect protection systems against the products of aerobic metabolism relying on GSH cycling, GSH itself also serves an indirect antioxidant function by protecting the amino acid cysteine against auto-oxidation (9Sundquist A.R. Fahey R.C. J. Biol. Chem. 1989; 264: 719-725Abstract Full Text PDF PubMed Google Scholar).Only three groups of prokaryotes, the Gram-negative cyanobacteria and purple bacteria and some Gram-positive streptococci and enterococci (10Sherrill C. Fahey R.C. J. Bacteriol. 1998; 180: 1454-1459Crossref PubMed Google Scholar, 11Patel M.P. Marcinkeviciene J. Blanchard J.S. FEMS Microbiol. Lett. 1998; 166: 155-163Crossref PubMed Google Scholar), produce GSH together with the recycling GR (12Fahey R.C. Sundquist A.R. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 1-53PubMed Google Scholar). Thus far, no significant GSH-dependent peroxidase activity has been reported for a GSH producing prokaryote. On the other hand, substantial thioltransferase activity is encountered indicating that in terms of oxygen shielding, GSH metabolism in prokaryotes does not serve a direct detoxification system for reactive oxygen intermediates but only maintains disulfides in the reduced state. Prokaryotic aerobes and pathogens require an array of antioxidant defense mechanisms to protect themselves against the reactive oxygen intermediates produced by the incomplete reduction of oxygen during respiration or by the antimicrobial response of the host phagocytes (13Fridovich I. Ann. N. Y. Acad. Sci. 1999; 893: 13-28Crossref PubMed Scopus (399) Google Scholar, 14Ochsner U.A. Vasil M.L. Alsabbagh E. Parvatiyar K. Hassett D.J. J. Bacteriol. 2000; 182: 4533-4544Crossref PubMed Scopus (211) Google Scholar). Detoxification of the freely diffusible hydrogen peroxide (H2O2), which in turn can be reduced further via the Fenton reaction to extremely reactive hydroxyl radicals, is completed by the action of catalases, heme- and manganese-containing peroxidases, and several members of the large multifunctional AhpC/TSA protein family, recently classified as peroxiredoxins (Prx). To date, it seems that all bacterial Prx enzymes obtain the necessary reducing equivalents from the thioredoxin reducing system itself or from a thioredoxin-like reducing system, because it was demonstrated recently that the flavoprotein component (AhpF) of the Salmonella typhimurium alkyl hydroperoxide reductase (AhpCF) system and bacterial thioredoxin reductase have very similar mechanistic properties (15Reynolds C.M. Poole L.B. Biochemistry. 2000; 39: 8859-8869Crossref PubMed Scopus (28) Google Scholar). Apparently, GSH is never involved in bacterial Prx-reduction.Chromatium species are anaerobic sulfur-oxidizing phototrophs that produce glutathione amide (GASH) (16Bartsch R.G. Newton G.L. Sherrill C. Fahey R.C. J. Bacteriol. 1996; 178: 4742-4746Crossref PubMed Google Scholar), a GSH derivative modified at the terminal glycine. An original anaerobic function for this GASH metabolism was proposed by Pott and Dahl (17Pott A.S. Dahl C. Microbiology. 1998; 144: 1881-1894Crossref PubMed Scopus (153) Google Scholar) and implies a possible involvement in the transfer of sulfide across the periplasmic membrane. When grown photoautotrophically on sulfide, GASH is present in its persulfide form (16Bartsch R.G. Newton G.L. Sherrill C. Fahey R.C. J. Bacteriol. 1996; 178: 4742-4746Crossref PubMed Google Scholar), supporting the hypothesis that the periplasmically formed persulfide becomes transported to the cytoplasm, where the GASH-bound sulfide is released by the action of a heterodisulfide reductase. Chromatium species extracts do show glutathione amide disulfide (GASSAG) reductase activity (16Bartsch R.G. Newton G.L. Sherrill C. Fahey R.C. J. Bacteriol. 