Publications

• Activation of methionine for transmethylation. II. The methionine-activating enzyme; studies on the mechanism of the reaction.

  CANTONI G.L., DURELL J.

  J Biol Chem 225:1033-1048(1957) [PubMed] [EuropePMC]

• Characterization of a plant S-adenosylmethionine synthetase from Acacia koa.

  Carrillo J.T., Borthakur D.

  The Acacia koa S-adenosylmethionine (SAM) synthetase was identified from transcriptome data and cloned into the T₇-expression vector pEt14b. Assays indicate a thermoalkaliphic enzyme which tolerates conditions up to pH 10.5, 55 °C and 3 M KCl. In vitro examples of plant SAM-synthetase a ... >> More

  The Acacia koa S-adenosylmethionine (SAM) synthetase was identified from transcriptome data and cloned into the T₇-expression vector pEt14b. Assays indicate a thermoalkaliphic enzyme which tolerates conditions up to pH 10.5, 55 °C and 3 M KCl. In vitro examples of plant SAM-synthetase activity are scarce, however this study provides supporting evidence that these extremophilic properties may actually be typical for this plant enzyme. Enzyme kinetic constants (K_m = 1.44 mM, K_cat = 1.29 s^-1, V_max 170 μM. min^-1) are comparable to nonplant SAM-synthetases except that substrate inhibition was not apparent at 10 mM ATP/L-methionine. Methods were explored in this study to reduce feedback inhibition, which is known to limit SAM-synthetase activity in vitro. Four single-point mutation variants of the Acacia koa SAM-synthetase were produced, each with varying degrees of reduced reaction rate, greater sensitivity to product inhibition and loss of thermophilic properties. Although an enhanced mutant was not produced, this study describes the first mutagenesis of a plant SAM-synthetase. Overcoming feedback inhibition was accomplished by the addition of organic solvent to enzyme assays. Acetonitrile, methanol or dimethylformamide, when included as 25% of the assay volume, improved total SAM production by 30-65%. << Less

  Plant Physiol Biochem 210:108618-108618(2024) [PubMed] [EuropePMC]

• S-Adenosylmethionine; a new intermediate formed enzymatically from L-methionine and adenosinetriphosphate.

  Cantoni G.L.

  J Biol Chem 204:403-416(1953) [PubMed] [EuropePMC]

• Cloning, expression, and functional characterization of the beta regulatory subunit of human methionine adenosyltransferase (MAT II).

  LeGros H.L., Halim A.-B., Geller A.M., Kotb M.

  MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function. The full-length cDNA that encodes the beta subunit of human MAT II was cloned and found to encode for a ... >> More

  MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function. The full-length cDNA that encodes the beta subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The beta subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native beta subunit protein, and to the rbeta protein. There is no cross-reactivity between the MAT II alpha(2) or beta subunits. None of the anti-beta subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no beta subunit protein. Interestingly, the rbeta subunit associated with E. coli as well as human MAT alpha subunits. This association changed the kinetic properties of both enzymes and lowered the K(m) of MAT for L-methionine. Together, the data show that we have cloned and expressed the human MAT II beta subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells. << Less

  J. Biol. Chem. 275:2359-2366(2000) [PubMed] [EuropePMC]

• Structure and function study of the complex that synthesizes S-adenosylmethionine.

  Murray B., Antonyuk S.V., Marina A., Van Liempd S.M., Lu S.C., Mato J.M., Hasnain S.S., Rojas A.L.

