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• W2001504192 abstract "The biological methyl donor S-adenosylmethionine (AdoMet) can exist in two diastereoisomeric states with respect to its sulfonium ion. The S configuration, (S,S)-AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the R form, producing (R,S)-AdoMet. As of yet, (R,S)-AdoMet has no known physiological function and may inhibit cellular reactions. In this study, we found two Saccharomyces cerevisiae enzymes that are capable of recognizing (R,S)-AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, identified previously as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine, respectively. We found here that Sam4 recognizes both (S,S)- and (R,S)-AdoMet, but that its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet, whereas no activity is seen with the S,S form. R,S-Specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)-AdoMet in these organisms. The biological methyl donor S-adenosylmethionine (AdoMet) can exist in two diastereoisomeric states with respect to its sulfonium ion. The S configuration, (S,S)-AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the R form, producing (R,S)-AdoMet. As of yet, (R,S)-AdoMet has no known physiological function and may inhibit cellular reactions. In this study, we found two Saccharomyces cerevisiae enzymes that are capable of recognizing (R,S)-AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, identified previously as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine, respectively. We found here that Sam4 recognizes both (S,S)- and (R,S)-AdoMet, but that its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet, whereas no activity is seen with the S,S form. R,S-Specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)-AdoMet in these organisms. The aging process, as well as several human diseases, has been linked to the accumulation of spontaneously damaged biomolecules. Cells have evolved several ways of dealing with these altered molecules, including degradation, excretion, and repair pathways (1Martindale J.L. Holbrook N.J. J. Cell. Physiol. 2002; 192: 1-15Crossref PubMed Scopus (1923) Google Scholar, 2Clarke S. Ageing Res. Rev. 2003; 2: 263-285Crossref PubMed Scopus (234) Google Scholar, 3Lombard D.B. Chua K.F. Mostoslavsky R. Franco S. Gostissa M. Alt F.W. Cell. 2005; 120: 497-512Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar, 4Friquet B. FEBS Lett. 2006; 580: 2910-2916Crossref PubMed Scopus (186) Google Scholar, 5Veiga da Cunha M. Jacquemin P. Delpierre G. Godfraind C. Theate I. Vertommen D. Clotman F. Lemaigre F. Devuyst O. Van Schaftingen E. Biochem. J. 2006; 399: 257-264Crossref PubMed Scopus (60) Google Scholar, 6Hernebring M. Brolen G. Aguilaniu H. Semb H. Nystrom T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7700-7705Crossref PubMed Scopus (115) Google Scholar). The balance between the formation of age-altered molecules and the pathways that limit their cellular accumulation has been described as a battle between chemistry and biochemistry, where chemistry ultimately wins (2Clarke S. Ageing Res. Rev. 2003; 2: 263-285Crossref PubMed Scopus (234) Google Scholar). Although enzymes that recognize damaged DNA (3Lombard D.B. Chua K.F. Mostoslavsky R. Franco S. Gostissa M. Alt F.W. Cell. 2005; 120: 497-512Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar) and proteins (1Martindale J.L. Holbrook N.J. J. Cell. Physiol. 2002; 192: 1-15Crossref PubMed Scopus (1923) Google Scholar, 2Clarke S. Ageing Res. Rev. 2003; 2: 263-285Crossref PubMed Scopus (234) Google Scholar, 5Veiga da Cunha M. Jacquemin P. Delpierre G. Godfraind C. Theate I. Vertommen D. Clotman F. Lemaigre F. Devuyst O. Van Schaftingen E. Biochem. J. 2006; 399: 257-264Crossref PubMed Scopus (60) Google Scholar) have been well characterized, this is not yet the case for spontaneously altered small molecules. Of the large number of metabolites that are produced and used by biological systems, many are unstable, degrading into forms that may have reduced function or that may be toxic. One pathway of small molecule degradation and cellular recognition has been described recently. Here, trans-aconitate formed spontaneously from the citric acid cycle intermediate cis-aconitate results in the inhibition of at least two steps in the cycle (7Lauble H. Kennedy M.C. Beinert H. Stout C.D. J. Mol. Biol. 1994; 237: 437-451Crossref PubMed Scopus (73) Google Scholar, 8Rebholz K.L. Northrop D.B. Arch. Biochem. Biophys. 