1996; 178: 4742-4746Crossref PubMed Google Scholar), and the involvement of GAR as the heterodisulfide reductase in the hypothesized sulfide transfer mechanism has to be considered because GSH persulfide reduction was established already for the bovine GR (18Moutiez M. Aumercier M. Parmentier B. Tartar A. Sergheraert C. Biochim. Biophys. Acta. 1995; 1245: 161-166Crossref PubMed Scopus (9) Google Scholar).Here we present the isolation and successful expression inEscherichia coli of the Chromatium gracile garBgene, which has permitted the characterization of the C. gracile GAR enzyme. Further, we provide evidence for the existence of a novel Prx-containing peroxidase system, probably widespread among Gram-negative pathogens, that is fueled by the GAR-dependent redox cycling of theChromatium-specific low molecular weight thiol GASH.EXPERIMENTAL PROCEDURESMaterialsRestriction enzymes were from New England Biolabs (Beverly, MA). T4 DNA ligase, the DIG DNA labeling kit, and the DIG luminescent detection kit were obtained from Roche Diagnostics. Taq DNA polymerase was from Amersham Pharmacia Biotech. Plasmid DNA was prepared on a 30-ml scale using the Qiagen (Crawley, UK) plasmid purification kit. The pGEM-T and pUC18 plasmids were from Promega(Madison, WI), and the pET11a plasmid was from Novagen (Madison, WI). DNA sequencing was performed using an ABI PRISM 377 sequence detection system (Applied Biosystems, Foster City, CA). An AKTA-design FPLC system (Amersham Pharmacia Biotech) was used for all chromatographic protein purification steps (all other chromatographic equipment was also purchased from Amersham Pharmacia Biotech). All other biochemical reagents were purchased from Sigma-Aldrich. A Uvikon 943 double beam UV-visible spectrophotometer (Kontron Instruments, Watford, UK) was used for the spectroscopic measurements.Strains and MediaC. gracile (DSM 1712) was grown on Pfenning's medium (19Pfenning N. Annu. Rev. Microbiol. 1993; 47: 1-29Crossref PubMed Scopus (6) Google Scholar), supplemented with 1% NaCl, by anaerobic photosynthesis at a temperature of 30 °C. E. coli strains were grown on LB medium (Life Technologies, Inc.) supplemented with 100 μg/ml carbeniciline when necessary. Strain XL-1 blue (New England Biolabs, Herefordshire, UK) was used as a recipient to detect α-complementation for pGEM-T derivatives on LB plates supplemented with 80 μm isopropyl-β-d-thiogalactoside and 32 μg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactoside. All expression plasmids were introduced into competent BL21(DE3) E. coli (Novagen).N-terminal Amino Acid Sequence DeterminationGAR was partially purified according to the method of Chung and Hurlbert (20Chung Y.C. Hurlbert R.E. J. Bacteriol. 1975; 123: 203-211Crossref PubMed Google Scholar). The partially purified enzyme sample was loaded onto an SDS-polyacrylamide gel, and after electrophoresis, the proteins were transferred onto a ProBlott membrane (Applied Biosystems) as described by Maniatis et al. (21Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 931-946Google Scholar). The membrane was stained with Coomassie Blue, and the protein band corresponding to the GAR enzyme was subjected to N-terminal sequence determination using a 477A pulsed liquid sequenator (Applied Biosystems). Sequencing reagents were from the same firm. Forty-nine residues were identified covering a 51-amino acid N-terminal stretch, TQHFDLIAIGGGSGGLAVAEKAAAFGKRVALIESKALGGTXVNVGXVPKKV. The same procedure was applied to verify the first seven amino acid residues of the partially purified recombinant Prx/Grx.SDS-PAGEProtein samples were subjected to reducing SDS-polyacrylamide gel electrophoresis (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206048) Google Scholar) and stained with Coomassie Blue or Silver (23Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3729) Google Scholar). The total protein concentration was determined by the method of Bradford (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213462) Google Scholar) using the Bio-Rad 500-0006 kit with bovine serum albumin as a standard.DNA TechniquesC. gracile genomic DNA was isolated according to the