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• W2040382520 abstract "The enzyme ThiI is common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli ThiI) found in this motif is essential for activity (Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J., and Mueller, E. G. (2000) J. Biol. Chem. 275, 8283–8286). In light of that finding and the report of the involvement of the protein IscS in the reaction (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561–16568), we proposed two mechanisms for the sulfur transfer mediated by ThiI, and both suggested possible involvement of the thiol group of another cysteine residue in ThiI. We have now substituted each of the cysteine residues with alanine and characterized the effect on activity in vivo and in vitro. Cys-108 and Cys-202 were converted to alanine with no significant effect on ThiI activity, and C207A ThiI was only mildly impaired. Substitution of Cys-344, the only cysteine residue conserved among all sequenced ThiI, resulted in the loss of function in vivo and a 2700-fold reduction in activity measured in vitro. We also examined the possibility that ThiI contains an iron-sulfur cluster or disulfide bonds in the resting state, and we found no evidence to support the presence of either species. We propose that Cys-344 forms a disulfide bond with Cys-456 during turnover, and we present evidence that a disulfide bond can form between these two residues in native ThiI and that disulfide bonds do form in ThiI during turnover. We also discuss the relevance of these findings to the biosynthesis of thiamin and iron-sulfur clusters. The enzyme ThiI is common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli ThiI) found in this motif is essential for activity (Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J., and Mueller, E. G. (2000) J. Biol. Chem. 275, 8283–8286). In light of that finding and the report of the involvement of the protein IscS in the reaction (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561–16568), we proposed two mechanisms for the sulfur transfer mediated by ThiI, and both suggested possible involvement of the thiol group of another cysteine residue in ThiI. We have now substituted each of the cysteine residues with alanine and characterized the effect on activity in vivo and in vitro. Cys-108 and Cys-202 were converted to alanine with no significant effect on ThiI activity, and C207A ThiI was only mildly impaired. Substitution of Cys-344, the only cysteine residue conserved among all sequenced ThiI, resulted in the loss of function in vivo and a 2700-fold reduction in activity measured in vitro. We also examined the possibility that ThiI contains an iron-sulfur cluster or disulfide bonds in the resting state, and we found no evidence to support the presence of either species. We propose that Cys-344 forms a disulfide bond with Cys-456 during turnover, and we present evidence that a disulfide bond can form between these two residues in native ThiI and that disulfide bonds do form in ThiI during turnover. We also discuss the relevance of these findings to the biosynthesis of thiamin and iron-sulfur clusters. 4-thiouridine pyridoxal 5′-phosphate dithiothreitol 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) IscS bearing the C-terminal His6tag encoded by pET29b polymerase chain reaction nickel-nitrilotriacetic acid The metabolism of many sulfur-containing biomolecules remains incompletely understood. Among the metabolic pathways requiring further elucidation are those leading to iron-sulfur clusters (1Zheng L.M. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, 2Agar J.N. Yuvaniyama D. Dean D.R. Johnson M.K. J. Inorg. Biochem. 1999; 74: 61Crossref Google Scholar, 3Agar J.N. Zheng L.M. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (116) Google Scholar, 4Yuvaniyama P. Agar J.N. Cash V.L. Johnson M.K. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (273) Google Scholar, 5Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar), biotin (6Bui B.T.S. Florentin D. Fournier F. Ploux O. Mejean A. Marquet A. FEBS Lett. 1998; 440: 226-230Crossref PubMed Scopus (110) Google Scholar, 7Gibson K.J. Pelletier D.A. Turner I.M. Biochem. Biophys. Res. Commun. 