1994; 312: 227-233Crossref PubMed Scopus (24) Google Scholar). trans-Aconitate is then recognized by a specific yeast methyltransferase; the methyl ester formed has reduced inhibitory properties (9Cai H. Strouse J. Dumlao D. Jung M.E. Clarke S. Biochemistry. 2001; 40: 2210-2219Crossref PubMed Scopus (25) Google Scholar). One of the crucial small molecule metabolites in all organisms is S-adenosyl-l-methionine (AdoMet) 2The abbreviations used are: AdoMet, S-adenosyl-l-methionine; HPLC, high performance liquid chromatography; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; AdoHcy, S-adenosyl-l-homocysteine.2The abbreviations used are: AdoMet, S-adenosyl-l-methionine; HPLC, high performance liquid chromatography; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; AdoHcy, S-adenosyl-l-homocysteine. (10Loenen W.A. Biochem. Soc. Trans. 2006; 34: 330-333Crossref PubMed Scopus (242) Google Scholar, 11Fontecave M. Atta M. Mulliez E. Trends Biochem. Sci. 2004; 29: 243-249Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 12Lu S.C. Int. J. Biochem. Cell Biol. 2000; 32: 391-395Crossref PubMed Scopus (360) Google Scholar). Second to ATP, it is probably the most widely used cofactor in nature (12Lu S.C. Int. J. Biochem. Cell Biol. 2000; 32: 391-395Crossref PubMed Scopus (360) Google Scholar, 13Cantoni G.L. Annu. Rev. Biochem. 1975; 44: 435-451Crossref PubMed Scopus (408) Google Scholar). Not only does it serve as the primary methyl donor, but it also functions as an amino, adenosyl, and ribosyl donor (11Fontecave M. Atta M. Mulliez E. Trends Biochem. Sci. 2004; 29: 243-249Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). It also plays a role in the formation of adenosyl radicals (14Jarrett J.T. Curr. Opin. Chem. Biol. 2003; 7: 174-182Crossref PubMed Scopus (72) Google Scholar) and as a precursor of polyamines (15Urdiales J.L. Medina M.A. Sanchez-Jimenez F. Eur. J. Gastroenterol. Hepatol. 2001; 13: 1015-1019Crossref PubMed Scopus (48) Google Scholar). AdoMet has been shown to be unstable in cells, forming a variety of degradation products. Internal cyclization can form homoserine lactone and 5′-methylthioadenosine; hydrolysis at the glycosidic bond can form adenine and S-pentosylmethionine; and racemization at the sulfonium ion can form the R,S diastereomer (16Wu S.E. Huskey W.P. Borchardt R.T. Schowen R.L. Biochemistry. 1983; 22: 2828-2832Crossref PubMed Scopus (66) Google Scholar, 17Creason G.L. Madison J.T. Thompson J.F. Phytochemistry. 1985; 24: 1151-1155Crossref Scopus (15) Google Scholar, 18Hoffman J.L. Biochemistry. 1986; 25: 4444-4449Crossref PubMed Scopus (153) Google Scholar). We have been particularly interested in this latter reaction because pathways for the utilization or metabolism of this diastereomer have not been described. As biosynthesized, AdoMet is in the S,S configuration, where the first S corresponds to the stereochemistry at the sulfonium ion and the second to that at the α-carbon (19Cornforth J.W. Reichard S.A. Talalay P. Carrell H.L. Glusker J.P. J. Am. Chem. Soc. 1977; 99: 7292-7300Crossref PubMed Scopus (48) Google Scholar, 20De La Haba G. Jamieson G.A. Mudd S.H. Richards H.H. J. Am. Chem. Soc. 1959; 81: 3975-3980Crossref Scopus (61) Google Scholar). The S,S form appears to be the biologically active species (21Cannon L.M. Butler F.N. Wan W. Zhou Z.S. Anal. Biochem. 2002; 308: 358-363Crossref PubMed Scopus (34) Google Scholar, 22Bentley R. Chem. Soc. Rev. 2005; 34: 609-624Crossref PubMed Scopus (392) Google Scholar), and a number of methyltransferases have been shown to use it exclusively (20De La Haba G. Jamieson G.A. Mudd S.H. Richards H.H. J. Am. Chem. Soc. 1959; 81: 3975-3980Crossref Scopus (61) Google Scholar, 21Cannon L.M. Butler F.N. Wan W. Zhou Z.S. Anal. Biochem. 