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W2051385199 abstract "Several plant species can tolerate high concentrations of selenium in the environment, and they accumulate organoselenium compounds. One of these compounds is Se-methylselenocysteine, synthesized by a number of species from the genus Astragalus (Fabaceae), like A. bisulcatus. An enzyme has been previously isolated from this organism that catalyzes methyl transfer fromS-adenosylmethionine to selenocysteine. To elucidate the role of the enzyme in selenium tolerance, the cDNA coding for selenocysteine methyltransferase from A. bisulcatus was cloned and sequenced. Data base searches revealed the existence of several apparent homologs of hitherto unassigned function. The gene for one of them, yagD from Escherichia coli, was cloned, and the protein was overproduced and purified. A functional analysis showed that the YagD protein catalyzes methylation of homocysteine, selenohomocysteine, and selenocysteine withS-adenosylmethionine and S-methylmethionine as methyl group donors. S-Methylmethionine was now shown to be also the physiological methyl group donor for the A. bisulcatus selenocysteine methyltransferase. A model system was set up in E. coli which demonstrated that expression of the plant and, although to a much lesser degree, of the bacterial methyltransferase gene increases selenium tolerance and strongly reduces unspecific selenium incorporation into proteins, provided thatS-methylmethionine is present in the medium. It is postulated that the selenocysteine methyltransferase under selective pressure developed from anS-methylmethionine-dependent thiol/selenol methyltransferase. Several plant species can tolerate high concentrations of selenium in the environment, and they accumulate organoselenium compounds. One of these compounds is Se-methylselenocysteine, synthesized by a number of species from the genus Astragalus (Fabaceae), like A. bisulcatus. An enzyme has been previously isolated from this organism that catalyzes methyl transfer fromS-adenosylmethionine to selenocysteine. To elucidate the role of the enzyme in selenium tolerance, the cDNA coding for selenocysteine methyltransferase from A. bisulcatus was cloned and sequenced. Data base searches revealed the existence of several apparent homologs of hitherto unassigned function. The gene for one of them, yagD from Escherichia coli, was cloned, and the protein was overproduced and purified. A functional analysis showed that the YagD protein catalyzes methylation of homocysteine, selenohomocysteine, and selenocysteine withS-adenosylmethionine and S-methylmethionine as methyl group donors. S-Methylmethionine was now shown to be also the physiological methyl group donor for the A. bisulcatus selenocysteine methyltransferase. A model system was set up in E. coli which demonstrated that expression of the plant and, although to a much lesser degree, of the bacterial methyltransferase gene increases selenium tolerance and strongly reduces unspecific selenium incorporation into proteins, provided thatS-methylmethionine is present in the medium. It is postulated that the selenocysteine methyltransferase under selective pressure developed from anS-methylmethionine-dependent thiol/selenol methyltransferase. selenocysteine polyacrylamide gel electrophoresis polymerase chain reaction kilobase pair(s) base pair Because of the chemical similarity of the elements sulfur and selenium, many organisms are unable to discriminate between the two in their metabolism. As a consequence, selenium is processed along the sulfur pathways and is incorporated unspecifically into low and high molecular weight compounds normally containing sulfur. The extent of replacement of sulfur by selenium depends on the ratio of the two elements in the environment and on the differential affinities of the sulfur pathway enzymes for their cognate substrate and the selenium-containing analog (for reviews, see Refs. 1Shrift A. Annu. Rev. Plant Physiol. 1969; 20: 475-494Crossref Google Scholar, 2Brown T.A. Shrift A. Biol. Rev. 1982; 57: 59-84Crossref Google Scholar, 3Läuchli A. Bot. Acta. 1993; 106: 455-468Crossref Scopus (196) Google Scholar, 4Stadtman T.C. Annu. Rev. Biochem. 