  S-Adenosylmethionine (SAMe) is the principal methyl donor of the cell and is synthesized via an ATP-driven process by methionine adenosyltransferase (MAT) enzymes. It is tightly linked with cell proliferation in liver and colon cancer. In humans, there are three genes, mat1A, mat2A and mat2B, whic ... >> More

  S-Adenosylmethionine (SAMe) is the principal methyl donor of the cell and is synthesized via an ATP-driven process by methionine adenosyltransferase (MAT) enzymes. It is tightly linked with cell proliferation in liver and colon cancer. In humans, there are three genes, mat1A, mat2A and mat2B, which encode MAT enzymes. mat2A and mat2B transcribe MATα2 and MATβ enzyme subunits, respectively, with catalytic and regulatory roles. The MATα2β complex is expressed in nearly all tissues and is thought to be essential in providing the necessary SAMe flux for methylation of DNA and various proteins including histones. In human hepatocellular carcinoma mat2A and mat2B genes are upregulated, highlighting the importance of the MATα2β complex in liver disease. The individual subunits have been structurally characterized but the nature of the complex has remained elusive despite its existence having been postulated for more than 20 years and the observation that MATβ is often co-localized with MATα2. Though SAMe can be produced by MAT(α2)4 alone, this paper shows that the V max of the MATα2β complex is three-to fourfold higher depending on the variants of MATβ that participate in complex formation. Using X-ray crystallography and solution X-ray scattering, the first structures are provided of this 258 kDa functional complex both in crystals and solution with an unexpected stoichiometry of 4α2 and 2βV2 subunits. It is demonstrated that the N-terminal regulates the activity of the complex and it is shown that complex formation takes place surprisingly via the C-terminal of MATβV2 that buries itself in a tunnel created at the interface of the MAT(α2)2. The structural data suggest a unique mechanism of regulation and provide a gateway for structure-based drug design in anticancer therapies. << Less

  IUCrJ 1:240-249(2014) [PubMed] [EuropePMC]

• Cloning expression and characterization of methionine adenosyltransferase in Leishmania infantum promastigotes.

  Reguera R.M., Balana-Fouce R., Perez-Pertejo Y., Fernandez F.J., Garcia-Estrada C., Cubria J.C., Ordonez C., Ordonez D.

  Methionine adenosyltransferase (MAT) catalyzes the synthesis of s-adenosylmethionine (AdoMet), a metabolite that plays an important role in a variety of cellular functions, such as methylation, sulfuration, and polyamine synthesis. In this study, genomic DNA from the protozoan parasite Leishmania ... >> More

  Methionine adenosyltransferase (MAT) catalyzes the synthesis of s-adenosylmethionine (AdoMet), a metabolite that plays an important role in a variety of cellular functions, such as methylation, sulfuration, and polyamine synthesis. In this study, genomic DNA from the protozoan parasite Leishmania infantum was cloned and characterized. L. infantum MAT, unlike mammalian MAT, is codified by two identical genes in a tandem arrangement and is only weakly regulated by AdoMet. L. infantum MAT mRNA is expressed as a single transcript, with the enzyme forming a homodimer with tripolyphosphatase in addition to MAT activity. Expression of L. infantum MAT in Escherichia coli proves that the MAT and tripolyphosphatase activities are functional in vivo. MAT shows sigmoidal behavior and is weakly inhibited by AdoMet, whereas tripolyphosphatase activity has sigmoidal behavior and is strongly activated by AdoMet. Plasmids containing the regions flanking MAT2 were fused immediately upstream and downstream of the luciferase-coding region and transfected into L. infantum. Subsequent examination of luciferase activity showed that homologous expression in L. infantum promastigotes was dramatically dependent on the presence of polypyrimidine tracts and a spliced leader junction site upstream of the luciferase gene, whereas downstream sequences appeared to have no bearing on expression. << Less

  J. Biol. Chem. 277:3158-3167(2002) [PubMed] [EuropePMC]

• Three differentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization.

  Schroeder G., Eichel J., Breinig S., Schroeder J.