2002; 308: 358-363Crossref PubMed Scopus (34) Google Scholar, 23Zappia V. Zydek-Cwick C.R. Schlenk F. Biochim. Biophys. Acta. 1969; 178: 185-187Crossref PubMed Scopus (20) Google Scholar, 24Borchardt R.T. Wu Y.S. J. Med. Chem. 1976; 19: 1099-1103Crossref PubMed Scopus (49) Google Scholar). However, the instability of the sulfonium center results in spontaneous racemization under biological conditions to form (R,S)-AdoMet (Fig. 1) (20De La Haba G. Jamieson G.A. Mudd S.H. Richards H.H. J. Am. Chem. Soc. 1959; 81: 3975-3980Crossref Scopus (61) Google Scholar). If only the S,S form is used, and the R,S form is constantly produced by racemization, the levels of biologically inactive (R,S)-AdoMet should build up over time in cells. This material may not only take up precious cell space, but may also be toxic (24Borchardt R.T. Wu Y.S. J. Med. Chem. 1976; 19: 1099-1103Crossref PubMed Scopus (49) Google Scholar, 25Beaudouin C. Haurat G. Laffitte J.A. Renaud B. J. Neurochem. 1993; 61: 928-935Crossref PubMed Scopus (12) Google Scholar). The racemization of AdoMet may also be a factor in its pharmacology when used as a nutritional supplement (SAMe) (26Bottiglieri T. Am. J. Clin. Nutr. 2002; 76: 1151S-1157SCrossref PubMed Google Scholar). These preparations generally contain from 20 to 40% of the R,S form (27Hanna G.M. Pharmazie. 2004; 59: 251-256PubMed Google Scholar), and there is uncertainty at present about the relative contribution of each form to the therapeutic effect and whether there may be any toxicity associated with the R,S form. There is some indirect evidence to suggest that (R,S)-AdoMet can be metabolized in cells. Based on steady-state calculations for cells at physiological temperature and pH, the ratio of (R,S)- to (S,S)-AdoMet has been calculated as 19:81 (18Hoffman J.L. Biochemistry. 1986; 25: 4444-4449Crossref PubMed Scopus (153) Google Scholar). However, the R,S form is undetectable in soybean extracts (17Creason G.L. Madison J.T. Thompson J.F. Phytochemistry. 1985; 24: 1151-1155Crossref Scopus (15) Google Scholar), and the R,S/S,S ratio in mouse liver (18Hoffman J.L. Biochemistry. 1986; 25: 4444-4449Crossref PubMed Scopus (153) Google Scholar) and rat brain (25Beaudouin C. Haurat G. Laffitte J.A. Renaud B. J. Neurochem. 1993; 61: 928-935Crossref PubMed Scopus (12) Google Scholar) has been measured as 3:97. Thus, there appears to be a mechanism(s) that keeps the intracellular (R,S)-AdoMet levels low. We were thus interested in an older report that suggested that (R,S)-AdoMet might be utilized by a yeast methyltransferase (23Zappia V. Zydek-Cwick C.R. Schlenk F. Biochim. Biophys. Acta. 1969; 178: 185-187Crossref PubMed Scopus (20) Google Scholar). In this work, homocysteine methyltransferase activity was observed at twice the rate when an S,S/R,S mixture of AdoMet was used compared with that seen with the S,S form at an equal total concentration (23Zappia V. Zydek-Cwick C.R. Schlenk F. Biochim. Biophys. Acta. 1969; 178: 185-187Crossref PubMed Scopus (20) Google Scholar). Previous studies have shown that there are at least two homocysteine methyltransferase enzymes present in Saccharomyces cerevisiae (28Neuhierl B. Thanbichler M. Lottspeich F. Bock A. J. Biol. Chem. 1999; 274: 5407-5414Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 29Thomas D. Becker A. Surdin-Kerjan Y. J. Biol. Chem. 2000; 275: 40718-40724Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). These are encoded by the SAM4 and MHT1 genes and utilize AdoMet and S-methylmethionine, respectively, as methyl donors (29Thomas D. Becker A. Surdin-Kerjan Y. J. Biol. Chem. 2000; 275: 40718-40724Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). It is not clear whether either of these enzymes was responsible for the utilization of (R,S)-AdoMet seen initially (23Zappia V. Zydek-Cwick C.R. Schlenk F. Biochim. Biophys. Acta. 1969; 178: 185-187Crossref PubMed Scopus (20) Google Scholar). We were intrigued by the apparent lack of cellular logic in encoding an AdoMet-utilizing homocysteine