Characterization of a full-length cDNA encoding human liver S-adenosylmethionine synthetase: tissue-specific gene expression and mRNA levels in hepatopathies.
ABSTRACT: The sequence of a full-length cDNA coding for human liver S-adenosylmethionine synthetase has been determined. It spans 3217 nucleotides and encodes a protein of 395 amino acid residues, with a calculated molecular mass of 43,647 Da. The structural features deduced from the amino acid sequence show a close similarity to those of the rat liver enzyme. The liver-specific S-adenosylmethionine synthetase gene appears to be present as a single copy in the genome, as revealed by Southern analysis. The occurrence of a single mRNA species for this enzyme has been determined by primer extension and Northern analysis. Among several human tissues examined, this gene is expressed only in the liver. Similar S-adenosylmethionine synthetase mRNA levels have been detected in biopsies from normal human liver and from patients with alcoholic cirrhosis and hepatocellular carcinoma. Based on these results, a possible mechanism of regulation of human liver S-adenosylmethionine synthetase is discussed.
Project description:The active site of rat liver S-adenosylmethionine synthetase was studied using 8-azido ATP, a photolabile analogue of ATP. Both forms of the enzyme, tetramer and dimer, could be labelled by using concentrations of the analogue similar to the KmATP values for each form, 350 microM and 1 mM respectively. Labelling of both S-adenosylmethionine synthetase forms with 8-azido [alpha-32P]ATP, followed by tryptic digestion and purification by HPLC, afforded one specifically labelled peptide in each case. Identification of the labelled peptide by amino acid analysis and peptide sequencing, and comparison with the enzyme sequence, indicated that the same peptide (267-286) was modified in both enzyme forms. The results are discussed on the basis of the high degree of similarity that this peptide shows in all the known S-adenosylmethionine synthetase sequences.
Project description:S-adenosylmethionine (AdoMet) synthetase (EC 184.108.40.206) is the enzyme that catalyzes the synthesis of AdoMet, a molecule important for all cellular organisms. We have cloned and characterized an AdoMet synthetase gene (sam1) from Pneumocystis spp. This gene was transcribed primarily as an approximately 1.3-kb mRNA which encodes a protein containing 381 amino acids in P. carinii or P. murina and 382 amino acids in P. jirovecii. sam1 was also transcribed as part of an apparent polycistronic transcript of approximately 5.6 kb, together with a putative chromatin remodeling protein homologous to Saccharomyces cerevisiae, CHD1. Recombinant Sam1, when expressed in Escherichia coli, showed functional enzyme activity. Immunoprecipitation and confocal immunofluorescence analysis using an antipeptide antibody showed that this enzyme is expressed in P. murina. Thus, Pneumocystis, like other organisms, can synthesize its own AdoMet and may not depend on its host for the supply of this important molecule.
Project description:We have examined the functional importance of the cysteine residues of rat liver S-adenosylmethionine synthetase. For this purpose the ten cysteine residues of the molecule were changed to serines by site-directed mutagenesis. Ten recombinant enzyme mutants were obtained by using a bacterial expression system. The same level of expression was obtained for the wild type and mutants, but the ratio of S-adenosylmethionine synthetase between soluble and insoluble fractions differed for some of the mutant forms. The immunoreactivity against an anti-(rat liver S-adenosylmethionine synthetase) antibody was equivalent in all the cases. Effects on S-adenosylmethionine synthetase activities were also measured. Mutants C57S, C69S, C105S and C121S showed decreased relative specific activity of 68, 85, 63 and 29%, respectively, compared with wild-type, whereas C312S resulted in an increase of 1.6-fold. Separation of tetramer and dimer forms for wild type and mutants was carried out by using phenyl-Sepharose columns. The dimer/tetramer ratio was calculated based on the activity and on the protein level estimated by immunoblotting. No monomeric forms of the enzyme were detected in any case. Comparison of dimer/tetramer ratios indicates the importance of cysteine-69 (dimer/tetramer protein ratio of 88 versus 10.2 in the wild type) in maintaining the oligomeric state of rat liver S-adenosylmethionine synthetase. Moreover, all the mutations carried out of cysteine residues between cysteine-35 and cysteine-105 altered the ratio between oligomeric forms.
Project description:A cDNA containing the complete coding sequence for rat liver S-adenosylmethionine synthetase was cloned into the prokaryotic expression vector pT7-7 and expressed in Escherichia coli BL21(DE3). A major additional band corresponding to a protein of 48 kDa was detected on SDS/PAGE after induction with isopropyl beta-D-thiogalactopyranoside. This protein was distributed in both the soluble and insoluble fractions and accounted for approx. 30% of the total bacterial protein. The soluble enzyme was fully active, as revealed by assays in vitro of S-adenosylmethionine synthetase activity. In addition, transformed bacteria exhibited highly increased levels of intracellular S-adenosylmethionine. Two active forms of the recombinant enzyme, with apparent molecular masses of 210 kDa and 110 kDa, were detected when cytosolic extracts of the transformed cells were fractionated by gel-filtration chromatography. It is concluded that the expressed S-adenosylmethionine synthetase polypeptide assemble as tetramers and dimers.
Project description:The metE gene, encoding S-adenosylmethionine synthetase (EC 220.127.116.11) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli.
