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• W1989511088 abstract "In Saccharomyces cerevisiae,expression of the ACR2 and ACR3 genes confers arsenical resistance. Acr2p is the first identified eukaryotic arsenate reductase. It reduces arsenate to arsenite, which is then extruded from cells by Acr3p. In this study, we demonstrate that ACR2complemented the arsenate-sensitive phenotype of an arsCdeletion in Escherichia coli. ACR2 was cloned into a bacterial expression vector and expressed in E. coli as a C-terminally histidine-tagged protein that was purified by sequential metal chelate affinity and gel filtration chromatography. Acr2p purified as a homodimer of 34 kDa. The purified protein was shown to catalyze the reduction of arsenate to arsenite. Enzymatic activity as a function of arsenate concentration exhibited an apparent positive cooperativity with an apparent Hill coefficient of 2.7. Activity required GSH and glutaredoxin as the source of reducing equivalents. Thioredoxin was unable to support arsenate reduction. However, glutaredoxins from both S. cerevisiae and E. coli were able to serve as reductants. Analysis ofgrx mutants lacking one or both cysteine residues in the Cys-Pro-Tyr-Cys active site demonstrated that only the N-terminal cysteine residue is essential for arsenate reductase activity. This suggests that during the catalytic cycle, Acr2p forms a mixed disulfide with GSH before being reduced by glutaredoxin to regenerate the active Acr2p reductase. In Saccharomyces cerevisiae,expression of the ACR2 and ACR3 genes confers arsenical resistance. Acr2p is the first identified eukaryotic arsenate reductase. It reduces arsenate to arsenite, which is then extruded from cells by Acr3p. In this study, we demonstrate that ACR2complemented the arsenate-sensitive phenotype of an arsCdeletion in Escherichia coli. ACR2 was cloned into a bacterial expression vector and expressed in E. coli as a C-terminally histidine-tagged protein that was purified by sequential metal chelate affinity and gel filtration chromatography. Acr2p purified as a homodimer of 34 kDa. The purified protein was shown to catalyze the reduction of arsenate to arsenite. Enzymatic activity as a function of arsenate concentration exhibited an apparent positive cooperativity with an apparent Hill coefficient of 2.7. Activity required GSH and glutaredoxin as the source of reducing equivalents. Thioredoxin was unable to support arsenate reduction. However, glutaredoxins from both S. cerevisiae and E. coli were able to serve as reductants. Analysis ofgrx mutants lacking one or both cysteine residues in the Cys-Pro-Tyr-Cys active site demonstrated that only the N-terminal cysteine residue is essential for arsenate reductase activity. This suggests that during the catalytic cycle, Acr2p forms a mixed disulfide with GSH before being reduced by glutaredoxin to regenerate the active Acr2p reductase. glutaredoxin thioredoxin thioredoxin reductase polymerase chain reaction base pair(s) 2-hydroxyethyl disulfide 3-(N-morpholino)propanesulfonic acid 2-(N-morpholino)ethanesulfonic acid polyacrylamide gel electrophoresis All organisms are constantly exposed to geochemical and anthropomorphic arsenic (1.Abernathy C.O. Liu Y.P. Longfellow D. Aposhian H.V. Beck B. Fowler B. Goyer R. Menzer R. Rossman T. Thompson C. Waalkes M. Environ Health Perspect. 1999; 107: 593-597Crossref PubMed Scopus (531) Google Scholar). Arsenic is a human carcinogen (2.Hayes R.B. Cancer Causes Control. 1997; 8: 371-385Crossref PubMed Scopus (369) Google Scholar) that is frequently present in high concentrations in drinking water (3.Smith A.H. Hopenhayn-Rich C. Bates M.N. Goeden H.M. Hertz-Picciotto I. Duggan H.M. Wood R. Kosnett M.J. Smith M.T. Environ. Health Perspect. 