Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: DrugBank:EXPT03052 (THF)
8,150 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protein product of the ADE3 gene of the yeast Saccharomyces cerevisiae has been identified as the cytoplasmic trifunctional C1-tetrahydrofolate (THF) synthase, which possesses 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5) activities. However, it has been suggested that the ADE3-encoded C1-THF synthase does not play a role in providing the enzymes involved in the generation of one-carbon intermediates in the biosynthesis of the purine bases but functions in maintaining the structural integrity of the enzyme complex involved in purine biosynthesis [Barlowe, C. K. & Appling, D. A. (1990) Mol. Cell. Biol. 10, 5679-5687]. This hypothesis is based on their finding that the presence of the full-length ADE3 C1-THF synthase, whether catalytically active or not, is correlated with the Ade+ phenotype. In contrast to their results, our deletion analysis of the ADE3 gene indicates that the presence of either the synthetase or dehydrogenase/cyclohydrolase domains of C1-THF synthase is enough to complement the adenine requirement in ade3 strains. These results are also consistent with those obtained in heterologous expression of spinach and Clostridium acidiurici monofunctional synthetases in ade3 strains. Heterologous expression studies show that the high synthetase activity may be correlated with the increased growth in medium lacking adenine. These results suggest that the catalytic activity of the C1-THF synthase is involved in purine biosynthesis.
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PMID:Function of yeast cytoplasmic C1-tetrahydrofolate synthase. 846 69

Methionine synthase enzymes catalyze methyl group transfer from 5-methyltetrahydrofolate to homocysteine to give methionine and tetrahydrofolate. Assays for this enzyme activity usually monitor transfer of a 14C-methyl group from the N5-position of methyltetrahydrofolate to homocysteine to produce 14C-methionine that must be purified by anion-exchange chromatography. Alternatively, tetrahydrofolate may be derivatized with a formylating agent under acidic conditions to produce methenyltetrahydrofolate. We report optimization of this reaction for assay of cobalamin-dependent methionine synthase to give an economical method for determining enzyme activity that does not require the use of radioactive compounds. By heating for 10 min in 1 N hydrochloric acid containing 12% formic acid, the enzymatic product tetrahydrofolate is converted into methenyltetrahydrofolate, which absorbs light at 350 nm, while residual substrate 5-methyltetrahydrofolate does not contribute to the absorbance at 350 nm. The assay allows the derivatized product to be characterized in situ with a minimal increase in volume upon acidification. The results of the spectrophotometric assay given here have been compared with the radioactive assay to confirm the validity of the derivatization under the assay conditions. We also report the extension of this assay method for use in activity measurements of cobalamin-independent methionine synthase.
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PMID:Characterization of nonradioactive assays for cobalamin-dependent and cobalamin-independent methionine synthase enzymes. 857 14

Serine is generally accepted as the major one-carbon donor in folate-mediated one-carbon metabolism in most cells. Previous work from our laboratory with the yeast Saccharomyces cerevisiae has demonstrated that glycine and formate can also provide one-carbon units. Under normal growth conditions, it is likely that cells utilize serine, glycine, formate, and perhaps other one-carbon donors simultaneously, but to differing degrees. In the present work, we have used 13C NMR to monitor how yeast cells distribute alternative, competing one-carbon sources into various pools. Cells were grown with [2-13C]glycine and unlabeled formate or folinic acid (leucovorin, 5-formyl-tetrahydrofolate) as competing one-carbon sources. The relative contribution of each one-carbon donor to the three oxidation states of the tetrahydrofolate-bound one-carbon pool [5-methyl-tetrahydrofolate (CH3-THF), 5,10-methylene-THF (CH2-THF), and 10-formyl-THF (10-CHO-THF)] was determined by analysis of two metabolic end products of one-carbon metabolism, choline and adenine. Glycine-derived 13C-labeled one-carbon units are incorporated into these two metabolites; dilution of the 13C indicates competition by the unlabeled one-carbon source. The results reveal that the contribution from formate, folinic acid, and glycine is different for each of the one-carbon pools. Formate competed most dramatically at the 10-CHO-THF pool, with decreasing competition into the CH2-THF and CH3-THF pools. In a mutant strain lacking cytosolic CH2-THF dehydrogenase activity, a distinct shift toward the use of glycine instead of formate as the source of one-carbon units for the more reduced pools (CH2-THF and CH3-THF) was observed, while 10-CHO-THF pools were not affected. In contrast, the formyl group of folinic acid competed almost exclusively at the 10-CHO-THF level, with barely detectable dilution of the CH2-THF and CH3-THF pools in wild-type cells. The mutant strain exhibited essentially identical results, confirming that 5-formyl-THF enters the active one-carbon pool at the level of 10-CHO-THF, presumably via 5,10-methenyl-THF. Furthermore, donation of one-carbon units by folinic acid was observed only when cells were depleted of THF by treatment with the dihydrofolate reductase inhibitor methotrexate. These results reveal that the state of equilibrium between one-carbon pools in a growing cell depends on the source of the one-carbon units. This work illustrates the power of 13C NMR for examining the in vivo utilization of alternative one-carbon donors under a variety of conditions.
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PMID:13C NMR analysis of the use of alternative donors to the tetrahydrofolate-dependent one-carbon pools in Saccharomyces cerevisiae. 857 65