  We describe the molecular and functional characterization of three closely related S-adenosyl-L-methionine synthetase (SAMS) isoenzymes from Catharanthus roseus (Madagascar periwinkle). The genes are differentially expressed in cell cultures during growth of the culture and after application of va ... >> More

  We describe the molecular and functional characterization of three closely related S-adenosyl-L-methionine synthetase (SAMS) isoenzymes from Catharanthus roseus (Madagascar periwinkle). The genes are differentially expressed in cell cultures during growth of the culture and after application of various stresses (elicitor, nutritional down-shift, increased NaCl). Seedlings revealed organ-specific expression and differential gene regulation after salt stress. A relationship analysis indicated that plant SAMS group in two main clusters distinguished by characteristic amino acid exchanges at specific positions, and this suggested differences in the enzyme properties or the regulation. SAMS1 and SAMS2 are of type I and SAMS3 is of type II. The properties of the isoenzymes were compared after heterologous expression of the individual enzymes, but no significant differences were detected in a) optima for temperature (37 to 45 degrees C) or pH (7 to 8.3); b) dependence on cations (divalent: Mg2+, Mn2+, Co2+; monovalent: K+, NH4+, Na+); c) K(m)s for ATP and L-methionine; d) inhibition by reaction products (S-adenosyl-L-methionine, PPi, Pi), by the reaction intermediate tripolyphosphate, and by the substrate analogues ethionine and cycloleucine; e) response to metabolites from the methyl cycle (L-homocysteine) or from related pathways (L-ornithine, putrescine, spermidine, spermine); f) native protein size (gel permeation chromatography). The results represent the first characterization of plant SAMS isoenzyme properties with individually expressed proteins. The possibility is discussed that the isoenzyme differences reflect specificities in the association with enzymes that use S-adenosyl-L-methionine. << Less

  Plant Mol. Biol. 33:211-222(1997) [PubMed] [EuropePMC]

• Identification of a highly diverged class of S-adenosylmethionine synthetases in the archaea.

  Graham D.E., Bock C.L., Schalk-Hihi C., Lu Z.J., Markham G.D.

  S-adenosylmethionine is the primary alkylating agent in all known organisms. ATP:L-methionine S-adenosyltransferase (MAT) catalyzes the only known biosynthetic route to this central metabolite. Although the amino acid sequence of MAT is strongly conserved among bacteria and eukarya, no homologs ha ... >> More

  S-adenosylmethionine is the primary alkylating agent in all known organisms. ATP:L-methionine S-adenosyltransferase (MAT) catalyzes the only known biosynthetic route to this central metabolite. Although the amino acid sequence of MAT is strongly conserved among bacteria and eukarya, no homologs have been recognized in the completed genome sequences of any archaea. In this study, MAT has been purified to homogeneity from the archaeon Methanococcus jannaschii, and the gene encoding it has been identified by mass spectrometry. The peptide mass map identifies the gene encoding MAT as MJ1208, a hypothetical open reading frame. The gene was cloned in Escherichia coli, and expressed enzyme has been purified and characterized. This protein has only 22 and 23% sequence identity to the E. coli and human enzymes, respectively, whereas those are 59% identical to each other. The few identical residues include the majority of those constituting the polar active site residues. Each complete archaeal genome sequence contains a homolog of this archaeal-type MAT. Surprisingly, three bacterial genomes encode both the archaeal and eukaryal/bacterial types of MAT. This identification of a second major class of MAT emphasizes the long evolutionary history of the archaeal lineage and the structural diversity found even in crucial metabolic enzymes. << Less

  J. Biol. Chem. 275:4055-4059(2000) [PubMed] [EuropePMC]

• NADP+ binding to the regulatory subunit of methionine adenosyltransferase II increases intersubunit binding affinity in the hetero-trimer.

  Gonzalez B., Garrido F., Ortega R., Martinez-Julvez M., Revilla-Guarinos A., Perez-Pertejo Y., Velazquez-Campoy A., Sanz-Aparicio J., Pajares M.A.

  Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, althoug ... >> More

  Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP(+) with a 1:1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP(+) binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells. << Less

  PLoS ONE 7:E50329-E50329(2012) [PubMed] [EuropePMC]

• Activation of methionine for transmethylation. III. The methionine-activating enzyme of Bakers' yeast.

  MUDD S.H., CANTONI G.L.

  J Biol Chem 231:481-492(1958) [PubMed] [EuropePMC]