1999; 254: 632-635Crossref PubMed Scopus (59) Google Scholar, 8Begley T.P. Xi J. Kinsland C. Taylor S. McLafferty F. Curr. Opin. Chem. Biol. 1999; 3: 623-629Crossref PubMed Scopus (85) Google Scholar), molybdopterin (9Rajagopalan K.V. Biochem. Soc. Trans. 1997; 25: 757-761Crossref PubMed Scopus (64) Google Scholar), lipoic acid (10Ollagnier-de Choudens S. Fontecave M. FEBS Lett. 1999; 453: 25-28Crossref PubMed Scopus (62) Google Scholar), thiamin (8Begley T.P. Xi J. Kinsland C. Taylor S. McLafferty F. Curr. Opin. Chem. Biol. 1999; 3: 623-629Crossref PubMed Scopus (85) Google Scholar, 11Begley T.P. Downs D.M. Ealick S.E. McLafferty F.W. Van Loon A. Taylor S. Campobasso N. Chiu H.J. Kinsland C. Reddick J.J. Xi J. Arch. Microbiol. 1999; 171: 293-300Crossref PubMed Scopus (235) Google Scholar), and sulfur-containing bases in RNA (12Grosjean H. Benne R. Modification and Editing of RNA. American Society for Microbiology, Washington, D. C.1998Crossref Google Scholar). The sulfur-containing nucleosides include 4-thiouridine (s4U),1 which is found at position 8 of some bacterial tRNA (Fig.1) and serves as a photosensor for near-UV light (12Grosjean H. Benne R. Modification and Editing of RNA. American Society for Microbiology, Washington, D. C.1998Crossref Google Scholar). The s4U undergoes a photoactivated 2 + 2 cycloaddition with cytidine 13 when the tRNA is exposed to light of a wavelength similar to the 334 nm absorbance maximum of s4U (13Favre A. Yaniv M. Michelson A.M. Biochem. Biophys. Res. Commun. 1969; 37: 266-271Crossref PubMed Scopus (119) Google Scholar, 14Bergstrom D.E. Leonard N.J. Biochemistry. 1971; 11: 1-9Crossref Scopus (90) Google Scholar, 15Favre A. Michelson A.M. Yaniv M. J. Mol. Biol. 1971; 58: 367-379Crossref PubMed Scopus (112) Google Scholar). The resulting cross-linked tRNA are poor aminoacylation substrates (16Carre D.S. Thomas G. Favre A. Biochimie (Paris). 1974; 56: 1089-1101Crossref PubMed Scopus (65) Google Scholar), and the accumulation of uncharged tRNA arrests growth by triggering the stringent response (17Ramabhadran T.V. Jagger J. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 59-63Crossref PubMed Scopus (108) Google Scholar, 18Kramer G.F. Baker J.C. Ames B.N. J. Bacteriol. 1988; 170: 2344-2351Crossref PubMed Google Scholar). Lipsett and co-workers (19Peterkofsky A. Lipsett M.N. Biochem. Biophys. Res. Commun. 1965; 20: 780-786Crossref PubMed Scopus (9) Google Scholar, 20Abrell J.W. Kaufman D.E. Lipsett M.N. J. Biol. Chem. 1971; 246: 294-301Abstract Full Text PDF PubMed Google Scholar) investigated the enzymology of s4U biosynthesis inEscherichia coli and reported that the overall reaction utilized cysteine as the sulfur donor and required ATP as a substrate. Lipsett and co-workers (20Abrell J.W. Kaufman D.E. Lipsett M.N. J. Biol. Chem. 1971; 246: 294-301Abstract Full Text PDF PubMed Google Scholar) concluded that two enzymes were required and that one of them also plays a role in thiamin biosynthesis and requires the cofactor PLP for activity (21Lipsett M.N. J. Biol. Chem. 1972; 247: 1458-1461Abstract Full Text PDF PubMed Google Scholar, 22Ryals J. Hsu R.-Y. Lipsett M.N. Bremer H. J. Bacteriol. 1982; 151: 899-904Crossref PubMed Google Scholar). By using a genetic screen based on the role of s4U as a photosensor (18Kramer G.F. Baker J.C. Ames B.N. J. Bacteriol. 1988; 170: 2344-2351Crossref PubMed Google Scholar,22Ryals J. Hsu R.-Y. Lipsett M.N. Bremer H. J. Bacteriol. 1982; 151: 899-904Crossref PubMed Google Scholar, 23Thomas G. Favre A. C. R. Seances Acad. Sci. Ser. D. 1977; 284: 1345-1347PubMed Google Scholar, 24Lipsett M.N. J. Bacteriol. 1978; 135: 993-997Crossref PubMed Google Scholar), the genetic loci of two genes required for s4U biosynthesis (named nuvA and nuvC) were mapped (22Ryals J. Hsu R.-Y. Lipsett M.N. Bremer H. J. Bacteriol. 1982; 151: 899-904Crossref PubMed Google Scholar, 23Thomas G. Favre A. C. R. Seances Acad. Sci. Ser. D. 1977; 284: 1345-1347PubMed Google Scholar, 24Lipsett M.N. J. Bacteriol. 