1996; 65: 83-100Crossref PubMed Scopus (808) Google Scholar). There are, however, metabolic systems in which biological discrimination takes place. The first one is the specific synthesis and insertion of selenocysteine into proteins, directed by a UGA codon in the respective mRNA (4Stadtman T.C. Annu. Rev. Biochem. 1996; 65: 83-100Crossref PubMed Scopus (808) Google Scholar, 5Hüttenhofer A. Böck A. Simons R.W. Grunberg-Manago M. RNA Structure and Function. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1998: 603-639Google Scholar). Biosynthesis of selenocysteine occurs in a tRNA-bound state and, therefore, separate from sulfur metabolism. The crucial step in the discrimination between sulfur and selenium seems to reside in the synthesis of the selenium donor molecule monoselenophosphate by the enzyme selenophosphate synthetase (for a review, see Ref. 4Stadtman T.C. Annu. Rev. Biochem. 1996; 65: 83-100Crossref PubMed Scopus (808) Google Scholar). Monoselenophosphate is also the selenium donor for the conversion of 2-thiouridine into 2-selenouridine in several tRNA species (6Wittwer A.J. Stadtman T.C. Arch. Biochem. Biophys. 1986; 248: 540-550Crossref PubMed Scopus (43) Google Scholar, 7Stadtman T.C. Annu. Rev. Biochem. 1990; 59: 111-127Crossref PubMed Scopus (354) Google Scholar). The second biological phenomenon, in which discrimination between selenium and sulfur occurs is selenium tolerance of plants that accumulate high amounts of organoselenium compounds (for reviews, see Refs. 1Shrift A. Annu. Rev. Plant Physiol. 1969; 20: 475-494Crossref Google Scholar, 2Brown T.A. Shrift A. Biol. Rev. 1982; 57: 59-84Crossref Google Scholar, 3Läuchli A. Bot. Acta. 1993; 106: 455-468Crossref Scopus (196) Google Scholar). The majority of these plants belongs to the genusAstragalus (Fabaceae) and they are characterized by the following: (i) the accumulation of high amounts of selenium, mostly in the form of Se-methylselenocysteine (8Trelease S.F. DiSomma A.A. Jacobs A.L. Science. 1960; 132: 618Crossref PubMed Scopus (31) Google Scholar, 9Shrift A. Virupaksha T.K. Biochim. Biophys. Acta. 1963; 71: 483-485Crossref PubMed Scopus (15) Google Scholar, 10Shrift A. Virupaksha T.K. Biochim. Biophys. Acta. 1965; 100: 65-75Crossref PubMed Scopus (50) Google Scholar); (ii) an increased tolerance to selenium (11Trelease S.F. Science. 1942; 95: 656-657Crossref PubMed Scopus (1) Google Scholar); and (iii) a greatly reduced incorporation of selenium into cellular proteins (12Brown T.A. Shrift A. Plant Physiol. 1981; 67: 1051-1053Crossref PubMed Google Scholar). Numerous studies on the specificity of the enzymes in sulfur metabolism of these plants have shown that they are also involved in the synthesis of organoselenium compounds (for review, see Ref. 2Brown T.A. Shrift A. Biol. Rev. 1982; 57: 59-84Crossref Google Scholar). A general mechanism explaining the high selenium tolerance of these plants was not apparent, however. A common feature of selenium accumulator plants is that tolerance is always paralleled by synthesis of selenium-containing compounds like Se-methylselenocysteine, γ-glutamyl-Se-methylselenocysteine, or selenocystathionine (2Brown T.A. Shrift A. Biol. Rev. 1982; 57: 59-84Crossref Google Scholar). For this reason, it was hypothesized that the basis of selenium tolerance may reside in the existence of enzymes scrutinizing the cellular pool of sulfur metabolites for selenium compounds and converting them to adducts that are non-proteinogenic (12Brown T.A. Shrift A. Plant Physiol. 1981; 67: 1051-1053Crossref PubMed Google Scholar, 13Virupaksha T.K. Shrift A. Biochim. Biophys. Acta. 1965; 107: 69-80Crossref PubMed Scopus (44) Google Scholar, 14Burnell J.N. Plant Physiol. 1981; 67: 316-324Crossref PubMed Google Scholar). Indeed, a methyltransferase could be purified recently from a selenium accumulator species, Astragalus bisulcatus, which specifically methylated selenocysteine with S-adenosylmethionine as methyl donor. The activity of this selenocysteine methyltransferase (SeCys1 methyltransferase) with l-cysteine was at least 3 orders of magnitude lower than with l-selenocysteine (15Neuhierl B. Böck A. Eur. J. Biochem. 