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At least 19 recordsLinked to original sources
PubMed1986

Purification and properties of rat lens methionine adenosyltransferase.

Methionine adenosyltransferase (MAT) has been partially purified from rat lenses using a combination of ammonium sulfate fractionation and hydrophobic chromatography on phenyl Sepharose columns. The partially purified enzyme resembles purified Type II MAT from non-hepatic tissues. The Km for methionine is 3.0 microM, and the Km for ATP is 80 microM. The enzyme is activated by potassium ions (25-50 mM), and inhibited by higher concentrations of potassium. A divalent cation (magnesium or manganese) is essential for activity. The Vmax with magnesium is about five times higher than with manganese, but the optimal manganese concentration is around 2.0 mM, compared with 10-20 mM for magnesium. The enzyme is active over a broad pH range, with optimal activity between pH 7.0 and 8.0. The enzyme is inhibited by all three of its products, phosphate, pyrophosphate, and S-adenosylmethionine. Individually phosphate and pyrophosphate are weak inhibitors, but in combination they show a marked synergistic inhibitory effect. Tripolyphosphate is also an effective inhibitor. The inhibition of the enzyme by the cataractogenic agent, dimethylsulfoxide, further confirmed the similarity to Type II MAT.

Animals↗
PubMed2000

Hysteretic behavior of methionine adenosyltransferase III. Methionine switches between two conformations of the enzyme with different specific activity.

Methionine adenosyltransferase III (MATIII) catalyzes S-adenosylmethionine (AdoMet) synthesis and, as part of its reaction mechanism, it also hydrolyzes tripolyphosphate. Tripolyphosphatase activity was linear over time and had a slightly sigmoidal behavior with an affinity in the low micromolar range. On the contrary, AdoMet synthetase activity showed a lag phase that was independent of protein concentration but decreased at increasing substrate concentrations. Tripolyphosphatase activity, which appeared to be slower than AdoMet synthesis, was stimulated by preincubation with ATP and methionine so that it matched AdoMet synthetase activity. This stimulation process, which is probably the origin of the lag phase, represents the slow transition between two conformations of the enzyme that could be distinguished by their different tripolyphosphatase activity and sensitivity to S-nitrosylation. Tripolyphosphatase activity appeared to be the rate-determining reaction in AdoMet synthesis and the one inhibited by S-nitrosylation. The methionine concentration necessary to obtain half-maximal stimulation was in the range of physiological methionine fluctuations. Moreover, stimulation of MAT activity by methionine was demonstrated in vivo. We propose that the hysteretic behavior of MATIII, in which methionine induces the transition to a higher specific activity conformation, can be considered as an adaptation to the specific functional requirements of the liver.

Acid Anhydride Hydrolases↗
PubMed1995

Molecular mechanisms of an inborn error of methionine pathway. Methionine adenosyltransferase deficiency.

Methionine adenosyltransferase (MAT) is a key enzyme in transmethylation, transsulfuration, and the biosynthesis of polyamines. Genetic deficiency of alpha/beta-MAT causes isolated persistent hypermethioninemia and, in some cases, unusual breath odor or neural demyelination. However, the molecular mechanism(s) underlying this deficiency has not been clearly defined. In this study, we characterized the human alpha/beta-MAT transcription unit and identified several mutations in the gene of patients with enzymatically confirmed diagnosis of MAT deficiency. Site-directed mutagenesis and transient expression assays demonstrated that these mutations partially inactivate MAT activity. These results establish the molecular basis of this disorder and allow for the development of DNA-based methodologies to investigate and diagnose hypermethioninemic individuals suspected of having abnormalities at this locus.

Amino Acid Metabolism, Inborn Errors↗
PubMed2004

Impaired liver regeneration in mice lacking methionine adenosyltransferase 1A.