1992; 97: 259-267Crossref PubMed Scopus (954) Google Scholar). Despite the health hazards of arsenic, no specific human arsenic detoxification genes have been identified. Recently a gene cluster,ACR1, ACR2, and ACR3, onSaccharomyces cerevisiae chromosome XVI, was shown to confer resistance to arsenate (As(V)) and arsenite (As(III)), the first such eukaryotic genes to be identified (4.Bobrowicz P. Wysocki R. Owsianik G. Goffeau A. Ulaszewski S. Yeast. 1997; 13: 819-828Crossref PubMed Scopus (190) Google Scholar). Acr3p catalyzes extrusion of the arsenite from cells, thus conferring resistance (5.Ghosh M. Shen J. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5001-5006Crossref PubMed Scopus (345) Google Scholar). However, to confer resistance to arsenate, cells must first reduce it to arsenite. Although arsenate is nonenzymatically reduced by GSH, the process is too slow to be biologically significant (6.Delnomdedieu M. Basti M.M. Otvos J.D. Thomas D.J. Chem. Biol. Interact. 1994; 90: 139-155Crossref PubMed Scopus (264) Google Scholar), necessitating enzymatic mechanisms for reduction. Several bacterial arsenate reductases have been identified (7.Ji G. Garber E.A.E. Armes L.G. Chen C.M. Fuchs J.A. Silver S. Biochemistry. 1994; 33: 7294-7299Crossref PubMed Scopus (124) Google Scholar, 8.Oden K.L. Gladysheva T.B. Rosen B.P. Mol. Microbiol. 1994; 12: 301-306Crossref PubMed Scopus (124) Google Scholar), but until recently, there were no known eukaryotic arsenate reductases. The S. cerevisiae ACR2 gene was shown to be required for high level arsenate resistance (4.Bobrowicz P. Wysocki R. Owsianik G. Goffeau A. Ulaszewski S. Yeast. 1997; 13: 819-828Crossref PubMed Scopus (190) Google Scholar), and disruption of ACR2resulted in arsenate sensitivity (9.Mukhopadhyay R. Rosen B.P. FEMS Microbiol. Lett. 1998; 168: 127-136Crossref PubMed Google Scholar). The product of theACR2 gene, the 130-residue Acr2p, is a member of a family of proteins that are totally unrelated to any bacterial arsenate reductases. Two S. cerevisiae homologues of Acr2p areYGR203W, a 148-residue protein of unknown function (GenBankTM accession number S0003435), andYMR036C (GenBankTM accession number S0004639), a member of the Cdc25A family of protein phosphotyrosyl phosphatases (10.Fauman E.B. Cogswell J.P. Lovejoy B. Rocque W.J. Holmes W. Montana V.G. Piwnica-Worms H. Rink M.J. Saper M.A. Cell. 1998; 93: 617-625Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). These three proteins have the consensus sequence HCX 5R, which corresponds to the phosphatase active site (11.Fauman E.B. Saper M.A. Trends Biochem Sci. 1996; 21: 413-417Abstract Full Text PDF PubMed Scopus (319) Google Scholar). This suggests that some commonality may exist in the enzymatic mechanism of an arsenate reductase and a phosphatase, both of which have oxyanionic substrates. We have previously shown that ACR2-disrupted yeast cells are sensitive to arsenate but resistant to arsenite (9.Mukhopadhyay R. Rosen B.P. FEMS Microbiol. Lett. 1998; 168: 127-136Crossref PubMed Google Scholar), the same phenotype an arsC deletion produces in E. coli (8.Oden K.L. Gladysheva T.B. Rosen B.P. Mol. Microbiol. 