1978; 135: 993-997Crossref PubMed Google Scholar). By using the same genetic screen, we identified the genethiI as essential for s4U formation (25Mueller E.G. Buck C.J. Palenchar P.M. Barnhart L.E. Paulson J.L. Nucleic Acids Res. 1998; 26: 2606-2610Crossref PubMed Scopus (86) Google Scholar) shortly after Downs and co-workers (26Webb E. Claas K. Downs D.M. J. Bacteriol. 1997; 179: 4399-4402Crossref PubMed Google Scholar) identified the same gene as essential for thiamin biosynthesis. We have cloned and overexpressedthiI from E. coli (25Mueller E.G. Buck C.J. Palenchar P.M. Barnhart L.E. Paulson J.L. Nucleic Acids Res. 1998; 26: 2606-2610Crossref PubMed Scopus (86) Google Scholar). Kambampati and Lauhon (27Kambampati R. Lauhon C.T. Biochemistry. 1999; 38: 16561-16568Crossref PubMed Scopus (102) Google Scholar) isolated another enzyme, IscS, that sufficed along with ThiI forin vitro generation of s4U in tRNA, and they have since confirmed (28Lauhon C.T. Kambampati R. J. Biol. Chem. 2000; 275: 20096-20103Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) thatiscS mutants lack s4U and are thiamin auxotrophs. 2The congruence of nuvA andnuvC to thiI and iscS remains unclear due to several apparent discrepancies between the chromosomal locations of the genes and the phenotypes of nuvA and nuvCmutants relative to thiI and iscS mutants.2The congruence of nuvA andnuvC to thiI and iscS remains unclear due to several apparent discrepancies between the chromosomal locations of the genes and the phenotypes of nuvA and nuvCmutants relative to thiI and iscS mutants. IscS is a NifS-like protein that functions in iron-sulfur cluster formation, is PLP-dependent, and proceeds through an enzymic persulfide intermediate (29Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 30Zheng L.M. White R.H. Cash V.L. Dean D.R. Biochemistry. 1994; 33: 4714-4720Crossref PubMed Scopus (354) Google Scholar). Based on sequence alignments that revealed similarity between the segment of ThiI around Cys-456 and other sulfurtransferases, we investigated the importance of Cys-456 for the function of ThiI and found that C456A ThiI was inactive both in vivo and in vitro (31Palenchar P.M. Buck C.J. Cheng H. Larson T.J. Mueller E.G. J. Biol. Chem. 2000; 275: 8283-8286Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The report of the role of IscS in s4U biosynthesis fit nicely with our independent conclusion that ThiI would proceed through a persulfide intermediate on Cys-456 by providing a source of S0 to form that persulfide group. Since our report, Kambampati and Lauhon (32Kambampati R. Lauhon C.T. J. Biol. Chem. 2000; 275: 10727-10730Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) have established that sulfur flows from cysteine to IscS to ThiI to s4U in tRNA. Based on all the evidence, we proposed two alternative mechanisms for the biosynthesis of s4U (Fig.2), and both immediately suggest a role for another cysteine residue in ThiI (31Palenchar P.M. Buck C.J. Cheng H. Larson T.J. Mueller E.G. J. Biol. Chem. 2000; 275: 8283-8286Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). E. coli ThiI has four cysteine residues other than Cys-456, and sequence alignments of known ThiI proteins (all from prokaryotes) reveal that only one cysteine residue, Cys-344 in the E. coli enzyme, is completely conserved, 3P. Palenchar and E. Mueller, unpublished observations.3P. Palenchar and E. Mueller, unpublished observations.suggesting that this amino acid serves a critical role. We now report that this supposition is borne out by our investigations of the role of the cysteine residues in ThiI. Unless otherwise stated, all materials were purchased from either Sigma or Fisher and used as provided. Sephadex G-10, Sephadex G-25 (DNA grade), and [35S]cysteine were purchased from Amersham Pharmacia Biotech. Nuclease P1, dithiothreitol (DTT), chloramphenicol, kanamycin, ATP, and Tris were purchased fromRoche Molecular Biochemicals. Wizard® Genomic DNA Purification and pGEM®-T Easy Vector System II kits, E. coli JM109 cells, calf intestinal alkaline phosphatase, and Taq DNA polymerase were purchased from Promega Corp. (Madison, WI). Competent BLR(DE3) pLysS cells and pET29b were purchased from Novagen (Madison, WI). ThethiI mutant E. coli VJS2890(DE3) contains a kanamycin resistance cassette inserted within thiI (33Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.J. Costello C.K. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). A Higgins analytical CLIPEUS C18 5-μm column (50 × 4.6 mm) was purchased from Bodman Industries (Aston, PA). The tRNA substrate was the in vitro transcript of E. colitRNAPhe, and we described the preparation in detail elsewhere (34Ramamurthy V. Swann S.L. Paulson J.L. Spedaliere C.J. Mueller E.G. J. Biol. Chem. 1999; 274: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Ni-NTA superflow resin, QIAquick Gel Extraction kits, and QIAprep Spin Miniprep kits were purchased from Qiagen (Chatsworth, CA). A BioCad SPRINT perfusion chromatography system (PE Biosystems, Foster City, CA) was used for protein purification by chromatography over Poros 20 HS resin (PE Biosystems). Oligonucleotides (OPC-purified grade) were purchased from The Great American Gene Co. (Ramona, CA). QuikChangeTM site-directed mutagenesis kits were purchased from Stratagene (La Jolla, CA), and a Robocycler Gradient 96 Thermocycler (Stratagene) was used for the PCR component of the site-directed mutagenesis. DNA sequencing was performed either at the University of Delaware Cell Biology Core Facility using a Long Readir 4200 DNA sequencer (Li-Cor, Inc., Lincoln, NE) or at the Delaware Biotechnology Institute and University of Delaware Center for Agricultural Biotechnology core facility using an ABI Prism model 377 DNA sequencer (PE Biosystems). The complete sequences of all plasmids described in this publication are posted at www.udel.edu/chem/mueller. To substitute cysteine residues in ThiI with alanine, the QuikChangeTM mutagenesis protocol was used with appropriate primers (TableI) as we described previously (34Ramamurthy V. Swann S.L. Paulson J.L. Spedaliere C.J. Mueller E.G. J. Biol. Chem. 1999; 274: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). All of the ThiI variants described in this paper contain the 20-amino acid N-terminal His6 tag provided by pET15b, but all amino acid positions are numbered in terms of native ThiI (with no N-terminal His6 tag). The parent plasmid was pBH113S, which has N49S thiI in pET15b; as described previously (31Palenchar P.M. Buck C.J. Cheng H. Larson T.J. Mueller E.G. J. Biol. Chem. 2000; 275: 8283-8286Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), this N49S mutation arose spontaneously during propagation of pBH113, which is wild-type thiI in pET15b (25Mueller E.G. Buck C.J. Palenchar P.M. Barnhart L.E. Paulson J.L. Nucleic Acids Res. 1998; 26: 2606-2610Crossref PubMed Scopus (86) Google Scholar). All of the ThiI variants described here are N-terminally His6-tagged and have the N49S change; for the sake of simplicity, we will refer to them as “ThiI” (specified as either wild type or by the substituted cysteine residue).Table IPrimers for the site-directed mutagenesis of ThiI and the cloning and corrective site-directed mutagenesis of IscSPurposePrimersThiIC108AG GAA GGC AAA ACC TTC GCC GTA CGC GTG AAG CGC(pBH144)GCG CTT CAC GCG TAC GGC GAA GGT TTT GCC TTC CC202AATG TTG ATG CGT CGC GGC GCC CGC GTG CAT TAC(pBH143)GTA ATG CAC GCG GGC GCC GCG ACG CAT CAA CATC207AC CGC GTG CAT TACGCC TTC TTT AAC CTC GGC GGC(pBH142)GCC GCC GAG GTT AAA GAA GGC GTA ATG CAC GCG GC344ACGC ACG ATG CCG GAA TAT GCT GGT GTG ATC TCC AAA AGC(pBH141)GCT TTT GGA GAT CAC ACC AGC ATA TTC CGG CAT CGT GCGIscSCloningAGACAT ATG AAA TTA CCG ATT TAT CTCCCG AGA ATT CTT AAT GAT GAG CCC ATT CGA TCorrect P244LG CGT TCC GGC ACT CTG CCT GTT CAC CAG ATC GC GAT CTG GTG AAC AGG CAG AGT GCC GGA ACG CCorrect penultimate codonGC ATC GAA TGG GCT CAT CAT TAA GAA TTC GAG CTC CGT CGCG ACG GAG CTC GAA TTC TTA ATG ATG AGC CCA TTC GAT GCThe plasmids encoding altered ThiI are named in parentheses beneath the amino acid change. The primers are broken into codons (upper primer) or their complements (lower primer), and the changed bases are underlined. The restriction sites specified by the primers for the cloning ofiscS are shown in italics. Open table in a new tab The plasmids encoding altered ThiI are named in parentheses beneath the amino acid change. The primers are broken into codons (upper primer) or their complements (lower primer), and the changed bases are underlined. The restriction sites specified by the primers for the cloning ofiscS are shown in italics. The overexpression, purification, and storage of each altered ThiI was accomplished using the methods that we have described in detail elsewhere (35Mueller E.G. Palenchar P.M. Protein Sci. 1999; 8: 2424-2427Crossref PubMed Scopus (47) Google Scholar). Protein expression was induced by addition of isopropyl-β-d-thiogalactopyranoside to the growth medium, and the cells were harvested 3 h later. Chromatography of cell extracts over Ni-NTA resin yielded essentially homogeneous ThiI, which was changed into appropriate buffer and used immediately or precipitated by addition of solid ammonium sulfate (to nominal 75% saturation) for storage at 4 °C. By using the methods that we have described elsewhere (35Mueller E.G. Palenchar P.M. Protein Sci. 1999; 8: 2424-2427Crossref PubMed Scopus (47) Google Scholar), extinction coefficients at 280 nm were determined, and far-UV CD spectra were recorded for each altered ThiI. The generation, isolation, and properties of the C456A ThiI have been described previously (31Palenchar P.M. Buck C.J. Cheng H. Larson T.J. Mueller E.G. J. Biol. Chem. 2000; 275: 8283-8286Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The effect of substituting the cysteine residues of ThiI on its activity in vivo was assessed in two ways. First, the thiI mutant VJS2890(DE3) was transformed with a plasmid encoding an altered ThiI, and the transformants were subjected to near-UV screening. The screening of cells producing each altered ThiI was performed twice, except for the cells expressing C202A and C207A ThiI, which were performed four times; qualitatively identical results were obtained in every case. Second, the UV spectrum of tRNA isolated from saturated cultures of the transformants was recorded and examined for the characteristic peak due to s4U (λmax ∼334 nm). The procedures for both of these in vivocharacterizations have been described in detail elsewhere (25Mueller E.G. Buck C.J. Palenchar P.M. Barnhart L.E. Paulson J.L. Nucleic Acids Res. 1998; 26: 2606-2610Crossref PubMed Scopus (86) Google Scholar). This assay is essentially the one that we have described previously (31Palenchar P.M. Buck C.J. Cheng H. Larson T.J. Mueller E.G. J. Biol. Chem. 2000; 275: 8283-8286Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) except that recombinant IscS·His6 was substituted for cell extracts. The generation of s4U was monitored by following the incorporation of 35S into s4U froml-[35S]cysteine. A typical assay mixture (350 μl) was 50 mm Tris-HCl buffer, pH 8.5, containing ATP (4 mm), pyridoxal 5′-phosphate (40 μm), magnesium chloride (5 mm), in vitro transcript of E. coli tRNAPhe (20 μm),l-[35S]cysteine (484 μm; 123 μCi/μmol), DTT (1 mm), IscS·His6 (4 nm), and ThiI (1 nm). For the C344A ThiI, the assays contained higher concentrations of the proteins, 4.8 μm IscS·His6 and 1.2 μm C344A ThiI. Reactions were initiated by the addition of recombinant ThiI and incubated at 37 °C. At various times, aliquots (100 μl) were removed, and [35S]s4U was quantitated by the method that we have described in detail elsewhere (31Palenchar P.M. Buck C.J. Cheng H. Larson T.J. Mueller E.G. J. Biol. Chem. 2000; 275: 8283-8286Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In this method, unreacted l-[35S]cysteine is removed by size-exclusion chromatography, and the tRNA is digested to nucleosides, which are resolved by reverse phase high pressure liquid chromatography (36Gehrke C.W. Kuo K.C. McCune R.A. Gerhardt K.O. Agris P.F. J. Chromatogr. 1982; 230: 297-308Crossref PubMed Scopus (141) Google Scholar, 37Buck M. Connick M. Ames B.N. Anal. Biochem. 