cetyl-trimethyl-ammonium-bromide method described by Ausubel et al. (25Ausubel F.M. Bocut R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Strohl K. Current Protocols in Molecular Biology. Greene Wiley, Interscience, New York1990: 241-242Google Scholar). A 150-base pair fragment was obtained from C. gracile genomic DNA via PCR amplification using degenerate primers based on the N-terminal amino acid sequence of endogenous GAR (forward primer, 5′-ACICARCAYTTYGAY-3′; reverse primer, 5′-CYTTYTTIGGIACRCA-3′). This PCR fragment was ligated into the pGEM-T vector, and the resulting construct (pGEM-GAR) was verified by sequence analysis. The pGEM-GAR construct was used as a template for PCR-based synthesis of a digoxigenin-labeled probe using the DIG DNA labeling kit. Therefore, a perfect match primer pair was designed (forward primer, 5′-ACCCAGCATTTCGACCTG-3′; reverse primer, 5′-CCTTCTTGGGCACGCAGC-3′).C. gracile subgenomic DNA fragments, generated by subjecting the genomic DNA to the action of various restriction enzymes, were screened with the digoxigenin-labeled probe in Southern hybridization experiments. A single signal corresponding to a fragment of ∼3.6 kilobases was obtained when the genomic DNA was initially cut withBamHI. A pUC18-subgenomic library of C. gracileDNA consisting of BamHI fragments between ∼3.4 and 3.9 kilobases was screened by colony hybridization with the digoxigenin-labeled oligonucleotide. The detection of transformants was performed with the nonradioactive DIG luminescent detection kit according to the manufacturer's instructions. Nucleotide sequence determination of one positive pUC18 derivative was initiated using the pUC18 universal primers M13F and M13R, and new primers were synthesized at ∼450 nucleotide intervals based on the results of previous sequencing.Synthesis of GASH and GASSAGGSH was synthesized on the Advanced ChemTech 90 peptide synthetizer (Louisville, KY) using theN-(9-fluorenyl)methoxycarbonyl (Fmoc) strategy (26Atherton E. Sheppard R.C. Solid Phase Peptide Synthesis: A Practical Approach. IRL press, Oxford1989Google Scholar) on a polyamide resin developed for the synthesis of peptide amides (0, 52 mm/g) (Rink Resin, Advanced ChemTech) (27Story S.C. Aldrich J.V. Int. J. Pept. Protein Res. 1992; 39: 87-92Crossref PubMed Scopus (23) Google Scholar). Fmoc-l-amino acids (3 eq = 1, 56 mm) were introduced into the chain as pre-activated pentafluorophenyl esters (OPfp) in the presence of a 3-fold excess of 1-hydroxybenzotriazole (1 eq). The introduced residues were Fmoc-Gly-OPfp, Fmoc-Cys(Trt)-OPfp, andN-α-t-butoxycarbonyl-Glu(OPfp)-α-t-butyl. The efficiency of coupling was always checked by the Kaiser test. Deprotection of the Fmoc groups was carried out with 20% piperidine inN,N-dimethylformamide for 15 min. After washings, the peptide was liberated with a 95:5 (v/v) solution of trifluoroacetic acid/1,2-ethanedithiol for 90 min under nitrogen. After filtration, the trifluoroacetic acid was removed under vacuum. The peptide mixture in water was treated with diethyl ether (1/1) to eliminate scavengers. GASSAG was prepared according to the method described by Bartsch et al. (16Bartsch R.G. Newton G.L. Sherrill C. Fahey R.C. J. Bacteriol. 1996; 178: 4742-4746Crossref PubMed Google Scholar) and purified by HPLC on a C18 reverse-phase column.Overexpression of GAR and Prx/GrxThe garB gene was PCR-amplified, allowing the incorporation of NdeI (forward primer, 5′-GGGAATTCCATATGACCCAGCATTTCG-3′) and BamHI (reverse primer, 5′-CACGGATCCTCAGGCCGC-3′) sites at the 5′ and 3′ termini of the gene, respectively. The PCR amplification of the garA gene also resulted in an NdeI/BamHI-bordered gene (forward primer, 5′-GGAATTCCATATGTTGCAAGATCG-3′; reverse primer, 5′-CACGGATCCTTAGGCGCTGGCGCGCTCC-3′). The amplified fragments were digested with NdeI and BamHI and were subsequently cloned into the NdeI/BamHI-digested expression plasmid pET11a. The resulting constructs (pET-GAR and pET-Prx/Grx), first