1994; 12: 301-306Crossref PubMed Scopus (124) Google Scholar). Native Acr2p produced in E. coli was found exclusively in inclusion bodies. In contrast, a maltose-binding protein-Acr2p chimera was soluble and exhibited a low level of arsenate reductase activity when supplemented with yeast cytosol. However, the activity was low, and the source of reducing equivalents unknown. In this study, we demonstrate that the S. cerevisiae ACR2gene conferred arsenate resistance in an arsenate-sensitive strainE. coli. Conditions were established to isolate and purify a six-histidine-tagged Acr2p from E. coli cytosol. The enzyme was shown to have the mass of a homodimer. The source of reducing equivalents was identified as GSH and glutaredoxin (Grx).1 Acr2p exhibited arsenate reductase activity when the S. cerevisiaeglutaredoxin Grx1p and GSH were supplied as electron donors. TheS. cerevisiae thioredoxin (Trx) was unable to substitute for Grx. In addition, any of the three E. coli glutaredoxins supported Acr2p-catalyzed arsenate reduction. Glutaredoxins have the active site Cys-Pro-Tyr-Cys. The N-terminal cysteine is required for both protein disulfide reduction and reduction of mixed protein-glutathione disulfides (12.Bushweller J.H. Åslund F. Wuthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (204) Google Scholar). The other cysteine residue is required for the former activity but not for the latter. Mutation of the codon for the C-terminal cysteine of the E. coliglutaredoxin Grx2 had no effect on Acr2p activity. In contrast, a cysteine-to-serine substitution in the N-terminal residue rendered Grx2 incapable of serving as a reductant to Acr2p-catalyzed arsenate reduction. These results indicate that a mixed Acr2p-SG disulfide is formed during the catalytic cycle. This report provides the first characterization of the enzymatic activity of a eukaryotic arsenate reductase. Strains and plasmids used in this study are described in Table I. Cells of E. coli were grown in a low phosphate medium (8.Oden K.L. Gladysheva T.B. Rosen B.P. Mol. Microbiol. 1994; 12: 301-306Crossref PubMed Scopus (124) Google Scholar) or Luria-Bertani medium (13.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) at the indicated temperatures supplemented with 10 μg/ml tetracycline or 50 or 125 μg/ml ampicillin, as appropriate. S. cerevisiae strains were grown at 30 °C in complete yeast extract-peptone-dextrose (14.Adams A. Gottschling D.E. Kaiser C. Stearns T. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1998Google Scholar) medium supplemented with 2% glucose. Alternatively, the minimal (14.Adams A. Gottschling D.E. Kaiser C. Stearns T. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1998Google Scholar) medium with 2% glucose or galactose supplemented with auxotrophic requirements was used.Table IStrains and plasmidsStrain/plasmidGenotypeRef. or sourceBacterial strains JM109recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi Δ(lac-proAB) F′ [traD36 proA+B+ lac1q ΔlacZ M15]13.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar W3110K12 F−IN(rrnD-rrnE)33.Bachmann B.J. Neidhardt F.C. Ingraham J.L. Low K.B. Magasanik B. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1987: 1190-1219Google Scholar WC3110K12 F−IN(rrnD-rrnE) ΔarsCThis study TOP10F− mcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15ΔlacX74 deoR recA1 araD139Δ(araA-leu)7697 galU galK rpsL endA1 nupGInvitrogenYeast strains W303–1BMat-α ade2–1 his3–11,15 leu2–3,112 ura3–1 trp-134.Bowman S. Ackerman S.H. Griffiths D.E. Tzagoloff A. J. Biol. Chem. 1991; 266: 7517-7523Abstract Full Text PDF PubMed Google Scholar RMIMat-α ade2–1 his3–11,15 leu2–3,112 ura3–1 trp-1 ACR2∷HIS39.Mukhopadhyay R. Rosen B.P. FEMS Microbiol. Lett. 1998; 168: 127-136Crossref PubMed Google ScholarPlasmids PYES2.0Multicopy, shuttle vector, Apr,URA3, gal1Invitrogen pGEM-TMulticopy E. coli cloning vector, AprPromega pET28bE. coli cloning and expression vector, KmrNovagen pLD55E. coli replicative and conjugative plasmid, tetAR, Apr17.Metcalf W.W. Jiang W. Daniels L.L. Kim S.K. Haldimann A. Wanner B.L. Plasmid. 