methyltransferase. Homocysteine methyltransferases function to make methionine, which is then the precursor of AdoMet. The source of the methyl group can be N5-methyltetrahydrofolate, S-methylmethionine, or betaine (12Lu S.C. Int. J. Biochem. Cell Biol. 2000; 32: 391-395Crossref PubMed Scopus (360) Google Scholar, 29Thomas D. Becker A. Surdin-Kerjan Y. J. Biol. Chem. 2000; 275: 40718-40724Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). If AdoMet itself were used as the donor, the reaction would appear to be a futile cycle, resulting in the hydrolysis of 3 ATP eq (Fig. 2). However, such a “futile cycle” could be justified if the AdoMet-dependent homocysteine methyltransferase utilized the R,S form rather than the S,S form. In this scenario, the inactive R,S form, resulting from the unwanted racemization of the S,S form, would be converted to methionine, which could in turn be converted to the active S,S form of AdoMet. Such a mechanism could explain the low cellular levels of (R,S)-AdoMet observed (17Creason G.L. Madison J.T. Thompson J.F. Phytochemistry. 1985; 24: 1151-1155Crossref Scopus (15) Google Scholar, 18Hoffman J.L. Biochemistry. 1986; 25: 4444-4449Crossref PubMed Scopus (153) Google Scholar, 25Beaudouin C. Haurat G. Laffitte J.A. Renaud B. J. Neurochem. 1993; 61: 928-935Crossref PubMed Scopus (12) Google Scholar). In this work, we demonstrate that Sam4 and Mht1 in S. cerevisiae are both in fact capable of using (R,S)-AdoMet as a methyl donor. Sam4 has a higher specificity for (R,S)- than for (S,S)-AdoMet, and Mht1 uses (R,S)-AdoMet in exclusion of the S,S form. Thus, these two enzymes may work to prevent the accumulation of (R,S)-AdoMet within cells. Preparation and Purification of (R,S)- and (S,S)-[3H]AdoMet— S-Adenosyl-l-[methyl-3H]methionine (referred to throughout as [3H]AdoMet; 1 mCi/ml, 79.0 Ci/mmol, in dilute HCl/ethanol (9:1, v/v) at pH 2–2.5; GE Healthcare) was diluted 10-fold in 0.1 m HCl and divided into two aliquots. One aliquot was incubated for 8 days at 37 °C, resulting in an R,S-enriched [3H]AdoMet sample, whereas the other was kept at –80 °C. An aliquot (100 μl) of each preparation was then fractionated with a cationexchange HPLC column (Whatman Partisil SCX, 10-μm bead diameter, 4.6 mm, inner diameter, × 250 mm) using a method similar to that described previously (18Hoffman J.L. Biochemistry. 1986; 25: 4444-4449Crossref PubMed Scopus (153) Google Scholar). Briefly, the column was equilibrated and eluted with 80% buffer A (0.7 ml of concentrated NH4OH added to 1000 ml of 20% acetonitrile in H2O and brought to pH 3.0 with 88% acetic acid) and 20% buffer B (buffer A adjusted to 50 mm (NH4)2SO4 and then to pH 3.0 with concentrated sulfuric acid). The column was eluted at room temperature at a flow rate of 1 ml/min. The absorbance of the eluate was monitored at 254 nm, and 500-μl fractions were collected. An aliquot (50 μl) of each fraction was mixed with 2 ml of fluor (Safety-Solve, Research Products International Corp., Mount Prospect, IL) to measure radioactivity. The fractions corresponding to the R,S peak of the incubated sample and to the S,S peak of the non-incubated sample were each pooled, divided into 200-μl aliquots, and stored at –80 °C. [3H]AdoMet concentrations were calculated using a specific activity of 79,000 cpm/pmol provided by the manufacturer. NMR Analysis of AdoMet Stereochemistry—AdoMet (chloride salt; purity of ∼70% with 1 mol/mol H2O and 4.6% methanol; Sigma) was dissolved in 0.1 m HCl. One aliquot was incubated at 37 °C for 160 h, whereas the other was not incubated. Both were then dried and dissolved in D2O to a final concentration of 10 mg/ml. The 1H NMR spectrum for 500 μl of each sample was determined using a Bruker ARX400 spectrometer operating at 400.13 MHz as described previously (27Hanna G.M. Pharmazie. 