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor. Of the two genes that encode MAT, MAT1A is mainly expressed in adult liver and MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic SAMe content and spontaneously develop hepatocellular carcinoma. The current study examined the influence of chronic hepatic SAMe deficiency on liver regeneration. Despite having higher baseline hepatic staining for proliferating cell nuclear antigen, MAT1A knockout mice had impaired liver regeneration after partial hepatectomy (PH) as determined by bromodeoxyuridine incorporation. This can be explained by an inability to up-regulate cyclin D1 after PH in the knockout mice. Upstream signaling pathways involved in cyclin D1 activation include nuclear factor kappaB (NFkappaB), the c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and signal transducer and activator of transcription-3 (STAT-3). At baseline, JNK and ERK are more activated in the knockouts whereas NFkappaB and STAT-3 are similar to wild-type mice. Following PH, early activation of these pathways occurred, but although they remained increased in wild-type mice, c-jun and ERK phosphorylation fell progressively in the knockouts. Hepatic SAMe levels fell progressively following PH in wild-type mice but remained unchanged in the knockouts. In culture, MAT1A knockout hepatocytes have higher baseline DNA synthesis but failed to respond to the mitogenic effect of hepatocyte growth factor. Taken together, our findings define a critical role for SAMe in ERK signaling and cyclin D1 regulation during regeneration and suggest chronic hepatic SAMe depletion results in loss of responsiveness to mitogenic signals.

Adenosine Triphosphate↗
PubMed2005

Effect of fasting on methionine adenosyltransferase expression and the methionine cycle in the mouse liver.

The effect of fasting on mouse liver methionine adenosyltransferase (MAT I/ III) expression and the regulation of methionine metabolism were investigated. The mRNA level, protein level, and activity of MAT I/III were increased by fasting for 10 or 16 h. In spite of the increase of MAT I/III activity, S-adenosylmethionine, the product of methionine due to MAT I/III, decreased. S-Adenosylhomocysteine, which is made from S-adenosylmethionine by its coupling to methyltransferase, increased as a result of fasting for 16 h. These results suggest that the total methylation reactions using S-adenosylmethionine are stimulated in the fasting mouse liver. However, the DNA methylation level was not changed by fasting for 16 h. Glutathione, which is made by the transsulfuration pathway from homocysteine, decreased due to fasting. Regulation of supplementation of S-adenosylmethionine may occur in the fasting mouse because MAT I/III activity increases and the flow to glutathione is decreased.

Animals↗
PubMed2000

Assignment of a single disulfide bridge in rat liver methionine adenosyltransferase.

Rat liver methionine adenosyltransferase incorporated 8 mol of N-ethylmaleimide per mol of subunit upon denaturation in the presence of 8 M urea, whereas 10 such groups were labelled when dithiothreitol was also included. This observation prompted a re-examination of the state of the thiol groups, which was carried out using peptide mapping, amino acid analysis and N-terminal sequencing. The results obtained revealed a disulfide bridge between Cys35 and Cys61. This disulfide did not appear to be conserved because cysteines homologous to residue 61 do not exist in methionine adenosyltransferases of other origins, therefore suggesting its importance for the differential aspects of the liver-specific enzyme.

Amino Acid Sequence↗
PubMed1981

Constitutive behavior of methionyl-tRNA synthetase compared to repressible behavior of methionine adenosyltransferase in mammalian cells.

Methionine adenosyltransferase, one of the two major enzymes utilizing methionine, is regulated by the levels of methionine in the growth medium (Jacobsen, S.J., Hoffman, R.M. and Erbe, R.W. (1980) J. Natl. Cancer Inst. 65, 1237--1244, and Caboche, M. and Mulsant, P. (1978) Somatic Cell Genet. 4, 407--421). We report here that methionyl-tRNA synthetase, unlike methionine adenosyltransferase, behaves in a constitutive manner with respect to the concentration of methionine in the culture medium. This behavior is seen in Chinese hamster ovary cells and in normal diploid and SV40-transformed human fibroblasts. Although the kinetics of regulation of methionine adenosyltransferase and methionyl-tRNA synthetase by exogenous methionine are clearly different, the levels of the two enzymes in the human cell lines are similar.

Amino Acyl-tRNA Synthetases↗
PubMed2000

Molecular genetics of hepatic methionine adenosyltransferase deficiency.