1996; 35: 1-13Crossref PubMed Scopus (373) Google Scholar pBAD/Myc-His A/CE. coli cloning and expression vectors, AprInvitrogen pBAD-ACR2pGEM-T-ACR2 was digested with NcoI andHindIII and inserted into theNcoI-HindIII site of pBAD/Myc-His AThis study pET-ArsCarsC gene from E. coliwas cloned into the NdeI-HindIII sites of pET28a8.Oden K.L. Gladysheva T.B. Rosen B.P. Mol. Microbiol. 1994; 12: 301-306Crossref PubMed Scopus (124) Google Scholar pYES-ArsCpET-ArsC was digested withNdeI and NotI. The NdeI site was made blunt, and the resulting fragment was inserted into pYES2.0 with a blunt BamHI end and a cohesive NotI endThis study pLD55-ΔarsCpLD55 with 800-bpSalI-BamHI insert containing 5′ and 3′ ends ofarsC with internal 400 bp deletedThis study pBAD-YGRX1pGEM-T-YGRX1 was digested with NcoI andEcoRI and inserted into the NcoI-EcoRI sites of pBAD/Myc-His CThis study pACR2–1393-bpACR2 gene was cloned by PCR from strain W303–1B into pET28b in frame with the C-terminal six-histidine tag9.Mukhopadhyay R. Rosen B.P. FEMS Microbiol. Lett. 1998; 168: 127-136Crossref PubMed Google Scholar pACR2–2496-bp ACR2–6H was cloned by PCR from pACR2–1 into pMAL-C2 in-frame with coding sequence for the N terminus of the mature form of the maltose-binding protein9.Mukhopadhyay R. Rosen B.P. FEMS Microbiol. Lett. 1998; 168: 127-136Crossref PubMed Google Scholar pACR2–3496-bp ACR2–6H was cloned into pYES2.0 from pACR2–2 by directional cloning9.Mukhopadhyay R. Rosen B.P. FEMS Microbiol. Lett. 1998; 168: 127-136Crossref PubMed Google Scholar pGEM-YGR2.4-kilobase fragment containing YGR203Wcloned into pGEM-TThis study yEP352E. coli-S. cerevisiae shuttle vector, Apr,URA3S. Ackerman yEP-PYGRYGR203Wcloned as 1257-bp HindIII-SphI fragment inserted into HindIII-SphI-digested yEP352This study pBAD-YTRX1pGEM-T-YTRX1 was digested with NcoI andHindIII and inserted into the NcoI andHindIII sites of pBAD-Myc-HisA.This study pBAD-YTRR1pGEM-T-YTRR1 was digested with NcoI andEcoRI and inserted into the NcoI andEcoRI sites of pBAD-Myc-HisC.This study pUC18-LEU2–8BglII fragment containingLEU2 of yEP13 was cloned into the BamHI site of pUC18S. Ackerman Open table in a new tab All nucleic acid modifying enzymes and restriction enzymes were obtained from Life Technologies, Inc. Plasmid isolation, DNA restriction endonuclease analysis, ligation, and transformation were performed as described (13.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Either Qiaprep Spin miniprep kit or Qiaquick gel extraction kit (Qiagen) was used to prepare plasmid DNA for restriction enzyme digestion, sequencing, and recovering DNA fragments from low melting point agarose gels. The sequence of each polymerase chain reaction (PCR) product was confirmed by DNA sequencing of the entire gene. Sequencing was performed using a Amersham Pharmacia Biotech Cy5 labeled autosequence kit and an ALFexpress apparatus by the method of Sanger et al. (15.Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52769) Google Scholar). The ACR2 gene from S. cerevisiae strain W303–1B genomic DNA was amplified by PCR to introduce a NcoI site at the 5′ end and aHindIII site at the 3′ end. The forward primer was 5′-CCATGGTAAGTTTCATAACGTC-3′, and the reverse primer was 5′-AAGCTTACCACTAACAATCAATTTAAGG-3′. A 30-cycle PCR (94 °C for 0.5 min, 55 °C for 0.5 min, and 72 °C for 1 min) was run with yeast genomic DNA. The 396-bp amplified product was cloned into pGEM-T. The resulting construct was digested with NcoI andHindIII and inserted into theNcoI-HindIII sites of pBAD/Myc-HisA in frame with