1983; 129: 1-13Crossref PubMed Scopus (118) Google Scholar); the level of 35S in the s4U is determined by in-line scintillation counting. Genomic DNA was purified from E. coli JM109 cells (Promega) using the Wizard Genomic DNA Purification protocol (Promega) according to the manufacturer's instructions. PCR amplification of iscS in the genomic DNA was achieved using appropriate primers (Table I), Taq DNA polymerase (Promega), and HotStart tubes (Molecular Bio-Products Inc., San Diego) according to the HotStart protocol. The primers (Table I) specified an NdeI site that includes the start ATG ofiscS and an EcoRI site that follows the TAA that terminates iscS. A Robocyler Gradient 96 thermal cycler (Stratagene) was used for the PCR, as described elsewhere (25Mueller E.G. Buck C.J. Palenchar P.M. Barnhart L.E. Paulson J.L. Nucleic Acids Res. 1998; 26: 2606-2610Crossref PubMed Scopus (86) Google Scholar). The PCR product was purified by agarose gel electrophoresis and recovered using the QIAquick gel extraction protocol (Qiagen). The isolated PCR product was ligated into pGEM-T using the pGEM-T Easy Vector System II (Promega) as specified by the manufacturer, and restriction analysis confirmed the generation of the target plasmid. This plasmid was digested with NdeI and EcoRI (New England Biolabs, Beverly, MA), and the iscS fragment was isolated by agarose gel electrophoresis and ligated into pET29b (Novagen) that had been opened with the same enzymes. The success of the construction was confirmed by restriction analysis, and the plasmid was named pBH400. Sequencing of iscS in pBH400 revealed two discrepancies from the published sequence as follows: a T → C transition in codon 244 that results in the substitution of proline for leucine and a deletion of the T in the penultimate codon that moves the stop codon specified by the “reverse” PCR primer out of frame and brings the C-terminal His6 tag encoded by pET29b into frame. IscS bearing the C-terminal His6 tag will be denoted IscS·His6. Both alterations of iscS in pBH400 were corrected using the QuikChangeTM protocol (Stratagene) with appropriate primers (Table I) as we have described previously (34Ramamurthy V. Swann S.L. Paulson J.L. Spedaliere C.J. Mueller E.G. J. Biol. Chem. 1999; 274: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The plasmid encoding L244P IscS (with no C-terminal His6 tag) is pBH401; the plasmid encoding IscS·His6 (with Leu-244 restored) is pBH402; the plasmid encoding native IscS (with Leu-244 restored and no C-terminal His6 tag) is pBH403. The procedure for overexpression and purification of IscS·His6 and L244P IscS·His6 was identical to that for the overexpression and purification of ThiI (35Mueller E.G. Palenchar P.M. Protein Sci. 1999; 8: 2424-2427Crossref PubMed Scopus (47) Google Scholar) except that cultures of BLR(DE3) pLysS/pBH402 or BLR(DE3) pLysS/pBH400 were grown and induced in LB medium containing kanamycin (30 μg/ml) and chloramphenicol (34 μg/ml). Isolated IscS·His6 (∼40 mg/liter of culture) was dialyzed against 50 mm potassium phosphate buffer, pH 7.5, containing magnesium chloride (5 mm), potassium chloride (100 mm), and EDTA (0.1 mm). The enzyme remains stable for weeks to months when stored at 4 °C in the same buffer at moderate concentration (3–4 mg/ml). By comparingA280 nm and the concentration of IscS measured by the biuret assay (35Mueller E.G. Palenchar P.M. Protein Sci. 1999; 8: 2424-2427Crossref PubMed Scopus (47) Google Scholar), we calculated ε280 nm = 25,400m−1 cm−1, which we now use to determine the concentration of IscS·His6. BLR(DE3) pLysS/pBH402 (encoding IscS·His6) and BLR(DE3) pLysS/pBH403 (encoding native IscS) was transformed with pBH113 (encoding wild-type ThiI) using the TransformAidTM protocol (MBI Fermentas, Hanover, MD). The joint overexpression of ThiI and IscS was accomplished as described for the overexpression of either protein alone. ThiI overexpressed with native IscS was cleanly separated from the latter by chromatography over Ni-NTA resin. ThiI overexpressed with IscS·His6 was co-purified with the latter by chromatography over Ni-NTA resin; the two proteins were then separated by chromatography over Poros 20 HS resin, eluting with a linear gradient (over 13 column volumes) of potassium chloride (0–1.5m) in 50 mm potassium phosphate buffer, pH 7.5, containing DTT (1 mm). The UV-visible spectrum was recorded after exchange of isolated ThiI into 50 mm potassium phosphate buffer, pH 7.5, containing magnesium chloride (5 mm), potassium chloride (100 mm), and EDTA (0.1 mm) by