Hepatic methionine adenosyltransferase (MAT) deficiency is caused by mutations in the human MAT1A gene that abolish or reduce hepatic MAT activity that catalyzes the synthesis of S-adenosylmethionine from methionine and ATP. This genetic disorder is characterized by isolated persistent hypermethioninemia in the absence of cystathionine beta-synthase deficiency, tyrosinemia, or liver disease. Depending on the nature of the genetic defect, hepatic MAT deficiency can be transmitted either as an autosomal recessive or dominant trait. Genetic analyses have revealed that mutations identified in the MAT1A gene only partially inactivate enzymatic activity, which is consistent with the fact that most hepatic MAT-deficient individuals are clinically well. Two hypermethioninemic individuals with null MAT1A mutations have developed neurological problems, including brain demyelination, although this correlation is by no means absolute. Presently, it is recommended that a DNA-based diagnosis should be performed for isolated hypermethioninemic individuals with unusually high plasma methionine levels to assess if therapy aimed at the prevention of neurological manifestations is warranted.

Adenosine Triphosphate↗
PubMed1998

Cloning and functional characterization of the 5'-flanking region of human methionine adenosyltransferase 2A gene.

Methionine adenosyltransferase (MAT) is a critical cellular enzyme which catalyzes the formation of S-adenosylmethionine, the principal methyl donor. In mammals, two different genes, MAT1A and MAT2A, encode for liver-specific and non-liver-specific MAT, respectively. We have cloned and characterized a 1.4-kb 5'-flanking region of the human MAT2A (GenBank Accession No. AF039088). Two major transcriptional start sites were identified by primer extension and S1 nuclease protection analysis; one was within 10 nucleotides downstream and the other was located at 158 nucleotides upstream from the consensus TATA box, respectively. The promoter is highly GC rich (75%) in the first 300 base pairs and contains several Sp-1 binding sites, a C/EBP, a HSF2, a STATx, a c-Myb, several v-Myb, and numerous GATA consensus binding sites. The human MAT2A promoter was able to efficiently drive luciferase expression in both Jurkat and 293 cells, but sequential deletion analysis of the promoter revealed that different regions of the promoter are important for cell-specific MAT2A expression.

Base Sequence↗
PubMed2000

Cloning and functional characterization of the 5'-flanking region of human methionine adenosyltransferase 1A gene.

Methionine adenosyltransferase (MAT) is an essential cellular enzyme which catalyses the formation of S-adenosylmethionine, the principal methyl donor and precursor for polyamines. In mammals, two different genes, MAT1A and MAT2A, encode for liver-specific and non-liver-specific MAT respectively. We previously described a switch in the MAT expression from MAT1A to MAT2A in human liver cancer, which offered the cancerous cell a growth advantage. Loss of MAT1A expression was due to lack of gene transcription. To study regulation of the MAT1A gene, we have cloned and characterized a 1.9 kb 5'-flanking region of the human MAT1A gene. One transcriptional start site, located 25 nt downstream from a consensus TATA box, was identified by primer extension and RNase protection assays. The promoter contains several consensus binding sites for CAAT enhancer binding protein (C/EBP) and hepatocyte-enriched nuclear factor (HNF), transcriptional factors important in liver-specific gene expression. The human MAT1A promoter was able to efficiently drive luciferase expression in Chang cells, a human liver cell line, but not in HeLa cells. Sequential deletion analysis of the promoter revealed two DNA regions upstream of the translational start site, -705 to -839 bp and -1111 to -1483 bp, which are involved in positive and negative gene regulation, respectively. Specific protein binding to these regions was confirmed by electrophoretic-mobility-shift and DNase I footprinting assays. Similar to the situation with the rat MAT1A, glucocorticoid treatment also increased human MAT1A expression and promoter activity in a dose- and time-dependent manner.

5' Untranslated Regions↗
PubMed1995

Chromosomal localization and catalytic properties of the recombinant alpha subunit of human lymphocyte methionine adenosyltransferase.