1996; 239: 235-238Crossref PubMed Scopus (148) Google Scholar). In the present communication we present the causal connection between synthesis of the SeCys-methyltransferase and selenium tolerance. The cDNA coding for this enzyme in A. bisulcatus has been cloned and shown to confer selenium tolerance when transferred toEscherichia coli, provided that the cognate methyl group donor is available. Moreover, we show that the enzyme belongs to a class of methyltransferases involved in the metabolism ofS-methylmethionine. E. coli strain JM109 (16Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-109Crossref PubMed Scopus (11450) Google Scholar) was used for general cloning purposes and as a source of chromosomal DNA for amplification of the yagDgene. SeCys-methyltransferase and YagD were overproduced in E. coli strain BL21(DE3) (17Studier F.W. Moffat B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4811) Google Scholar). E. coli strain MTD1, carrying an in frame deletion in the yagD gene, was constructed from strain KL19 (18Low B. Proc. Natl. Acad. Sci. U. S. A. 1968; 60: 160-167Crossref PubMed Scopus (276) Google Scholar) by deleting nucleotides 270–548 of the yagD open reading frame (19Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5989) Google Scholar) according to the method of Hamilton et al. (20Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). A. bisulcatus seeds were from the Western Regional Plant Introduction Station, Pullman, WA (reference number PI 372510). Seeds were germinated according to Brown and Shrift (12Brown T.A. Shrift A. Plant Physiol. 1981; 67: 1051-1053Crossref PubMed Google Scholar), and seedlings were grown at 25 °C under continuous illumination. Conditions for plant cell culture of A. bisulcatus have been described previously (15Neuhierl B. Böck A. Eur. J. Biochem. 1996; 239: 235-238Crossref PubMed Scopus (148) Google Scholar). E. coli cells were grown aerobically in LB medium (21Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar) or in M9 minimal medium (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Cultures for overproduction of SeCys-methyltransferase and YagD were grown in overproduction medium composed of 3% (w/v) tryptone, 1% yeast extract, and 1% NaCl. The ability of E. coli strains to detoxify selenium in the presence of S-methylmethionine was tested in M9 minimal medium containing 0.8% glucose. The concentrations of sulfate and selenate ions were adjusted to 100 and 50 μm, respectively. 2-ml cultures with this medium were supplemented with serial 1:2 dilutions of S-methylmethionine and inoculated in a ratio of 1:20 (v/v) with cultures that had been adjusted to anA 600 of 0.02. A culture withoutS-methylmethionine supplementation was used as a control. Cells were grown aerobically at 37 °C for 24 h, after which theA 600 was determined. Growth experiments were performed in at least two independent experiments. Thein vivo incorporation of 75Se into E. coli under aerobic growth conditions was followed with the method described by Cox et al. (23Cox J.C. Edwards E.S. DeMoss J.A. J. Bacteriol. 1981; 145: 1317-1324Crossref PubMed Google Scholar), replacing L broth by M9 minimal medium containing 0.8% glucose and 50 μg/ml chloramphenicol where appropriate. [75Se]sodium selenite (specific activity, 10 mCi/mmol) was present at 0.3 μm. Cells were harvested by centrifugation after reaching an A 600 of 0.25–0.4 and lysed in SDS sample buffer by heating to 95 °C for 10 min. The supernatant of the subsequent centrifugation (10 min at 14,000 × g and room temperature) was used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were dried on filter paper and autoradiographed overnight on a PhosphorScreen (Molecular Dynamics, Krefeld, Germany). Standard recombinant DNA techniques were employed according to Sambrook et al. (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA fragments were amplified by PCR using Goldstar polymerase (Eurogentech, Belgium) or Pfu polymerase (Stratagene) and primer concentrations in the range of 0.4–0.6 pmol/μl. The positive selection vector pUKE was constructed in the following way: first, a 1.3-kb fragment containing the kanamycin resistance gene was produced from pUC4 KSAC (Pharmacia, Freiburg, Germany) byPstI restriction and treatment with Klenow polymerase