the C-terminal Myc epitope and a six-histidine-residue tag, creating plasmid pBAD-ACR2. The GRX1 gene from S. cerevisiae strain W303–1B genomic DNA was amplified by PCR to introduce a NcoI site at the 5′ end and an EcoRI site at the 3′ end. The forward primer was 5′-CCATGGTATCTCAAGAAACTATC-3′, and the reverse primer was 5′-GAATTCATTTGCAAGAATAGGTTCTAAC-3′. A 30-cycle PCR (94 °C for 1.0 min, 55 °C for 0.5 min, and 72 °C for 1.2 min) was run with yeast genomic DNA. The 330-bp amplified fragment was cloned into pGEM-T. The resulting plasmid was digested with NcoI andEcoRI and inserted into the NcoI-EcoRI sites of pBAD/Myc-HisC in frame with the C-terminal Myc epitope and a six-histidine-residue tag, creating plasmid pBAD-YGRX1. A 2.4-kilobase pair fragment of yeast genomic DNA containingYGR203W was amplified by PCR using a forward primer, 5′-CTCATTGTCCTGCTCTTC-3′, that hybridizes with a region 614 bp upstream of YGR203W and a reverse primer, 5′-CTTGTAATGTCCGTACAGC-3′, that hybridizes to a region 1318 bp downstream of the gene. The fragment was ligated into vector pGEM-T, creating pGEM-YGR. The resulting plasmid was digested with HindIII andSphI, which cut 533 bp upstream and 278 bp downstream ofYGR203W, respectively. The resulting 1257-bp fragment was then ligated to HindIII-SphI-digested yEP352, a multicopy yeast-E. coli shuttle vector, creating plasmid yEP-PYGR. The thioredoxin gene TRX1 from S. cerevisiaestrain W303–1B genomic DNA was amplified by PCR to introduce aNcoI site at the 5′ end and a HindIII site at the 3′ end. The forward primer was 5′-CCATGGTTACTCAATTCAAAACTGC-3′, and the reverse primer was 5′-AAGCTTAGCATTAGCAGCAATGGCTTGC-3′. A 30-cycle PCR (94 °C for 1.5 min, 55 °C for 0.5 min, and 72 °C for 2.5 min) was run with yeast genomic DNA. The 318-bp amplified product was cloned into p-GEM-T, and the resulting plasmid was digested with NcoI and HindIII and inserted into theNcoI and HindIII sites of pBAD/Myc-HisA, in-frame with the C-terminal Myc epitope and a six-histidine-residue tag, creating plasmid pBAD-YTRX1. The thioredoxin reductase (Trr) gene TRR1 from S. cerevisiae strain W303–1B genomic DNA was amplified by PCR to introduce a NcoI site at the 5′ end and a EcoRI site at the 3′ end. The forward primer was 5′-CCATGGTTCACAACAAAGTTAC-3′, and the reverse primer was 5′-GAATTCTTCTAGGGAAGTTAAGTATTTC-3′. A 30-cycle PCR (94 °C for 1 min, 55 °C for 0.5 min, and 72 °C for 1.2 min) was run with yeast genomic DNA. The 966-bp amplified product was cloned into p-GEM-T, and the resulting plasmid was digested with NcoI andEcoRI and inserted into the NcoI-EcoRI sites of pBAD/Myc-HisC, in frame with the C-terminal Myc epitope and a six-histidine-residue tag, creating plasmid pBAD-YTRR1. Disruption ofYGR203W was carried out by a one-step method (16.Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2033) Google Scholar). Plasmid pGEM-YGR was digested with BamHI, which removes a 147-bp fragment from the open reading frame of the gene. The linearized fragment was made blunt using large fragment of DNA polymerase I and ligated with a 2.8-kilobase pair XbaI-SmaI fragment from plasmid pUC18-LEU2–8 containing the LEU2gene, in which the XbaI site of the fragment had been made blunt using a large fragment of DNA polymerase I before ligation. The resulting plasmid was digested with HindIII, and the 4.9-kilobase pair fragment was isolated and transformed into yeast strain W303–1B, producing the YGR203W-disrupted strain. Verification of the YGR203W disruption was confirmed by PCR using a forward primer 5′-CTCATTGTCCTGCTCTTC-3′ that hybridizes with a region 614 bp upstream of YGR203W and a reverse primer 5′-AAGCTTACGCCACAGATCGGGTAG-3′ that hybridizes with the 3′ end ofYGR203W. Disruption of the chromosomal arsCgene that confers arsenate resistance in E. coli was carried out by allelic replacement (17.Metcalf W.W. Jiang W. Daniels L.L. Kim S.K. Haldimann A. Wanner B.L. Plasmid. 