Human lymphocyte methionine adenosyltransferase (HuLy MAT) consists of heterologous subunits alpha and beta. The cDNA sequence of the alpha subunit of HuLy MAT from Jurkat leukemic T cells was identical to that of the human kidney alpha subunit and highly homologous to the sequence of the extrahepatic MAT from other sources. The 3'-untranslated sequence was found to be highly conserved, suggesting that it may be important in regulating the expression of MAT. The extrahepatic alpha subunit unit of MAT was found to be expressed also in human liver, and no differences were found in the sequence of the alpha subunit from normal and malignant T cells. The sequence of two unspliced introns found in the cDNA clones from the Jurkat library enabled us to isolate genomic clones harboring the human extrahepatic alpha subunit gene and to localize it to the centromere on chromosome arm 2p, an area that corresponds to band 2p11.2. Expression of the alpha subunit cDNA in Escherichia coli yielded two peptides with the immunoreactivity and mobilities of authentic alpha/alpha' subunits from HuLy. The Km of the recombinant alpha subunit was 80 microM, which is 20-fold higher than found for the (alpha alpha')x beta y holoenzyme purified from leukemic lymphocytes and 4-10-fold higher than found for the normal lymphocyte enzyme. The data suggest that the alpha/alpha' subunits mediate the enzyme catalytic activity and that the beta subunit may be a regulatory subunit of extrahepatic MAT.

Adult↗
PubMed1997

Dominant inheritance of isolated hypermethioninemia is associated with a mutation in the human methionine adenosyltransferase 1A gene.

Methionine adenosyltransferase (MAT) I/III deficiency, characterized by isolated persistent hypermethioninemia, is caused by mutations in the MAT1A gene encoding MAT(alpha)1, the subunit of major hepatic enzymes MAT I ([alpha1]4) and III([alpha1]2). We have characterized 10 MAT1A mutations in MAT I/III-deficient individuals and shown that the associated hypermethioninemic phenotype was inherited as an autosomal recessive trait. However, dominant inheritance of hypermethioninemia, also hypothesized to be caused by MAT I/III deficiency, has been reported in two families. Here we show that the only mutation uncovered in one of these families, G, is a G-->A transition at nt 791 in exon VII of one MAT1A allele that converts an arginine at position 264 to a histidine (R264H). This single allelic R264H mutation was subsequently identified in two hypermethioninemic individuals in an additional family, C. Family C members were also found to inherit hypermethioninemia in a dominant fashion, and the available affected members analyzed carried the single allelic R264H mutation. Substitution of R-264 with histidine (R264H, the naturally occurring mutant), leucine (R264L), aspartic acid (R264D), or glutamic acid (R264E) greatly reduced MAT activity and severely impaired the ability of the MAT(alpha)1 subunits to form homodimers essential for optimal catalytic activity. On the other hand, when lysine was substituted for R-264 (R264K), the mutant alpha1 subunit was able to form dimers that retain significant MAT activity, suggesting that amino acid 264 is involved in intersubunit salt-bridge formation. Cotransfection studies show that R264/R264H MAT(alpha)1 heterodimers are enzymatically inactive, thus providing an explanation for the R264H-mediated dominant inheritance of hypermethioninemia.

Adolescent↗
PubMed2001

Regulation of the human MAT2B gene encoding the regulatory beta subunit of methionine adenosyltransferase, MAT II.

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet), a key molecule in transmethylation reactions and polyamine biosynthesis. The MAT II isozyme consists of a catalytic alpha2 and a regulatory beta subunit. Down-regulation of the MAT II beta subunit expression causes a 6-10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the beta subunit expression is regulated, we cloned the MAT2B gene, determined its organization, characterized its 5'-flanking sequences, and elucidated the in vitro and in vivo regulation of its promoter. Transcription of the MAT2B gene initiates at position -203 relative to the translation start site. Promoter deletion analysis defined a minimal promoter between positions +52 and +93 base pairs, a GC-rich region. Inclusion of the sequences between -4 and +52 enhanced promoter activity; this was primarily because of an Sp1 recognition site at +9/+15. The inclusion of sequences up to position -115 provided full activity; this was attributed to a TATA at -32. The Sp1 site at position +9 was key for the formation of protein.DNA complexes. Mutation of both the Sp1 site at +9 and the TATA at -32 reduced promoter activity to its minimal level. Supershift assays showed no effect of the anti-Sp1 antibody on complex formation, whereas the anti-Sp3 antibody had a strong effect on protein.DNA complex formation, suggesting that Sp3 is one of the main factors binding to this Sp1 site. Chromatin immunoprecipitation assays supported the involvement of both Sp1 and Sp3 in complexes formed on the MAT2B promoter. The data show that the 5'-untranslated sequences play an important role in regulating the MAT2B gene and identifies the Sp1 site at +9 as a potential target for modulating MAT2B expression, a process that can have a major effect on intracellular AdoMet levels.