verified by nucleotide sequencing, were then introduced into competent BL21(DE3) E. coli. For the preparation of the enzyme, one colony was used to inoculate 50 ml of LB medium containing 100 μg/ml carbeniciline, and the culture was incubated for 10 h. The culture was used to inoculate 4 liters of LB medium containing the antibiotic at a ratio of 10 ml/liter. When theA 600 value of the culture reached the value 0.7–1.0, isopropyl-β-d-thiogalactoside was added to a final concentration of 1 mm. Incubation was continued for 15 h at 37 °C, after which the cells were harvested by centrifugation (4000 × g) for 15 min, resuspended in 60 ml of sonication buffer (50 mm sodium phosphate buffer, pH 7.2, containing 1 mm EDTA), and stored at −80 °C overnight. The cells were broken using a Branson sonicator with four 30-s bursts of 45 watts with 30-s intervals.Protein PurificationGARThe crude extract from a GAR-producing culture was clarified by centrifugation at 15,000 × g for 30 min at 4 °C and fractionated with solid (NH4)2SO4. The 20–60% saturation precipitate was dissolved in 20 ml of buffer A (50 mmsodium phosphate buffer, pH 7.0, containing l mm EDTA and 1m (NH4)2SO4). The enzyme solution (2 ml/run) was applied onto a butyl-Sepharose packed HR 16/10 column (Amersham Pharmacia Biotech) equilibrated with buffer A. After loading, the resin was washed with 7 column volumes (CV) of the same buffer. The elution of GAR occurred with a decreasing step gradient of (NH4)2SO4 (step 1, 1000–450 mm in 8 CV; step 2, 450–375 mm in 10 CV; step 3, 375–0 mm in 1 CV) at the rate of 2 ml/min. The enzyme eluted during step 3 and the GAR-containing fractions of 10 runs were pooled, concentrated using 15-ml Vivaspin filters (molecular mass cutoff of 5 kDa), and dialyzed against 20 mm Tris-HCl, pH 7.0. The concentrate was further purified on the ResourceQ anion exchanger (Amersham Pharmacia Biotech) equilibrated with 20 mm Tris-HCl, pH 7.0. The yellow enzyme was eluted with a 30-CV linear gradient from 0 to 1 m NaCl in 20 mm Tris-HCl, pH 7.0, at a rate of 4 ml/min. The protein eluted at ∼0.49 m NaCl. Finally, the pooled fractions were concentrated and loaded onto a Hiload 16/60 Sephadex 75 gel-sizing column (Amersham Pharmacia Biotech) previously equilibrated with 10 mm Tris-HCl, pH 7.0. Elution occurred up-flow with 10 mm Tris-HCl, pH 7.0, at a flow rate of 2 ml/min. SDS-PAGE analysis of the pooled yellow-colored fractions indicated that GAR was purified to homogeneity. The gel-sizing protocol was also applied for native molecular mass determination. Molecular mass standards were derived from a gel filtration calibration kit (Amersham Pharmacia Biotech) containing ribonuclease A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), aldolase (158 kDa), and Dextran 2000 (2000 kDa).The absorption coefficient for the oxidized GAR prosthetic flavin was determined as described by Macheroux (28Macheroux P. Chapman S.K. Reid G.A. Flavoprotein Protocols. Humana Press, Tokawa, NJ1999: 1-7Google Scholar), except the cofactor was released from thermally denatured GAR. The concentration of homogeneous GAR was determined spectroscopically at 461 nm by using an absorption coefficient of 11.0 mm−1cm−1/active site.Prx/GrxThe cell debris of a Prx/Grx-containing crude extract was precipitated by centrifugation at 15,000 ×g for 30 min at 4 °C, and the supernatant was fractionated with solid (NH4)2SO4. The 30–70% saturation precipitate was dissolved in 20 ml of buffer B (50 mm sodium phosphate buffer, pH 7.4, containing 1 mm EDTA and 1.5 m(NH4)2SO4. The enzyme solution (2 ml/run) was then loaded onto an octyl-Sepharose packed HR 16/10 column (Amersham Pharmacia Biotech) equilibrated with buffer B. After loading, the resin was washed with the same buffer for 10 CV. The protein was eluted with a decreasing step gradient of (NH4)2SO4 (step 1, 1500–450 mm in 8 CV; step 2, 450–375 mm in 15 CV; step 3, 375–0 mm in 1 CV) at a rate of 2 ml/min. Fractions