1996; 35: 1-13Crossref PubMed Scopus (373) Google Scholar). Chromosomal DNA from E. coli strain W3110 was amplified as a 400-bp fragment by PCR using a forward primer, 5′-GTCGACCTGGCGTACTCAACGTGCTGGC-3′, that hybridizes with a region 386 bp upstream of the arsC gene and a reverse primer, 5′-AAGCTTGTAATGTTGCTCATATCAGTATCTC-3′, that hybridizes with a region that includes the first 14 bp from the 5′ end ofarsC. The primers added a SalI and aHindIII site at the 5′ and 3′ ends of the fragment, respectively. Using the same genomic DNA, a second PCR fragment of 400 bp was cloned using a forward primer, 5′-AAGCTTCGCCTGAAATAAAGCGGCGATATC-3′, that hybridizes with a region that includes the last 12 bp from the 3′ end of arsC and a reverse primer, 5′-GGATCCTTCTCTGATAGTGTGTGAAGT-3′, that hybridizes to a region 388 bp downstream of arsC. The second set of primers added a HindIII and a BamHI site at the 5′ and 3′ ends of the fragment, respectively. A 30-cycle PCR (94 °C for 1 min, 55 °C for 0.5 min, and 72 °C for 1 min) was run with E. coli genomic DNA. The respective products were cloned into pGEM-T. The first plasmid was digested with SalI andHindIII, and the second plasmid was digested withBamHI and HindIII. The fragments were then co-ligated into plasmid pLD55 that had been digested withBamHI and SalI, creating plasmid pLD55-▵arsC, in which 400 bp of the 426-bp arsCgene had been deleted. To replace the wild type chromosomalarsC gene with the deletion, plasmid pLD55-▵arsC was transformed into E. coli strain W3110. The transformants were grown on plates containing 15 μg/ml of tetracycline and 2.5 mm sodium pyrophosphate to select for integrants harboring the plasmid-encoded tetAR genes. Tetracycline-resistant colonies were then grown on tetracycline-sensitive-selective agar plates for selection of plasmid-free segregants. Colonies growing on tetracycline-sensitive-selective plates (17.Metcalf W.W. Jiang W. Daniels L.L. Kim S.K. Haldimann A. Wanner B.L. Plasmid. 1996; 35: 1-13Crossref PubMed Scopus (373) Google Scholar) were simultaneously screened for Tcs, Aps, and arsenate sensitivity. Disruption of arsC in the resulting strain, designated WC3110, was confirmed by PCR. The arsC gene from plasmid pET-ArsC (8.Oden K.L. Gladysheva T.B. Rosen B.P. Mol. Microbiol. 1994; 12: 301-306Crossref PubMed Scopus (124) Google Scholar) was cloned in the yeast-E. coli shuttle vector pYES2.0. Plasmid pET-ArsC was digested with NdeI and made blunt by using large fragment of DNA polymerase I. The linearized plasmid was then digested withNotI and purified. Plasmid pYES2.0 was first was first digested with BamHI and then made blunt using large fragment of DNA polymerase I. The linearized plasmid was digested withNotI and purified. The two linear fragments were ligated together to create plasmid pYES-ArsC. Cultures of E. coli strains W3110 and WC3110 bearing the indicated plasmids were grown overnight in low phosphate medium (8.Oden K.L. Gladysheva T.B. Rosen B.P. Mol. Microbiol. 