2004; 59: 251-256PubMed Google Scholar). Isoaspartyl Protein Methyltransferase Assay—Human recombinant l-isoaspartyl protein methyltransferase (0.13 μg, specific activity of 20,990 pmol/min/mg of protein) was incubated with 0.016 pmol of either (R,S)- or (S,S)-[3H]AdoMet in the presence or absence of 3.5 nmol of the isoaspartyl-containing peptide KASA(isoD)LAKY (California Peptide Research, Inc., Napa, CA). The reaction was buffered in 50 mm BisTris-HCl (pH 6.4) in a final volume of 40 μl. After incubation for 45 min at 37 °C, methylation of the peptide was determined as described previously (30Lowenson J.D. Clarke S. J. Biol. Chem. 1991; 266: 19396-19406Abstract Full Text PDF PubMed Google Scholar). Yeast Strains—Table 1 lists the yeast strains used in this study. The sam4– and mht1– single knock-out mutant strains were generated by the Saccharomyces Genome Deletion Project (www-sequence.stanford.edu/group/yeast_deletion_project/available.html) and purchased from Invitrogen. The sam4–/ mht1– double knock-out strain was created by mating the MATα strain of sam4– with the MATa strain of mht1– and selecting for cells able to grow on lysine- and methionine-deficient plates. The resulting diploid strain was induced to sporulate, and the haploid spores containing both gene deletions were identified by screening on kanamycin plates for the non-parental ditype. The deletion of both the SAM4 and MHT1 genes was confirmed by PCR analysis using flanking TAG1 and TAG2 primers (www-sequence.stanford.edu/group/yeast_deletion_project/PCR_strategy.html).TABLE 1Yeast strainsStrainGenotypeSourceBY4741MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0SGDPaStrains prepared by the Saccharomyces Genome Deletion Project (SGDP) and purchased from Invitrogen.mht1- (BY4741)BY4741, Δyll062c::KanrSGDPaStrains prepared by the Saccharomyces Genome Deletion Project (SGDP) and purchased from Invitrogen.BY4742MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0SGDPaStrains prepared by the Saccharomyces Genome Deletion Project (SGDP) and purchased from Invitrogen.sam4- (BY4742)BY4742, Δypl273w::KanrSGDPaStrains prepared by the Saccharomyces Genome Deletion Project (SGDP) and purchased from Invitrogen.mht1- (BY4742)BY4742, Δyll062c::KanrSGDPaStrains prepared by the Saccharomyces Genome Deletion Project (SGDP) and purchased from Invitrogen.sam4-/mht1-MATα Δypl273w::Kanr Δyll062c::Kanr LYS+ MET+This studya Strains prepared by the Saccharomyces Genome Deletion Project (SGDP) and purchased from Invitrogen. Open table in a new tab Extract Preparation—Yeast extracts were prepared by modification of a previous method (31Porras-Yakushi T.R. Whitelegge J.P. Miranda T.B. Clarke S. J. Biol. Chem. 2005; 280: 34590-34598Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Briefly, a single colony was inoculated in 6 ml of YPD medium (1% Bacto-yeast extract, 2% Bacto-peptone, and 2% dextrose) and incubated overnight with shaking at 30 °C. One ml of the culture was then transferred to 250 ml of YPD medium and grown to an absorbance of 1.0 at 600 nm. Cells were pelleted at 5000 × g for 5 min and washed twice with 5 ml of water. The cell pellet volume was estimated, and 2 volumes of lysis buffer (0.1 m sodium phosphate (pH 7.0) and 1 mm phenylmethylsulfonyl fluoride) and 1 volume of baked zirconium beads (BioSpec Products, Inc., Bartlesville, OK) were added. Cells were then lysed via six cycles of alternate 1-min periods of vortexing and incubating on ice. The lysate was removed from the beads and centrifuged at 14,000 × g for 50 min at 4 °C. The supernatant was then divided into aliquots of 500 μl and kept at –80 °C until needed. The total protein concentration for each lysate was determined using the method of Lowry et al. (32Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) after precipitation in 10% trichloroacetic acid and using bovine serum albumin as a standard. Arabidopsis seed extract prepared as described previously (33Villa S.T. Xu Q. Downie A.B. Clarke S.G. Physiol. Plant. 2006; 128: 581-592Crossref PubMed Scopus (23) Google Scholar) was provided by Sarah Villa (UCLA). Mouse liver and brain extracts were prepared as described previously (34Miranda T.B. Lowenson J.D. Clarke S. FEBS Lett. 2004; 577: 181-186Crossref PubMed Scopus (10) Google Scholar) and provided by Jonathan Lowenson (UCLA). An extract of Caenorhabditis elegans prepared as described previously (35Kagan R.M. Clarke S. Biochemistry. 