containing Prx/Grx, as assessed by activity measurements and SDS-PAGE analysis, were pooled during step 3, concentrated, and dialyzed against buffer C (20 mm Tris-HCl, pH 7.5, 0.1 mm EDTA). The dialyzed protein solution was applied onto a ResourceQ column pre-equilibrated with buffer C. The column was washed with 5 CV of buffer C, and the protein was eluted with a 25-CV linear salt gradient (0.0–1.0 m NaCl in buffer C) at a flow rate of 3.5 ml/min. Prx/Grx eluted at 0.22 m NaCl. SDS-PAGE analysis indicated the presence of the 27.5-kDa Prx/Grx enzyme together with a few contaminating protein bands.Mass DeterminationMass spectral analysis of the proteins was performed using nanospray ionization on a hybrid quadrupole time-of-flight mass spectrometer (Micromass, Whytenshawe, UK). The protein sample was diluted to ∼5 pmol/μl in acetonitrile/water/formic acid (1.1:0.01); 3 μl of the dilutions was loaded in a coated borosilicate needle (Protana, Odense, Denmark). The needle was placed into the quadrupole time-of-flight source, and after breaking the tip, a voltage of 1350 V was applied. To determine the native molecular weight and the type of quaternary structure, the solvent was replaced by water. The source temperature was 30 °C in all cases. The mass spectrometer was calibrated independently using NaCsI. Mass spectral identification of the prosthetic flavin of GAR was performed under the same conditions as described for the subunit mass determination, except the negative ion mode of analysis was applied.Enzyme AssayGARGAR activity was measured by two methods. For all determinations involving NADH, the GASSAG-dependent NADH oxidation was monitored at 340 nm (ε = 6.22 mm−1 cm−1) in 125 mmpotassium phosphate buffer, pH 7.1, containing 0.1 mm EDTA. However, in case NADPH was added as the source of reducing equivalents, monitoring GAR activity was based on the coupling of GASSAG reduction to the reduction of 5,5-dithio-bis-2-nitrobenzoate, which is measured at 412 nm (ε = 13.6 mm−1cm−1). Therefore, 500 μm5,5-dithio-bis-2-nitrobenzoate was added to the enzyme solution immediately before initiating the reaction. In all cases, the reaction was started with the simultaneous addition of substrate and reductant to the quartz cuvette containing the enzyme solution. All reactions were carried out at 25 °C. The steady-state kinetic data were analyzed by fitting them to the equation v =V max [S]/(K m + [S]).Prx/GrxPrx/Grx-dependent hydroperoxide reduction was demonstrated in a reconstitution assay by coupling the hydroperoxide-dependent GASH oxidation to the GASSAG-dependent NADH oxidation, again monitored at 340 nm. Unless otherwise stated, the reconstitution assay contained 0.4 units of GAR, 150 μm NADH, 500 μm GASH, 50 μg/ml partially purified Prx/Grx, and 100 μmH2O2 or small alkyl hydroperoxides in 0.5 ml of 125 mm potassium phosphate buffer, pH 7.9, and 0.1 mm EDTA. The reaction was started with the addition of the hydroperoxide. No blank rate of NADH oxidation was observed during the reconstitution assay devoid of hydroperoxide. In the specificity study, GASH, GAR, and NADH were replaced by yeast GR (0.4 units), GSH (500 μm), and NADPH (150 μm), respectively.RESULTSNucleotide and Amino Acid Sequence AnalysisThe isolated garB gene-containingBamHI fragment (Fig. 1) consists of 3519 base pairs and comprises three open reading frames (ORF): garA, garB, and an ORF encoding a polypeptide that is 67% identical to a hypothelical E. coliprotein. The GAR enzyme-encoding garB gene, 1392 base pairs long, translates to a polypeptide of 463 amino acids. However, in accordance with the situation for the E. coli (29Scrutton N.S. Berry A. Perham R.N. Biochem. J. 1987; 245: 875-880Crossref PubMed Scopus (41) Google Scholar) andPlasmodium falciparum GR enzymes (30Farber P.M. Becker K. Muller S. Schirmer R.H. Franklin R.M. Eur. J. Biochem. 1996; 239: 655-661Crossref PubMed Scopus (54) Google Scholar) and for theTrypanosoma congolense trypanothione reductase (31Sullivan F.X. Shames S.L. Walsh C.T. Biochemistry. 