size-exclusion chromatography over a spin column of Sephadex G-25 equilibrated in the same buffer. Samples of ThiI were subjected to DTNB titrations under both native and denaturing conditions. The enzyme samples were fresh preparations that had been isolated using buffers to which no reductant had been added. Any thiol-bearing components of the cell extracts (either small or macro-molecules) should have been removed by the chromatography over Ni-NTA and the exchange (by size-exclusion chromatography or dialysis) of ThiI-bearing column fractions into 50 mm potassium phosphate buffer, pH 7.5, containing magnesium chloride (5 mm), potassium chloride (100 mm), and EDTA (0.1 mm). The protein concentration ranged from 4.7 to 16.6 μm, and DTNB (20 eq relative to ThiI) was added as a solution (10 mm) in the same buffer. Control incubations were prepared by adding the same amount of DTNB to the same buffer (with no protein). The final mixtures (400 μl) were incubated 20–30 min at room temperature, and the intensely yellow dianion of 5-thio-2-nitrobenzoic acid was quantified by subtracting the A412 nm of the control incubation from the A412 nm of each sample and using an extinction coefficient of 13,600 m−1cm−1 (38Dawson R.M.C. Elliott D.C. Elliott W.H. Jones K.M. Data for Biochemical Research. 3rd Ed. Oxford Science Publications, Oxford1986: 388Google Scholar). To denature the wild-type ThiI, solid guanidinium chloride (∼0.23 g, ∼2.4 mmol) was added to the samples and dissolved to achieve a final guanidinium concentration of ∼4.6m; the volume and A412 nm values were measured after 20 min at room temperature. All titrations were performed in duplicate except for the C456A ThiI under native conditions, which was performed in quadruplicate. Titrations of ThiI with 1 eq of DTNB were performed under native conditions as described for titration with 20 eq of DTNB. All replicate experiments returned values within 5% of each other. To test for disulfide bond formation in ThiI during turnover, the s4U generation assay was run without DTT, with a reduced concentration of cysteine (which can reduce disulfide bonds), and in phosphate buffer to allow separation of IscS and ThiI by cation-exchange chromatography. The assay (1 ml) consisted of 150 mm potassium phosphate buffer, pH 8.5, containing ATP (4 mm), PLP (40 μm), tRNA (18 μm), cysteine (70 μm), IscS (24 μm), and ThiI (6 μm). After 5 h at 37 °C, ThiI was separated from IscS and other reaction components by chromatography over Poros 20 HS resin as described above except that DTT was omitted from the buffers. The ThiI eluted in two fractions, which were combined (1 ml; 2–3 μm), concentrated in a Microcon-10 device, and subjected to DTNB titrations under denaturing conditions as described above. As a control for oxidation under these conditions, ThiI was also incubated in the absence of tRNA and cysteine and subjected to the same work up. To measure the extent of s4U formation under these conditions, a duplicate reaction containing [35S]cysteine was run in parallel; 0.73 ± 0.02 eq (relative to ThiI) of s4U was generated. Assuming that turnover results in the formation of one disulfide bond in ThiI, the expected number of free thiol groups in ThiI isolated after turnover is (0.73) (3 thiol groups) + (0.27) (5 thiol groups) = 3.5 thiol groups. The PCR-based cloning ofiscS from E. coli genomic DNA resulted in the generation of an IscS overexpression plasmid based on pET29b (pBH400). Sequencing revealed a deletion in the penultimate codon ofiscS during the PCR, resulting in a frameshift that fused a C-terminal His6 tag to IscS (denoted IscS·His6). In addition, a proline residue was encoded by codon 244 rather than the leucine specified by the sequence in the genomic data base. Overexpressed and purified L244P IscS·His6 proved fully competent for in vitroassay of ThiI activity. Site-directed mutagenesis was used to generate overexpression plasmids for Is" @default.
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• W2040382520 title "The Role of the Cysteine Residues of ThiI in the Generation of 4-Thiouridine in tRNA" @default.
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