Project description:The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in methionine metabolism, by protein kinase C (PKC) phosphorylation has been studied. Both enzyme forms, tetramer and dimer, are phosphorylated by this kinase in the same residue, Thr-342, of the sequence. Phosphorylation of the dimer leads to its dissociation, with production of a fully-active monomer. The kinetics of the monomer have been studied, and a KmMet of 931.9 microM, a KmATP of 708 microM and a Vmax of 66.8 nmol/min/mg have been calculated. Alkaline phosphatase treatment of both enzyme forms (tetramer and dimer) produces a reduction in their activity with no change in the oligomeric state. On the other hand, PKC phosphorylation of the alkaline phosphatase-treated AdoMet synthetase forms leads to the dissociation of the dimer to produce a monomer. Rephosphorylation occurs again in the same residue, Thr-342, of the sequence. The significance of AdoMet synthetase regulation by PKC phosphorylation is further discussed.
Project description:S-adenosylmethionine (AdoMet) lies at an intersection of nucleotide and amino acid metabolism and performs a multitude of metabolic functions. AdoMet formation is catalyzed by S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase (MAT)), which is a target for development of anticancer and antimicrobial agents. High affinity MAT inhibitors have been found through computational docking of more than 200000 compounds for predicted binding to the crystallographically defined nucleotide binding region of the enzyme's active site. Two of the top scoring candidate compounds had IC(50) values less than 10 nM, more than 10000-fold lower than the substrates' K(M) values. The compounds are structurally unrelated to the natural ligands of the enzyme. The enzyme is protected from inhibition by ATP, but not by methionine, consistent with binding at the adenosyl region of the active site. These results validate in silico screening as a robust approach to the discovery of inhibitors of this chemotherapeutically relevant enzyme.
Project description:A genetically engineered strain of Pichia pastoris expressing S-adenosylmethionine synthetase gene from Saccharomyces cerevisiae under the control of AOX 1 promoter was developed. Induction of recombinant strain with 1% methanol resulted in the expression of SAM2 protein of?~?42 kDa, whereas control GS115 showed no such band. Further, the recombinant strain showed 17-fold higher enzyme activity over control. Shake flask cultivation of engineered P. pastoris in BMGY medium supplemented with 1% L-methionine yielded 28 g/L wet cell weight and 0.6 g/L S-adenosylmethionine, whereas control (transformants with vector alone) with similar wet cell weight under identical conditions accumulated 0.018 g/L. The clone cultured in the bioreactor containing enriched methionine medium showed increased WCW (117 g/L) as compared to shake flask cultures and yielded 2.4 g/L S-adenosylmethionine. In spite of expression of SAM 2 gene up to 90 h, S-adenosylmethionine accumulation tended to plateau after 72 h, presumably because of the limited ATP available in the cells at stationery phase. The recombinant P pastoris seems promising as potential source for industrial production of S-adenosylmethionine.
Project description:BACKGROUND:Exposure to ethanol abuse and severe oxidative stress are risk factors for hepatocarcinoma. The aim of this study was to evaluate the effects of S-adenosylmethionine (SAMe) and its combinations with taurine and/or betaine on the level of glutathione (GSH), a powerful antioxidant in the liver, in acute hepatotoxicity induced by ethanol. METHODS:To examine the effects of SAMe and its combinations with taurine and/or betaine on ethanol-induced hepatotoxicity, AML12 cells and C57BL/6 mice were pretreated with SAMe, taurine, and/or betaine, followed by ethanol challenge. Cell viability was detected with an MTT assay. GSH concentration and mRNA levels of GSH synthetic enzymes were measured using GSH reductase and quantitative real-time reverse transcriptase-PCR. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured with commercially available kits. RESULTS:Pretreatment of SAMe, with or without taurine and/or betaine, attenuated decreases in GSH levels and mRNA expression of the catalytic subunit of glutamate-cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis, in ethanol-treated cells and mice. mRNA levels of the modifier subunit of GCL and glutathione synthetase were increased in mice treated with SAMe combinations. SAMe, taurine, and/or betaine pretreatment restored serum ALT and AST levels to control levels in the ethanol-treated group. CONCLUSIONS:Combinations of SAMe with taurine and/or betaine have a hepatoprotective effect against ethanol-induced liver injury by maintaining GSH homeostasis.
Project description:Chinese hamster ovary cells were stably transfected with rat liver S-adenosylmethionine synthetase cDNA. As a result, S-adenosylmethionine synthetase activity increased 2.3-fold, an effect that was accompanied by increased S-adenosylmethionine, a depletion of ATP and NAD levels, elevation of the S-adenosylmethionine/S-adenosylhomocysteine ratio (the methylation ratio), increased DNA methylation and polyamine levels (spermidine and spermine), and normal GSH levels. By contrast, the transfected cells showed normal growth curves and morphology. Exposure to an oxidative stress by the addition of H2O2 resulted in a greater consumption of ATP and NAD in the transfected cells than in the wild-type cells. In turn, cell killing by H2O2 was greater in the transfected cells than in the wild-type cells. This killing of Chinese hamster ovary cells by H2O2 involved the activation of poly(ADP-ribose) polymerase with the resultant loss of NAD and ATP. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerse, but not the antioxidant N,N'-diphenylphenylenediamine, prevented the killing of Chinese hamster ovary cells by H2O2 and maintained the contents of NAD and ATP. The results of this study indicate that a moderate activation of the synthesis of S-adenosylmethionine leads to ATP and NAD depletion and to a greater sensitivity to cell killing by oxidative stress.