1989; 28: 4986-4992Crossref PubMed Scopus (58) Google Scholar), the N-terminal amino acid sequence analysis of the endogenous as well as of the recombinant GAR revealed that the initiator methionine is post-translationally deleted. Comparison with E. coli GR (Fig. 2) and Crithidia fasciculata trypanothione reductase (32Aboagye-Kwarteng T. Smith K. Fairlamb A.H. Mol. Microbiol. 1992; 6: 3089-3099Crossref PubMed Scopus (44) Google Scholar) reveals 49 and 34.5% amino acid sequence identity, respectively, and emphasizes the conservation of the motifs essential for binding of the prosthetic group and substrates in the C. gracile enzyme.Figure 2Alignment of the amino acid sequences ofC. gracile GAR , E. coli GR (44Greer S. Perham R.N. Biochemistry. 1986; 25: 2736-2742Crossref PubMed Scopus (135) Google Scholar), human erythrocyte GR (43Krauth-Siegel R.L. Blatterspiel R. Saleh M. Schiltz E. Schirmer R.H. Untucht-Grau R. Eur. J. Biochem. 1982; 121: 259-267Crossref PubMed Scopus (156) Google Scholar), and E. coli lipoamide dehydrogenase βαβ-fold region of the NADH-binding domain (49Stephens P.E. Lewis H.M. Darlison M.G. Guest J.R. Eur. J. Biochem. 1983; 135: 519-527Crossref PubMed Scopus (166) Google Scholar). Secondary structures are derived from the crystallographic analysis of the E. coli enzyme (59Mittl P.R. Schulz G.E. Protein Sci. 1994; 3: 799-809Crossref PubMed Scopus (76) Google Scholar) and exemplified via the ESPript 1.9 program. Identical residues at the same position in all the aligned sequences are in reverse contrast. Residues interacting with the disulfide substrate in the human enzyme are marked with an arrow, and the residues that interact with the C termini of the disulfide substrate are additionally marked with a star. The characteristic βαβ-fold region of NAD(P)H-binding domains is underlined, and the residues involved in the discrimination of the dinucleotide cofactor are marked with a square.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The garA and garB genes are separated by 185 base pairs and are transcribed in the same direction. A computer search for putative promoter sequences points to the cotranscription of the two ORFs. The enzyme deduced from garA comprises 247 amino acids and seems to be chimeric, having an N-terminal part that is significantly homologous to the Prx family enzymes (33Cheong N.E. Choi Y.O. Lee K.O. Kim W.Y. Jung B.G. Chi Y.H. Jeong J.S. Kim K. Cho M.J. Lee S.Y. Plant Mol. Biol. 1999; 40: 825-834Crossref PubMed Scopus (56) Google Scholar, 34Verdoucq L. Vignols F. Jacquot J.P. Chartier Y. Meyer Y. J. Biol. Chem. 1999; 274: 19714-19722Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) and a C-terminal domain that is Grx-like (35Nordstrand K. Slund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 1999; 286: 541-552Crossref PubMed Scopus (112) Google Scholar). Computer alignment of the entire Brassica rapa CPrxII, a Prx peroxidase that has been shown to catalyze the reduction of hydrogen peroxide with the use of electrons from the thioredoxin system (33Cheong N.E. Choi Y.O. Lee K.O. Kim W.Y. Jung B.G. Chi Y.H. Jeong J.S. Kim K. Cho M.J. Lee S.Y. Plant Mol. Biol. 1999; 40: 825-834Crossref PubMed Scopus (56) Google Scholar), with amino acid residues 1–163 of the garA deduced chimeric protein reveals 39% identity and striking homology around the N-terminal cysteine residue, which is believed to be the site of oxidation by peroxi" @default.
• W2081097951 created "2016-06-24" @default.
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• W2081097951 creator A5026427081 @default.
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• W2081097951 date "2001-06-01" @default.
• W2081097951 modified "2023-10-16" @default.
• W2081097951 title "Characterization of Glutathione Amide Reductase from Chromatium gracile" @default.
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