1994; 12: 301-306Crossref PubMed Scopus (124) Google Scholar). The cells were diluted 100-fold in the same medium containing 0.2% arabinose and varying amounts of sodium arsenate and allowed to grow at 20 °C for an additional 48 h. Growth was estimated from the absorbance at 600 nm. Cultures of S. cerevisiae strains W303–1B and RM1 bearing the indicated plasmids were grown overnight at 30 °C in minimal medium containing 2% galactose supplemented with 0.2 mg/ml each of histidine and/or uracil, as appropriate. The cells were then diluted to an A 600 of 0.1 into same medium containing varying amounts sodium arsenate and allowed to grow for an additional 24 h. To determine the conditions for production of Acr2p in E. coli, a culture of strain TOP10 bearing plasmid pBAD-ACR2 was grown at 37 °C in 300 ml of LB medium containing 50 μg/ml of ampicillin to anA 600 of 0.5. The culture was then divided into three 100-ml aliquots and induced by addition of 0.02% L(+)-arabinose (final concentration). The cultures were allowed to grow for an additional 3 h at 37 °C, 6 h at 30 °C, or 10 h at 20 °C. A sample (1 ml) of each was harvested by centrifugation at 3,000 × g for 10 min. The cell pellets were suspended in 0.1 ml of SDS sample buffer and incubated for 10 min in a boiling water bath. The remainder of each culture was harvested and washed once with Buffer A (10 mm Tris-HCl, 0.1 m KCl, pH 7.5). The cells were suspended in 4 ml of Buffer B (50 mmMOPS, pH 7.5, containing 20 mm imidazole, 0.5 mNaCl, 10 mm β-mercaptoethanol, and 20% glycerol) and lysed by a single passage through a French pressure cell at 20,000 p.s.i. Diisopropylfluorophosphate (2.5 μl/g) was added immediately after lysis. The lysate was diluted to 6 ml with Buffer B and centrifuged at 100,000 × g for 1 h at 4 °C. The pellet was suspended in 6 ml of Buffer B. Portions of each of the inclusion bodies and cytosols were mixed with 4× SDS sample buffer and incubated at 37 °C for 10 min. Samples were analyzed by SDS-PAGE (18.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) on 15% polyacrylamide gels. The proteins were transferred overnight onto a nitrocellulose membrane at 25 V and probed with a monoclonal antibody to the six-histidine tag (CLONTECH) using anti-mouse whole IgG (Sigma) as the secondary antibody. Immunoblotting was performed using an enhanced chemiluminescence assay (NEN Life Science Products) and exposed on x-ray film at room temperature according to the directions provided byCLONTECH. E. coli glutaredoxins were purified as described previously (19.Shi J. Vlamis-Gardikas A. Åslund F. Holmgren A. Rosen B.P. J. Biol. Chem. 1999; 274: 36039-36042Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). For purification of the S. cerevisiae Grx1p, E. coli strain TOP10 bearing pBAD-YGRX1 was grown in 2 liters of LB medium containing 50 μg/ml ampicillin with shaking at 37 °C. At anA 600 nm of 0.5, L(+)-arabinose was added to a final concentration of 0.002% as inducer, and the culture was grown for an additional 4 h at 37 °C. The cells were washed once with Buffer A. The cells were suspended in Buffer B at a ratio of 5 ml of buffer/g of wet cells and lysed by a single passage through a French pressure cell at 20,000 p.s.i. Diisopropylfluorophosphate (2.5 μl/g of wet cells) was added to the lysate immediately after lysis. The lysate was centrifuged at 100,000 × g for 60 min at 4 °C, and the supernatant solution was loaded at a flow rate of 0.5 ml/min onto a 7-ml Ni2+-NTA column preequilibrated with Buffer B. The column was then washed with 250 ml of Buffer B followed by elution with 125 ml of Buffer C (50 mm MOPS, pH 7.5, containing 200 mm imidazole, 0.5 m NaCl, 10 mm β-mercaptoethanol, and 20% glycerol). Fractions containing Grx1p were identified by SDS-PAGE (18.