Serine Transhydroxymethylase. Affinity of Tetrahydrofolate Compounds for the Enzyme and Enzyme-Glycine Complex<sup>*</sup>

Schirch, LaVerne; Ropp, Marjorie

doi:10.1021/bi00853a039

Cited by 85 publications

(56 citation statements)

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“…The spectral effects, shown in Figure 4, observed upon binding of H4-folate to the enzyme-glycine complexes are also observed when either 5-methyl-or 5-formyl-H4-folate replaces H4-folate (41). The large increase in absorbance at 500 nm upon formation of the enzyme-glycine-folate complex has been used as a rapid, accurate, and sensitive method for determining the dissociation constants of substrates and inhibitors for both the amino acid and H4-folate binding pockets.…”

Section: I N U T E Smentioning

confidence: 84%

“…The large increase in absorbance at 500 nm upon formation of the enzyme-glycine-folate complex has been used as a rapid, accurate, and sensitive method for determining the dissociation constants of substrates and inhibitors for both the amino acid and H4-folate binding pockets. These studies have shown a synergistic increase in affinity for the active site between the amino acid and H4-folate (41).…”

Section: I N U T E Smentioning

confidence: 95%

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Serine Hydroxymethyltransferase

Schirch

1982

Advances in Enzymology - And Related Areas of Molecular Biology

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Renews56 Sabo, D. L., and Fischer, E. H., Chemical properties ofE. coli lysine decarboxylase including a segment of its pyridoxal 5'-phosphate binding site. Biochemistry 13 (1974) 670 6. 57 Schirch, L., Serine hydroxymethylase. Meth. Enzymol. 17 (1971) 335-40. 58 Schirch, L., Serine hydroxymethyltransferase. Adv. Enzymol. 53 (1982) 83 112. 59 Shlyapnikov, S.V., Myasnikov, A.N., Severin, E.S., Myagkova, M. A., Demedkina, T. V., Torchinskii, Yu. M., and Braunstein, A. E., Primary structure of cytoplasmic aspartate aminotransferase from chicken heart. IV Structure of cyanogen bromide peptides and the complete amino acid sequence. Bioorg. Khim. 6 (1980) Glasgow, Glasgow G12 8QQ (Scotland) Abstract. The harmonization of biosynthetic pathways with organic reaction mechanisms has relied heavily on stereochemical studies. The field of biosynthesis of pyrrolizidine alkaloids exemplifies these connections through a wide range of common organic reactions including oxidation, condensation, and decarboxylation. Further, the applications of biogenetic concepts and enzyme-catalysed reactions to synthesis are illustrated. The results are exciting, not only for their intrinsic scientific interest, but because they point the way to using plant enzymes to recognise structurally modified biosynthetic intermediates and hence open routes to the synthesis of new compounds that would otherwise be difficult to obtain.

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Section: I N U T E Smentioning

confidence: 84%

Section: I N U T E Smentioning

confidence: 95%

Serine Hydroxymethyltransferase

Schirch

1982

Advances in Enzymology - And Related Areas of Molecular Biology

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“…30 and 38) or the two methylenetetrahydrofolate reductase genes (MET12 and MET13; Ref. 39). However, when the fau1 disruption was combined with disruptions of two genes in purine biosynthesis, a striking methionine deficiency was observed, and it appeared to be related to the purine intermediate, AICAR.…”

Section: Discussionmentioning

confidence: 99%

Cloning and Characterization of Methenyltetrahydrofolate Synthetase from Saccharomyces cerevisiae

Holmes¹,

Appling

2002

Journal of Biological Chemistry

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The folate derivative 5-formyltetrahydrofolate (folinic acid; 5-CHO-THF) was discovered over 40 years ago, but its role in metabolism remains poorly understood. Only one enzyme is known that utilizes 5-CHO-THF as a substrate: 5,10-methenyltetrahydrofolate synthetase (MTHFS). A BLAST search of the yeast genome using the human MTHFS sequence revealed a 211-amino acid open reading frame (YER183c) with significant homology. The yeast enzyme was expressed in Escherichia coli, and the purified recombinant enzyme exhibited kinetics similar to previously purified MTHFS. No new phenotype was observed in strains disrupted at MTHFS or in strains additionally disrupted at the genes encoding one or both serine hydroxymethyltransferases (SHMT) or at the genes encoding one or both methylenetetrahydrofolate reductases. However, when the MTHFS gene was disrupted in a strain lacking the de novo folate biosynthesis pathway, folinic acid (5-CHO-THF) could no longer support the folate requirement. We have thus named the yeast gene encoding methenyltetrahydrofolate synthetase FAU1 (folinic acid utilization). Disruption of the FAU1 gene in a strain lacking both 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase isozymes (ADE16 and ADE17) resulted in a growth deficiency that was alleviated by methionine. Genetic analysis suggested that intracellular accumulation of the purine intermediate AICAR interferes with a step in methionine biosynthesis. Intracellular levels of 5-CHO-THF were determined in yeast disrupted at FAU1 and other genes encoding folate-dependent enzymes. In fau1 disruptants, 5-CHO-THF was elevated 4-fold over wild-type yeast. In yeast lacking MTHFS along with both AICAR transformylases, 5-CHO-THF was elevated 12-fold over wild type. 5-CHO-THF was undetectable in strains lacking SHMT activity, confirming SHMT as the in vivo source of 5-CHO-THF. Taken together, these results indicate that S. cerevisiae harbors a single, nonessential, MTHFS activity. Growth phenotypes of multiply disrupted strains are consistent with a regulatory role for 5-CHO-THF in one-carbon metabolism and additionally suggest a metabolic interaction between the purine and methionine pathways.Although 5-formyl-tetrahydrofolate (5-CHO-THF 1 ; folinic acid or leucovorin) is the most stable derivative of the reduced folates (1), it does not have a known direct role as a one-carbon donor. Leucovorin has been employed extensively as a rescue agent in chemotherapeutic protocols, but the physiological role of this folate derivative remains poorly understood. 5-CHO-THF can arise nonenzymatically from the hydrolysis of 5,10-methenyl-THF (5,10-CH ϩ -THF) under mild conditions (2). For this reason, it was considered likely that the 5-CHO-THF detected in cell extracts was an artifact of preparation rather than a normal component of folate metabolism. More recent studies designed to prevent the possibility of artifactual conversion concluded that 5-CHO-THF is indeed a naturally occurring metabolite (3). The biological source of 5-CHO-THF, however,...