Animals↗
PubMed2002

Role of an intrasubunit disulfide in the association state of the cytosolic homo-oligomer methionine adenosyltransferase.

Recombinant rat liver methionine adenosyltransferase has been refolded into fully active tetramers (MAT I) and dimers (MAT III), using as a source chaotrope-solubilized aggregates resulting from specific washes of inclusion bodies. The conditions of refolding, dialysis in the presence of 10 mm dithiothreitol or 10 mm GSH with 1 mm GSSG, allowed the production of both isoforms, the nature of the redox agent determining the capacity of the final product (MAT I/III) to interconvert. Refolding in the presence of 10 mm dithiothreitol yielded mainly MAT III in a concentration-dependent equilibrium with the homotetramer MAT I. However, refolding in the presence of the redox pair GSH/GSSG resulted in a stable MAT I and III mixture. Blockage of dimer-tetramer interconversion has been found related to the production of a single intramolecular disulfide in methionine adenosyltransferase during the GSH/GSSG folding process. The residues involved in this disulfide have been identified by mass spectrometry and using a set of single cysteine mutants as cysteines 35 and 61. In addition, a kinetic intermediate in the MAT I dissociation to MAT III has been detected. The physiological importance of these results is discussed in light of the structural and regulatory data available.

Animals↗
PubMed1984

Synthesis of S-adenosylethionine by the gamma isozyme of methionine adenosyltransferase from Friend erythroleukemic cells.

Methionine adenosyltransferase (adenosine 5'-triphosphate-L-methionine S-adenosyltransferase, EC 2.5.1.6) was found to occur only as the gamma isozyme in Friend erythroleukemic cells. Enzyme activity in a dialyzed 105,000 X g supernatant was linear for at least 30 min and dependent on the amount of protein added. The Km for methionine was 10.6 microM. In the presence of 10% dimethyl sulfoxide, the enzyme activity was slightly inhibited and dithiothreitol was not required for maximum activity. These properties identify the methionine adenosyltransferase in Friend erythroleukemic cells as the gamma isozyme. L-Ethionine served as a substrate with a Km of 30 microM, and competitively inhibited the enzyme activity with a Ki of 150 microM. Friend erythroleukemic cells grown in the presence of ethionine accumulated S-adenosylethionine, which was dose and time dependent. Therefore, the gamma isozyme of methionine adenosyltransferase from Friend erythroleukemic cells utilize L-ethionine as substrate, resulting in an accumulation of S-adenosylethionine. These studies provide a mechanism by which ethionine through S-adenosylethionine may alter the methylation of DNA in Friend erythroleukemic cells.

Animals↗
PubMed1993

Methionine adenosyltransferase: structure and function.

Methionine adenosyltransferase (MAT), a key enzyme in metabolism, catalyzes the synthesis of one of the most important and pivotal biological molecules, S-adenosyl-methionine. In every organism studied thus far, MAT exists in multiple forms; most are encoded by related, but distinct genes. Molecular and immunological studies revealed the presence of considerable conservation in the structure of MAT from different species; however, the various MAT isozymes differ in their physical and kinetic properties in ways that allow them to be regulated differently. Recent studies suggest that human MAT is composed of nonidentical subunits that can assume multiple states of aggregation, each with different kinetic characteristics. The tissue distribution of MAT isozymes and the ability of cells within the same tissue to switch between the different forms of MAT suggest that this mode of regulation is important for cellular function and differentiation. Therefore, understanding the regulation and structure-function relationship of this fascinating enzyme should help us clarify its role in biology and may provide us with tools to effectively manipulate its activity in clinical situations such as cancer, autoimmunity and organ transplantation.