in the presence of all four nucleoside triphosphates. It was cloned into plasmid pUC19 (16Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-109Crossref PubMed Scopus (11450) Google Scholar), from which the ampicillin resistance gene had been removed by AvaII/AatII restriction and subsequent treatment with Klenow polymerase in the presence of all four nucleoside triphosphates. The resulting plasmid, containing the promoter of the kanamycin resistance gene in the opposite orientation with respect to the lac promoter, was designated pUKB. In the second step, the sequence coding for the EcoRI restriction endonuclease from plasmid pKGS (24Kuhn I. Stephenson F.H. Boyer H.W. Greene J. Gene (Amst.). 1986; 44: 253-263Crossref Scopus (35) Google Scholar) was amplified by PCR with primersEcoN (5′-TGTCTAATAAAAAACAGTC-3′, identical with nucleotides 2–20 of the EcoRI open reading frame) and EcoC (5′-TCACTTAGATGTAAGCTG-3′, complementary to nucleotides 817–834 of the open reading frame). The fragment was cloned into pUKB cleaved withEcoRI and treated with Klenow polymerase. The resulting plasmid, conferring sensitivity to the presence of 0.5 mmisopropylthiogalactoside in the medium (24Kuhn I. Stephenson F.H. Boyer H.W. Greene J. Gene (Amst.). 1986; 44: 253-263Crossref Scopus (35) Google Scholar), was named pUKE. Total RNA was extracted from 5 g of A. bisulcatus cells using hot acidic phenol as described by Aiba et al. (25Aiba H. Adhya S. de Crombrugghe B. J. Biol. Chem. 1981; 256: 11905-11910Abstract Full Text PDF PubMed Google Scholar) with the following modifications: the phenol solution originally described was replaced by a 1:1 (v/v) mixture of liquefied, unbuffered phenol and an 8m guanidinium chloride solution containing 0.3m sodium acetate. After heat treatment (20 min at 65 °C) and cooling to 4 °C, phase separation was achieved by addition of 0.5 volumes of chloroform, vigorous mixing, and centrifugation. After repeating the extraction with the aqueous phase, RNA was precipitated by addition of isopropyl alcohol to a final concentration of 50% (v/v) and sedimented by centrifugation. Poly(A)+ RNA was enriched on an oligo(dT)-cellulose matrix (Pharmacia, Freiburg, Germany), following the protocol provided by the manufacturer. Double-stranded cDNA was synthesized with the method of Gubler (26Gubler U. Nucleic Acids Res. 1988; 16: 2726Crossref PubMed Scopus (60) Google Scholar). A linker molecule produced by hybridization of the oligonucleotides Link1 (5′-AAGCTTGGTACCCGGG-3′) and 5′-phosphorylated Link2 (5′-AATTCCCGGGTACCAAGCTT-3′) was ligated to the cDNA, and molecules >400 bp were enriched using a “size sep 400” spin column (Pharmacia, Freiburg, Germany). The cDNA sequence coding for SeCys-methyltransferase fromA. bisulcatus was cloned by a PCR approach. First, SeCys-methyltransferase purified from A. bisulcatus (15Neuhierl B. Böck A. Eur. J. Biochem. 1996; 239: 235-238Crossref PubMed Scopus (148) Google Scholar) was hydrolyzed with endoprotease Endo-LysC, and peptides were purified and sequenced by Edman degradation. From these sequences degenerate oligonucleotides were derived and used as primers for PCR with cDNA from A. bisulcatus as a template. With primers 38MT34 (5′-ATIGC(AG)TC(AG)TAIGT(CT)TCICC-3′; where I indicates inosine) and 38MT66/2 (5′-GA(AG)GCICA(AG)GCITA(CT)GC-3′), a 270-bp fragment could be amplified, cloned into pUC19, and sequenced. From this sequence, two oligonucleotides with divergent orientation on the cDNA (38MT-1, 5′-CCATCCTTAGAGGTAAACGC-3′ and 38MT-2, 5′-ATCTGATACTTCTGCTTAAG-3′) were derived. They were used as primers for PCR with cDNA that had been hydrolyzed with HindIII and circularized by ligation at low concentration (<1 μg DNA/ml) as a template. From this amplification reaction, a 0.5-kb fragment could be cloned in pUKE and sequenced, which provided approximately 200 bp each of new sequence information in 5′ and 3′ direction. Using primers 38MT-2 and 38MT3 (5′-ATTATGTTCTCCTCTTCCAG-3′), PCR amplification was repeated with non-hydrolyzed, circularized cDNA as a template. A 0.75-kb fragment was cloned into pUKE and sequenced. This provided information on the 3′ end of the cDNA with a putative stop codon, although a poly(A) sequence was not found. The 