1995; 34: 10794-10806Crossref PubMed Scopus (42) Google Scholar) was provided by Tara Gomez (UCLA). Drosophila embryonic extract prepared as described previously (36Fellner T. Piribauer P. Ogris E. Methods Enzymol. 2003; 366: 187-203Crossref PubMed Scopus (8) Google Scholar) was provided by Thomas Fellner (UCLA). Homocysteine Methyltransferase Assays—A modification of the method of Shapiro et al. (37Shapiro S.K. Yphantis D.A. Almenas A. J. Biol. Chem. 1964; 239: 1551-1556Abstract Full Text PDF PubMed Google Scholar) was used. Unless indicated otherwise, 0.2 pmol of purified (R,S)- or (S,S)-[3H]AdoMet was incubated with or without the specified amount of dl-homocysteine (Sigma) in 0.15 m sodium phosphate (pH 7.0) with ∼0.1 mg of extract protein in a total volume of 200 μl. The temperature of incubation was dependent on the type of extract used: 37 °C for mouse; 30 °C for yeast, Drosophila and C. elegans; and 40 °C for Arabidopsis. At the end of each reaction incubation, [3H]methionine was separated from [3H]AdoMet by cation-exchange chromatography on Dowex 50WX8-400 columns (0.5 cm, inner diameter, × 2 cm; Sigma). Prior to use, the Dowex resin was washed alternately with 1 m HCl and 1 m NaOH, each step being separated by an H2O wash. This washing sequence was repeated a second time, after which the resin was finally equilibrated with 0.1 m sodium phosphate (pH 7.0). After the reaction mixtures were allowed to flow through the columns, the columns were eluted with 2 ml of H2O, and the total effluent was collected in scintillation vials, mixed with 15 ml of Safety-Solve fluor, and counted on a Beckman LS6500 counter. The amount of [3H]methionine produced was calculated from this radioactivity using a specific activity of 79,000 cpm/pmol. Preparation of (R,S)- and (S,S)-[3H]AdoMet—In preparing (R,S)-AdoMet, we took advantage of the fact that (S,S)-AdoMet can spontaneously racemize to the R,S form in vitro under physiological conditions to produce an S,S/R,S mixture that is roughly 50:50 (16Wu S.E. Huskey W.P. Borchardt R.T. Schowen R.L. Biochemistry. 1983; 22: 2828-2832Crossref PubMed Scopus (66) Google Scholar, 17Creason G.L. Madison J.T. Thompson J.F. Phytochemistry. 1985; 24: 1151-1155Crossref Scopus (15) Google Scholar, 18Hoffman J.L. Biochemistry. 1986; 25: 4444-4449Crossref PubMed Scopus (153) Google Scholar). In Fig. 3, we compare the 1H NMR spectra of a commercial preparation of AdoMet with and without incubation to induce racemization, which was performed at pH 1.0 to minimize the formation of other AdoMet degradation products such as methylthioadenosine and homoserine lactone (18Hoffman J.L. Biochemistry. 1986; 25: 4444-4449Crossref PubMed Scopus (153) Google Scholar). Clear peaks associated with the methyl group of AdoMet were found at 2.93 ppm in (S,S)-AdoMet and at 2.89 ppm in (R,S)-AdoMet (27Hanna G.M. Pharmazie. 2004; 59: 251-256PubMed Google Scholar). Under our conditions, we obtained a 49:51 mixture that was close to the equilibrium distribution. We then used these conditions to generate (R,S)-[3H]AdoMet from a preparation of (S,S)-[3H]AdoMet (Fig. 4). We were able to cleanly separate the isotopically labeled R,S and S,S forms by cation-exchange HPLC (Fig. 4) (cf. Ref. 18Hoffman J.L. Biochemistry. 1986; 25: 4444-4449Crossref PubMed Scopus (153) Google Scholar). To confirm the identity of the labeled (R,S)- and (S,S)-AdoMet samples obtained from the HPLC purification, we tested their ability to serve as substrates for a typical AdoMet-dependent methyltransferase that would be expected to use the S,S form. Fig. 5 shows that the human recombinant l-isoaspartyl protein repair methyltransferase uses purified (S,S)-[3H]AdoMet essentially to completion (94%), whereas no use is made of purified (R,S)-[3H]AdoMet.FIGURE 4Purification of (S,S)- and (R,S)-[3H]AdoMet. (S,S)-[3H]AdoMet and R,S-enriched [3H]AdoMet were fractionated by cation-exchange chromatography using a Partisil SCX HPLC column (4.6 mm, inner diameter, ×250 mm). R,S-Enriched [3H]AdoMet was prepared by incubation