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), pooled, and concentrated using a Millipore Ultrafree-15 BIOMAX-5K centrifugal filter (Millipore) at 2000 × g. Trx1p and Trr1p with C-terminal histidine tags were purified by Ni2+-NTA chromatography by essentially the same procedure as Grx1p. For purification of Acr2p, cells of E. coli strain TOP10 bearing pBAD-ACR2 were grown in 4 liters of LB medium containing 50 μg/ml ampicillin with shaking at 37 °C. At anA 600 nm of 0.5, L(+)-arabinose was added to a final concentration of 0.02% as inducer, and the culture was grown for an additional 10 h at 20 °C. The cells were washed once with Buffer A, suspended in Buffer B at a ratio of 5 ml of buffer/g of wet cells, and lysed by a single passage through a French pressure cell at 20,000 p.s.i. Diisopropylfluorophosphate (2.5 μl/g of wet cells) was added to the lysate immediately after lysis. The lysate was centrifuged at 100,000 × g for 60 min at 4 °C, and the supernatant solution was loaded at a flow rate of 0.5 ml/min onto a Ni2+-NTA column preequilibrated with Buffer B. The column was then washed with 350 ml of Buffer B followed by elution with 125 ml of Buffer C. Fractions containing Acr2p identified by SDS-PAGE, and fractions containing purified Acr2p were pooled and concentrated. The concentrated protein from the Ni2+-NTA column was applied to a 1.5-cm-diameter column filled to 75 cm with Sephacryl S-100 (Amersham Pharmacia Biotech) preequilibrated with Buffer D (50 mm MOPS, pH 6.5, containing 0.5 m NaCl, 10 mm β-mercaptoethanol, 20% glycerol, and 0.5 mm EDTA), eluted with the same buffer, pooled, and concentrated. All purified proteins were stored at −70 °C until use. Protein concentrations were determined from the absorbance at 280 nm using the following extinction coefficients for yeast proteins: Acr2p, 14,300m−1 cm−1; Grx1p, 5360 m−1cm−1; Trx1p, 9700m−1 cm−1; Trr1p, 23,380 m−1cm−1. Extinction coefficients were calculated by the method of Gill and von Hippel (20.Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar). The extinction coefficients for E. coli glutaredoxins were described previously (19.Shi J. Vlamis-Gardikas A. Åslund F. Holmgren A. Rosen B.P. J. Biol. Chem. 1999; 274: 36039-36042Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Arsenate reductase activity was assayed using a coupled assay as described previously (19.Shi J. Vlamis-Gardikas A. Åslund F. Holmgren A. Rosen B.P. J. Biol. Chem. 1999; 274: 36039-36042Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The assay buffer contained 50 mm MOPS, 50 mm MES, pH 6.5, 0.1 mg/ml bovine serum albumin, 0.4 mm NADPH, 15 nm yeast glutathione reductase (Calbiochem), 1 mm GSH, and 5 μm Acr2p. Reduction of 2-hydroxyethyldisulfide was used to ensure functioning of the coupling system. Sodium arsenate and glutaredoxins were added as indicated. Reductase activity was monitored at 340 nm and expressed as nmol of NADPH oxidized per mg of Acr2p using a molar extinction coefficient of 6200 for NADPH. The data were analyzed using SigmaPlot v. 5.0. TheS. cerevisiae ACR2 and E. coli arsC genes each confer arsenate resistance in their respective organisms. Acr2p and ArsC are totally unrelated, with no sequence similarity, and it was not known whether either yeast or E. coli has cofactors that would allow function of the heterologous reductase. It was of interest, therefore, to examine whether arsC could complement aS. cerevisiae strain in which ACR2 was disrupted and whether ACR2 could complement the arsenate-sensitive phenotype of an E. coli arsC disruption. The arsenate-sensitive S. cerevisiae strain RM1 was transformed with pYES-ArsC, which carries a wild type arsC gene under control of the GAL1 promoter. In presence of 2% galactose,arsC conferred arsenate resistance (Fig.1 A). When galactose was replaced" @default.
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