5′ end of the cDNA was cloned by first amplifying circularized cDNA with oligonucleotides 38MT-1 and 38MT-2 as primers. Products >1 kb were gel-purified and used as templates for a second amplification with Link2 and 38MT11 (5′-TTGCTTCAAATGCAAGCAGG-3′). From the amplified products, a 0.6-kb DNA fragment could be cloned into pUKE and sequenced, which contained a putative ATG start codon and 57 bp of 5′-untranslated sequence. Plasmids for the overproduction of SeCys-methyltransferase and YagD protein were constructed from plasmid pT7-7 (27Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 262: 1074-1078Crossref Scopus (2452) Google Scholar); the open reading frames were amplified by PCR with Pfu polymerase (primers: 38MT5, 5′-TTACTAGTAGATTTGTTTGC-3′ and 38MT8, 5′-ATGTCGTCGCCATTGATAAC-3′ for SeCys-methyltransferase; EcMTN, 5′-ATGTCGCAGAATAATCCG-3′ and EcMTC, 5′-TCAGCTTCGCGCTTTTAAC-3′ for YAGD) and cDNA from A. bisulcatus or chromosomal DNA from E. coli JM109 for SeCys-methyltransferase and YagD, respectively. Amplified DNA fragments were treated with Klenow polymerase in the presence of dATP, dGTP, and dCTP. They were ligated into pT7-7 which had been cleaved withNdeI/SmaI and treated with Klenow polymerase under the same conditions as the PCR fragments. The vector containing the methyltransferase gene (smtA) from A. bisulcatus was named p7AMT and that carrying the yagDgene from E. coli was designated p7EMT. A derivative of pT7-7 containing the trxA gene from E. coli under the control of the T7 promoter was constructed as follows: the trxA gene was isolated from pSM1 (28Müller S. Senn H. Gsell B. Vetter W. Baron C. Böck A. Biochemistry. 1994; 33: 3404-3412Crossref PubMed Scopus (148) Google Scholar) byEcoRI restriction, treatment with Klenow polymerase, and restriction with HindIII. pT7-7 was cleaved withSalI, DNA ends were made flush with Klenow polymerase, the plasmid was re-cleaved with HindIII, and the trxAfragment was inserted by ligation. The resulting plasmid was named pT77T. The overexpression plasmid p7AMTT, containing the sequences coding for SeCys-methyltransferase from A. bisulcatus and for thioredoxin from E. coli in an artificial operon, was prepared by transferring the sequence coding for the SeCys-methyltransferase from p7AMT into pT77T via the XbaI restriction sites of the plasmids. Vectors for constitutive production of SeCys-methyltransferase and YagD in E. coli were constructed from pACYC184 (29Chang A.C.Y. Cohen S.N. J. Bacteriol. 1987; 134: 1141-1156Crossref Google Scholar). The plasmid was cleaved with EcoRV andHincII and blunt-end XbaI fragments from plasmids p7AMT or p7EMT, containing the open reading frames for SeCys-methyltransferase or YagD, respectively, were inserted by ligation so that the corresponding genes could be expressed constitutively via the tetracycline promoter. The resulting plasmids were designated pACAMTT and pACEMTT, respectively. Sequences of proteins similar to that from SeCys-methyltransferase were obtained via internet from The Institute of Genomic Research 2Information available on-line at the following address: http://www.tigr.org. and the National Institutes of Health. 3Information available on-line at the following address: http://www.ncbi.nlm.nih.gov. Sequence alignments were produced with MegAlign (version 0.97). SDS-PAGE was performed according to Laemmli (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar). Crude extracts from plant tissues and cultured cells of A. bisulcatus for SDS-PAGE were obtained by mixing plant material in a 1:1 ratio (w/v) with SDS sample buffer. The mixture was frozen in liquid nitrogen, boiled for 10–15 min, and centrifuged. The supernatant was used for gel electrophoresis. For the generation of a polyclonal antiserum directed against SeCys-methyltransferase purified from A. bisulcatus, a rabbit was immunized by intradermal injection of the protein in a custom immunization program at Eurogentec (Belgium). In immunoblotting experiments (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), the polyclonal anti-SeCys-methyltransferase antiserum was used in a 1:3000–1:5000 dilution employing the enhanced chemiluminescence system from Boehringer Mannheim. The YagD protein was