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“…This absorbance shows saturation kinetics with most reduced folate substrates, including H 4 PteGlu n , N 5 -CHO-H 4 PteGlu n , and N 5 -CH 3 -H 4 PteGlu n (Schirch and Ropp, 1967;Stover and Schirch, 1991). The binding of substrates to rabbit cytosolic SHMT is a sequential random mechanism.…”

Section: Physical Properties Of Zmshmt(delsig)mentioning

confidence: 96%

Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited by Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases

et al. 2007

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ABSTRACT:Serine hydroxymethyltransferase (SHMT) provides activated onecarbon units required for the biosynthesis of nucleotides, protein, and methyl group by converting serine and tetrahydrofolate to glycine and N 5 ,N 10 -methylenetetrahydrofolate. It is postulated that SHMT activity is associated with the development of methotrexate resistance and the in vivo activity of SHMT is regulated by the binding of N 5 -CHO-THF, the rescue agent in high-dose methotrexate chemotherapy. The aim of this study is to advance our understanding of the folate-mediated one-carbon metabolism in zebrafish by characterizing zebrafish mitochondrial SHMT. The cDNA encoding zebrafish mitochondrial SHMT was cloned, overexpressed in Escherichia coli, and purified with a three-step purification protocol. Similarities in structural, physical, and kinetic properties were revealed between the recombinant zebrafish mitochondrial SHMT and its mammalian orthologs. Surprisingly, leucovorin significantly inhibits the aldol cleavage of serine catalyzed by zebrafish cytosolic SHMT but inhibits to a lesser extent the reaction catalyzed by the mitochondrial isozyme. This is, to our knowledge, the first report on zebrafish mitochondrial folate enzyme as well as the differential inhibition of leucovorin on these two SHMT isoforms. Western blot analysis revealed tissue-specific distribution with the highest enrichment present in liver for both cytosolic and mitochondrial SHMTs. Intracellular localization was confirmed by confocal microscopy for both mitochondrial and cytosolic SHMTs. Unexpectedly, the cytosolic isoform was observed in both nucleus and cytosol. Together with the previous report on zebrafish cytosolic SHMT, we suggest that zSHMTs can be used in in vitro assays for folate-related investigation and antifolate drug discovery.Folates carry the chemically activated single carbons at N 5 and/or N 10 positions and are required for the biosynthesis and metabolism of nucleic acid, protein, amino acid, methyl group, neurotransmitter, and vitamins. Its vital role in nucleotide biogenesis has led to the development of many anticancer drugs targeting folate-requiring enzymes. Among them, methotrexate (MTX) is one of the most widely used anticancer agents to date. It blocks de novo nucleotide synthesis by depleting reduced tetrahydrofolates mainly through inhibition of dihydrofolate reductase (DHFR) and thymidylate synthase (Fig. 1, enzymes 2 and 3, respectively). However, resistance to MTX often emerges and becomes the major impediment to its curative potential.To overcome this obstacle and prevent MTX-associated toxicity, high-dose MTX combined with leucovorin rescue is administered and has become an important regimen in the treatment of a variety of cancers (Frei et al., 1980). Despite these preventive measures, MTXinduced resistance and toxicity continue to occur, although infrequently. Mechanisms including elevated DHFR, decreased thymidylate synthase, impaired folate/antifolate transportation, and decreased polyglutamylation on MTX have been pr...

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Serine Transhydroxymethylase. Affinity of Tetrahydrofolate Compounds for the Enzyme and Enzyme-Glycine Complex^*

Cited by 85 publications

References 10 publications

Serine Hydroxymethyltransferase

Serine Hydroxymethyltransferase

Cloning and Characterization of Methenyltetrahydrofolate Synthetase from Saccharomyces cerevisiae

Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited by Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases

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Serine Transhydroxymethylase. Affinity of Tetrahydrofolate Compounds for the Enzyme and Enzyme-Glycine Complex*

Cited by 85 publications

References 10 publications

Serine Hydroxymethyltransferase

Serine Hydroxymethyltransferase

Cloning and Characterization of Methenyltetrahydrofolate Synthetase from Saccharomyces cerevisiae

Serine Hydroxymethyltransferase Isoforms Are Differentially Inhibited by Leucovorin: Characterization and Comparison of Recombinant Zebrafish Serine Hydroxymethyltransferases

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Serine Transhydroxymethylase. Affinity of Tetrahydrofolate Compounds for the Enzyme and Enzyme-Glycine Complex^*