4 The Adenosyltransferases

Mudd, S. Harvey

doi:10.1016/s1874-6047(08)60064-5

Cited by 16 publications

(6 citation statements)

References 56 publications

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“…These studies led us to propose that the ␤ subunit regulates MAT II activity by lowering the K m for L-Met and rendering the enzyme more resistant to feedback inhibition by its products (31). The data presented here, as well as those presented in Halim et al, 2 provide direct evidence that these hypotheses are correct and confirm the regulatory role of the ␤ subunit of MAT II.…”

Section: Discussionsupporting

confidence: 81%

“…The mode of interaction between the MAT II ␣2 and ␤ subunits remains to be determined; however, our recent studies suggest that the two subunits spontaneously associate. 2 The association between the human MAT II ␤ subunit and the E. coli MAT ␣ subunit is quite interesting because it may provide important clues as to the structural requirements for the association between the ␣ and ␤ subunits of MAT. The association between the MAT II subunits has a strong effect on the MAT kinetic properties, and we believe that this may be an important means by which the enzyme activity is regulated in vivo and a way to alter AdoMet levels to meet cellular requirements and regulate cell function.…”

Section: Discussionmentioning

confidence: 99%

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Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

Legros¹,

Halim²,

Geller³

et al. 2000

Journal of Biological Chemistry

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MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic ␣ 2 /␣ 2 subunits and a noncatalytic ␤ subunit, believed to have a regulatory function. The full-length cDNA that encodes the ␤ 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 ␤ 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 ␤ subunit protein, and to the r␤ protein. There is no cross-reactivity between the MAT II ␣ 2 or ␤ subunits. None of the anti-␤ subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no ␤ subunit protein. Interestingly, the r␤ subunit associated with E. coli as well as human MAT ␣ 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 ␤ 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.Methionine adenosyltransferase (MAT; S-adenosyl-L-methionine synthetase, EC 2.5.1.6) 1 is an essential enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet) from L-methionine (L-Met) and ATP (1, 2). AdoMet is the major methyl group donor, participating in the methylation of proteins, DNA, RNA, phospholipids, and other small molecules (reviewed in Refs. 3-5). In addition, AdoMet is the ultimate source of the propylamine moiety used in polyamine biosynthesis, and it serves as co-factor for other key enzymes in the one-carbon metabolism pathway (3-5). MAT is present in all living species, including thermophilic archaebacteria, plants, yeast, and mammals (reviewed in Refs. 4 and 6 -8). Interestingly, most species have more than one MAT isozyme (6).In mammals, it is now established that there are at least two MAT isozymes (9 -12). MAT I/III is expressed only in liver and has a catalytic subunit designated ␣ 1 that is encoded by the MAT1A gene (8, 9, 13-16). MAT I and MAT III represent different oligomeric forms of the ␣ 1 subunit -MAT III is a dimer, and MAT I is a tetramer of the ␣ 1 subunit (9, 17-19). MAT I and MAT III differ considerably in their physical, kinetic, and regulatory properties (8,9,20). The MAT II isozyme is expressed in all tissues, including the liver, and has been studied in many tissues including erythrocytes, lymphocytes, brain, kidney, testis, and fetal liver (11, 20 -27).We have been characterizing the human MAT II from human lymphocytes (22, 28 -31) 2 and were able to show that the form present in activated lymphocytes consists of distinct subunits (22...

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Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

Legros¹,

Halim²,

Geller³

et al. 2000

Journal of Biological Chemistry

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“…1 (ATP⅐L-methionine S-adenosyltransferase) (EC 2.5.1.6) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet) (1,2). AdoMet is the major methyl donor in transmethylation reactions and the propylamine donor in the biosynthesis of polyamines (3)(4)(5).…”

Section: Methionine Adenosyltransferase (Mat)mentioning

confidence: 99%

Regulation of the Human MAT2B Gene Encoding the Regulatory β Subunit of Methionine Adenosyltransferase, MAT II

LeGros

Halim

Chamberlin³

et al. 2001

Journal of Biological Chemistry

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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 ␣2 and a regulatory ␤ subunit. Down-regulation of the MAT II ␤ subunit expression causes a 6 -10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the ␤ 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 protein⅐DNA 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 protein⅐DNA 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. Methionine adenosyltransferase (MAT)1 (ATP⅐L-methionine S-adenosyltransferase) (EC 2.5.1.6) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet) (1, 2). AdoMet is the major methyl donor in transmethylation reactions and the propylamine donor in the biosynthesis of polyamines (3-5). Furthermore, AdoMet participates as a co-factor in key metabolic pathways (3-5). Most species studied to date have more than one MAT isozyme (6). In mammals, the two major MAT isozymes are designated MAT I/III and MAT II (7-10). The MAT I/III isozymes are, respectively, a tetramer and dimer of a catalytic ␣1 subunit that is expressed only in liver (7, 11-13). The MAT II isozyme is expressed in all tissues including the liver (9, 14 -21). Previous studies from our group (15, 22) have determined that MAT II from human leukemic T and B cells, as well from activated human lymphocytes, is a hetero-oligomer that consists of ␣2 (53 kDa), ␣2Ј (51 kDa), and ␤ (38 kDa) subunits. The ␣2/␤ hetero-oligomeric composition of MAT II was also determined in bovine brain, Ehrlich ascites tumor, and calf thymus (15,19). The ␣2 subunit is responsible for the enzyme catalytic activity and is post-translationally modifie...

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“…S-Adenosylmethionine synthetase (ATP: L-methionine Sadenosyltransferase, EC 2.5.1.6, SAM synthetase) is the only enzyme known to catalyze the biosynthesis of S-adenosylmethionine (SAM) from L-methionine (Met) and ATP (1). SAM is an important metabolic intermediate in all living cells as a donor of methyl groups in transmethylation reactions (2,3) and a precursor molecule in aminopropylation and transulfuration pathways (4).…”

Section: Introductionmentioning

confidence: 99%

Expression of Secreted His-Tagged S-adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization, One-Step Purification, and Immobilization

Luo¹,

Yuan²,

Luo³

et al. 2008

Biotechnol. Prog.

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S-Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S-adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomyces cereVisiae was successfully produced at high level (∼200 mg/L) by the recombinant methylotrophic yeast Pichia pastoris. The secreted His 6 -tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35°C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from DL-Met and ATP.

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4 The Adenosyltransferases

Cited by 16 publications

References 56 publications

Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

Regulation of the Human MAT2B Gene Encoding the Regulatory β Subunit of Methionine Adenosyltransferase, MAT II

Expression of Secreted His-Tagged S-adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization, One-Step Purification, and Immobilization

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