at 37 °C for 8 days at pH 1.0. Fractions (1 ml) were collected, and 50 μl was counted in 2 ml of scintillation fluor. □, radioactivity from the S,S sample; ○, radioactivity from the R,S-enriched sample. Fractions corresponding to the S,S and R,S peaks were pooled and stored at –80 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Confirmation of the configuration of purified (S,S)- and (R,S)-[3H]AdoMet by their utilization by the protein l-isoaspartyl O-methyltransferase. Human recombinant l-isoaspartyl protein methyltransferase activity was measured in the presence and absence of the peptide substrate KASA(isoD)LAKY using 0.016 pmol of either (S,S)- or (R,S)-[3H]AdoMet prepared as described in the legend to Fig. 4. Assays were done in triplicate, and error bars represent S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Yeast Sam4 and Mht1 Catalyze R,S-Dependent Homocysteine Methyltransferase Activity—We first investigated whether the extracts of wild-type S. cerevisiae could use (R,S)-AdoMet as a methyl donor in the conversion of homocysteine to methionine. We found that this was the case; after 60 min of incubation, we could quantitatively convert (R,S)-[3H]AdoMet to a neutral species consistent with [3H]methionine (Fig. 6). We found a similar result for (S,S)-[3H]AdoMet, indicating that both diastereomers could be utilized as methyl donors for yeast homocysteine methyltransferases. Control reactions in the absence of homocysteine revealed little or no methylation activity (Fig. 6), confirming the specificity of the assay. We also confirmed that the product was [3H]methionine in each case by co-chromatography of the product with a methionine standard by thin-layer chromatography using silica plates and a solvent system of n-butyl alcohol/acetic acid/water (4:1:1) (data not shown). Two homocysteine methyltransferases have been described in yeast, the products of the SAM4 and MHT1 genes (29Thomas D. Becker A. Surdin-Kerjan Y. J. Biol. Chem. 2000; 275: 40718-40724Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Thus, we measured the contribution of each of these enzymes to (R,S)-AdoMet utilization. When extracts of a sam4– knock-out strain were incubated with (S,S)-[3H]AdoMet, we found no homocysteine-dependent methyltransferase activity (Fig. 6), suggesting that (S,S)-AdoMet utilization in homocysteine methylation in yeast is limited to the Sam4 enzyme. On the other hand, homocysteine-dependent methyltransferase activity with (R,S)-[3H]AdoMet, although decreased, was still present (Fig. 6). This result provides the first direct evidence that the “degraded” R,S form of AdoMet may be utilized in a specific methyltransferase reaction. This result also suggests that, although Sam4 is responsible for most of the (R,S)-AdoMet-dependent homocysteine methyltransferase activity, there is at least one additional activity present that can utilize the R,S diastereomer, but not the S,S diastereomer, of AdoMet for methionine synthesis. We thus proceeded to determine whether Mht1 is responsible for all or part of the Sam4-independent homocysteine methyltransferase activity. We first analyzed activity in an extract of an mht1– deletion strain. We detected little change in activity with either (R,S)- or (S,S)-[3H]AdoMet, confirming the major role of the Sam4 enzyme. We then prepared a sam4–/ mht1– double knock-out strain. Extracts of this strain were found to be incapable of catalyzing homocysteine methylation with either (R,S)- or (S,S)-[3H]AdoMet (Fig. 6). This experiment demonstrates that the utilization of (R,S)-AdoMet for methionine synthesis in the sam4– extract is due to the Mht1 protein. This protein was demonstrated previously to catalyze homocy" @default.
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• W2001504192 title "Recognition of Age-damaged (R,S)-Adenosyl-L-methionine by Two Methyltransferases in the Yeast Saccharomyces cerevisiae" @default.
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• W2001504192 doi "https://doi.org/10.1074/jbc.m610029200" @default.
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