overproduced in E. coli BL21(DE3) containing the plasmid p7EMT. Cells were grown in 8 liters of overproduction medium containing 100 μg/ml ampicillin in a 10-liter laboratory fermenter (Braun, Melsungen, Germany) at 37 °C and maximal aeration. At an A 600 of 1.5, overproduction was started by the addition of isopropylthiogalactoside to a final concentration of 50 μm. Incubation was continued until the mass production in the culture had ceased, thereafter cells were harvested by centrifugation, washed with C-Buffer (25 mm Tris/Cl, 10 mm magnesium acetate, 1 mm EDTA, and 2 mm dithiothreitol, pH 7.5), frozen in liquid nitrogen, and stored at −20 °C. All steps of the purification of YagD were performed at 0–4 °C. After each step, fractions were analyzed by SDS-PAGE followed by Coomassie staining, and those containing the YagD protein were pooled. For the preparation of a crude extract, 20 g of cells were resuspended in 40 ml of C-buffer containing 1 mmphenylmethylsulfonyl fluoride and lysed by three passages through a French pressure cell at 13,000 p.s.i. The soluble (S100) fraction was obtained by centrifugation (30 min at 30,000 × g, supernatant = S30, and 2 h at 100,000 × g, supernatant = S100) and adjusted to 30% ammonium sulfate saturation. The pellet of the following centrifugation was resuspended in a small volume of C-buffer and dialyzed overnight against 2 liters of C-buffer. Proteins were chromatographed on a 1.6 × 10-cm Q-Sepharose column (Pharmacia, Germany), using a gradient from 0 to 500 mm NaCl in C-buffer over 20 column volumes. YagD was precipitated overnight from 50% ammonium sulfate, sedimented by centrifugation, and dissolved in a small volume of C-buffer containing 150 mm KCl. The protein was chromatographed on a 1.6 × 60-cm Superdex 75 gel filtration column (Pharmacia, Germany). Fractions that appeared pure in an SDS gel were pooled, and the YagD protein was precipitated from 50% ammonium sulfate overnight. For long term storage, the preparation was dialyzed against 500 ml of C-buffer containing 50% glycerol and kept at −20 °C. The purification of SeCys-methyltransferase from cultured A. bisulcatus cells has been described previously (15Neuhierl B. Böck A. Eur. J. Biochem. 1996; 239: 235-238Crossref PubMed Scopus (148) Google Scholar). Overproduction of SeCys-methyltransferase in E. coli was performed with strain BL21(DE3) transformed with plasmids pUBS520 (31Tanaka H. Soda K. Methods Enzymol. 1987; 143: 240-243Crossref PubMed Scopus (38) Google Scholar) and p7AMTT as described for the YagD protein. Cells were lysed, and the soluble fraction was prepared as described for the YagD protein. The purification protocol for the recombinant protein was similar to the purification from cultured A. bisulcatuscells, with the following modifications: after ammonium sulfate precipitation, hydrophobic interaction chromatography on the phenyl-Sepharose column was used first, followed by gel filtration on Superdex 75. Fractions containing SeCys-methyltransferase were chromatographed on a 1.6 × 10-cm Q-Sepharose column using a gradient from 0–500 mm KCl in C-buffer over 20 column volumes. Apparently pure fractions were pooled, whereas fractions containing contaminating proteins were further purified on a MonoQ column (Pharmacia, Germany) using the same gradient as before. However, when elution of SeCys-methyltransferase started, the gradient was held at the same KCl concentration until all protein was eluted. Fractions containing pure protein were combined with the pool from Q-Sepharose chromatography, and SeCys-methyltransferase was precipitated from 70% ammonium sulfate overnight. For long term storage, SeCys-methyltransferase was treated as described for the YagD protein. The direct assay used for the determination of SeCys-methyltransferase activity has been described (15Neuhierl B. Böck A. Eur. J. Biochem. 