Animals↗
PubMed2001

Biochemical basis for the dominant inheritance of hypermethioninemia associated with the R264H mutation of the MAT1A gene. A monomeric methionine adenosyltransferase with tripolyphosphatase activity.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main alkylating agent in living cells. Additionally, in the liver, MAT is also responsible for up to 50% of methionine catabolism. Humans with mutations in the gene MAT1A, the gene that encodes the catalytic subunit of MAT I and III, have decreased MAT activity in liver, which results in a persistent hypermethioninemia without homocystinuria. The hypermethioninemic phenotype associated with these mutations is inherited as an autosomal recessive trait. The only exception is the dominant mild hypermethioninemia associated with a G-A transition at nucleotide 791 of exon VII. This change yields a MAT1A-encoded subunit in which arginine 264 is replaced by histidine. Our results indicate that in the homologous rat enzyme, replacement of the equivalent arginine 265 by histidine (R265H) results in a monomeric MAT with only 0.37% of the AdoMet synthetic activity. However the tripolyphosphatase activity is similar to that found in the wild type (WT) MAT and is inhibited by PP(i). Our in vivo studies demonstrate that the R265H MAT I/III mutant associates with the WT subunit resulting in a dimeric R265H-WT MAT unable to synthesize AdoMet. Tripolyphosphatase activity is maintained in the hybrid MAT, but is not stimulated by methionine and ATP, indicating a deficient binding of the substrates. Our data indicate that the active site for tripolyphosphatase activity is functionally active in the monomeric R265H MAT I/III mutant. Moreover, our results provide a molecular mechanism that might explain the dominant inheritance of the hypermethioninemia associated with the R264H mutation of human MAT I/III.

Acid Anhydride Hydrolases↗
PubMed2000

The crystal structure of tetrameric methionine adenosyltransferase from rat liver reveals the methionine-binding site.

Most of the transmethylation reactions use the same methyl donor, S-adenosylmethionine (SAM), that is synthesised from methionine and ATP by methionine adenosyltransferase (MAT). In mammals, two MAT enzymes have been detected, one ubiquitous and another liver specific. The liver enzyme exists in two oligomeric forms, a tetramer (MAT I) and a dimer (MAT III), MAT I being the one that shows a higher level of affinity for methionine but a lower SAM synthesis capacity. We have solved the crystal structure of rat liver MAT I at 2.7 A resolution, complexed with a methionine analogue: l-2-amino-4-methoxy-cis-but-3-enoic acid (l-cisAMB). The enzyme consists of four identical subunits arranged in two tight dimers that are related by crystallographic 2-fold symmetry. The crystal structure shows the positions of the relevant cysteine residues in the chain, and that Cys35 and Cys61 are perfectly oriented for forming a disulphide link. This result leads us to propose a hypothesis to explain the control of MAT I/III exchange and hence, the effects observed on activity. We have identified the methionine-binding site into the active-site cavity, for the first time. The l-cisAMB inhibitor is stacked against Phe251 aromatic ring in a rather planar conformation, and its carboxylate group coordinates a Mg(2+), which, in turn, is linked to Asp180. The essential role of the involved residues in MAT activity has been confirmed by site-directed mutagenesis. Phe251 is exposed to solvent and is located in the beginning of the flexible loop Phe251-Ala260 that is connecting the N-terminal domain to the central domain. We postulate that a conformational change may take place during the enzymatic reaction and this is possibly the reason of the unusual two-step mechanism involving tripolyphosphate hydrolysis. Other important mechanistic implications are discussed on the light of the results. Moreover, the critical role that certain residues identified in this study may have in methionine recognition opens further possibilities for rational drug design.

Acid Anhydride Hydrolases↗