1996; 239: 235-238Crossref PubMed Scopus (148) Google Scholar). In short, assays were carried out with purified enzyme in an anaerobic hood under an atmosphere of 97% N2 and 3% H2 to avoid oxidation of the substrates, which were pre-reduced for at least 30 min with a 10-fold molar excess of sodium borohydride. Reactions (total volume of 15 μl) consisted of 50 mm sodium citrate buffer, pH 6.0, 10 mm magnesium acetate, 2 mmdithiothreitol, 1 mm EDTA, 1 mm methyl acceptor substrate, and the appropriate amount of protein. After 5 min of preincubation at 30 °C, the reaction was started by addition of [methyl- 14C]S-adenosylmethionine (specific activity, 59.3 mCi/mmol, NEN Life Science Products), and incubation was continued at 30 °C. 2.5-μl samples were withdrawn from the reaction mixture at the appropriate times, pipetted into 2.5 μl of glacial acetic acid to stop the reaction, and spotted onto silica gel 60 thin layer chromatography plates in two 2.5-μl portions. Se-[methyl- 14C]selenocysteine was separated from [methyl- 14C]S-adenosylmethionine by developing in 1-butanol:acetic acid:H2O = 4:1:1. After autoradiography, quantification was carried out by densitometry with the aid of a PhosphorImager (Molecular Dynamics). Calibration was done by spotting serial dilutions of [methyl- 14C]S-adenosylmethionine stock solutions onto thin layer chromatography plates and by autoradiography on the same screen. The same assay was used for the YagD protein, including 100 μg/ml bovine serum albumin in the reaction mixture and changing the pH to 6.5. Reaction velocities with methyl donor substrates other than [methyl- 14C]S-adenosylmethionine were measured indirectly by [14C]iodoacetamide derivatization of remaining selenocysteine; reaction mixtures and conditions were the same as in the assay with [methyl- 14C]S-adenosylmethionine. At appropriate times, 2.5 μl from the reaction were withdrawn and transferred into 2.5 μl of a solution containing a 10-fold molar excess of [14C]iodoacetamide (adjusted to a specific radioactivity of 0.2 mCi/mmol) and kept at room temperature in the dark for 30 min. The mixtures were transferred to silica gel thin layer chromatography plates and treated as described for the [methyl- 14C]S-adenosylmethionine assay (15Neuhierl B. Böck A. Eur. J. Biochem. 1996; 239: 235-238Crossref PubMed Scopus (148) Google Scholar). Serial dilutions of selenocysteine were treated likewise and used as standards. The rates of blank reactions without enzyme were subtracted to correct for spontaneous oxidation and non-enzymatic methylation of selenocysteine. As in the direct assay, all experiments were conducted at least in duplicate. l-Selenocysteine was synthesized froml-3-chloroalanine according to Tanaka and Soda (31Tanaka H. Soda K. Methods Enzymol. 1987; 143: 240-243Crossref PubMed Scopus (38) Google Scholar) and was donated by S. Müller (Munich, Germany).dl-Selenohomocysteine was a gift from A. Holmgren (Stockholm, Sweden). All other chemicals were from commercially available sources. A newly constructed positive selection vector (pUKE) was used for cloning of the cDNA sequence coding for the SeCys-methyltransferase from A. bisulcatus. It was derived from pUC19 by exchanging the ampicillin resistance gene for the kanamycin resistance gene from plasmid pUC4KSAC and by ligating into the multiple cloning site the gene (endo) for theEcoRI restriction endonuclease, so that its expression was under the control of the lac promoter. Since production of the EcoRI enzyme is toxic for E. coli K-12 strains (24Kuhn I. Stephenson F.H. Boyer H.W. Greene J. Gene (Amst.). 1986; 44: 253-263Crossref Scopus (35) Google Scholar), this vector allows direct selection for plasmid molecules that contain an insert in the multiple cloning site 5′ of theendo gene. Using oligodeoxyribonucleotides derived from internal peptide sequences of the SeCys-methyltransferase and cDNA from A. bisulcatus, a 1.4-kb cDNA containing the gene (smtA) coding for this protein was cloned by PCR in several steps (see “Experimental Procedures”). Its nucleotide sequence was determined (Fig. 1); it codes for a putative protein of 3" @default.
W2051385199 created "2016-06-24" @default.
W2051385199 creator A5026352903 @default.
W2051385199 creator A5031863434 @default.
W2051385199 creator A5038987239 @default.
W2051385199 creator A5041718533 @default.
W2051385199 date "1999-02-01" @default.
W2051385199 modified "2023-10-11" @default.
W2051385199 title "A Family of S-Methylmethionine-dependent Thiol/Selenol Methyltransferases" @default.
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