C1-tetrahydrofolate (THF) synthase is a eukaryotic trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. Although the 10-formyl-THF synthetase reaction (a reversible ATP-dependent formylation of THF) has been studied extensively, little is known about specific residues involved in the catalytic mechanism. In this study, we have examined the role of a highly conserved aspartate residue, Asp449 of yeast cytoplasmic C1-THF synthase. Asp449 is part of a putative folate binding site found in many proteins that bind 10-formyl-THF. The corresponding aspartate has been identified as a critical catalytic residue in Escherichia coli and human GAR transformylase, which catalyzes a 10-formyl-THF-dependent formyl transfer. In order to determine if Asp449 has a similar catalytic role in the 10-formyl-THF synthetase reaction, three mutant proteins were produced by site-directed mutagenesis in which Asp449 of yeast cytoplasmic C1-THF synthase was changed to Asn, Glu, or Ala. The mutant proteins were expressed in yeast, purified, and characterized with respect to kinetic properties and enzyme stability. All three of the mutant enzymes retained substantial 10-formyl-THF synthetase activity, indicating that Asp449 is not a critical catalytic residue. However, our data suggest that it does play a role in folate binding, probably by contributing to the proper conformation of the active site. Thus, these results suggest that the 10-formyl-THF binding site differs significantly between the GAR transformylase and 10-formyl-THF synthetase families, and that the conserved aspartate plays different roles in the two enzymes.
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PMID:Site-directed mutagenesis of a highly conserved aspartate in the putative 10-formyl-tetrahydrofolate binding site of yeast C1-tetrahydrofolate synthase. 880 78

10-Formyl-7,8-dihydrofolic acid (10-HCO-H2folate) was prepared by controlled air oxidation of 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate). The UV spectra of the 10-HCO-H2folate preparation has lambda max. 234, 333 nm and lambda min. 301 nm at pH 7.4, and lambda max. 257, 328 nm and lambda min. 229, 307 nm at pH 1. 1H-NMR spectroscopy of 10-HCO-H2folate (in 2H2O; 300 MHz) suggested a pure compound and gave resonances for one formyl group proton, two protons on C-7 and C-9, and no evidence for a C-6 proton, which is consistent with the structure proposed. The spectral properties indicated that the 10-HCO-H2folate preparation is not appreciably contaminated with 10-HCO-H4folate, 5,10-methenyltetrahydrofolic acid (5,10-CH = H4folate) or 10-formylfolic acid (10-HCO-folate). The above data establish that the 10-HCO-H2folate prepared here is authentic. In contrast, a folate with a UV spectrum having lambda max. 272 nm and lambda min. 256 nm at pH 7, which was prepared by 2,6-dichloro-indophenol oxidation of 10-HCO-H4folate and reported to be 97% pure [Baram, Chabner, Drake, Fitzhugh, Sholar and Allegra (1988) J. Biol. Chem. 263, 7105-7111], is apparently not 10-HCO-H2folate. 10-HCO-H2folate is utilized by Jurkat-cell (human T-cell leukaemia) and chicken liver aminoimidazolecarboxamide ribonucleotide transformylase (AICAR T'ase; EC 2.1.2.3) in the presence of excess 5-amino-imidazole-4-carboxamide ribotide (AICAR) resulting in the appearance of approximately 1 mol of H2folate product for each mol of AICAR formylated. The present 10-HCO-H2folate preparation had a kinetic advantage over 10-HCO-H4folate resulting from a difference of approx. 5-fold in K(m) values when both folates were used as cofactors for Jurkat-cell and rat bone marrow AICAR T'ase. No substantial kinetic advantage was observed using chicken liver AICAR T'ase. 10-HCO-H2folate had little or no activity with Jurkat-cell or chicken liver glycinamide ribonucleotide transformylase (GAR T'ase, EC 2.1.2.2). The existence in vivo of 10-HCO-H2folate is suggested in mammals by several reports of detectable amounts of radiolabelled 10-HCO-folate in bile and urine after administration of radiolabelled folic acid.
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PMID:Cofactor role for 10-formyldihydrofolic acid. 894 66

A bifunctional enzyme that catalyzes the conversion of formyltetrahydrofolate to methylene-tetrahydrofolate (5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylene tetrahydrofolate dehydrogenase), has been subcloned from a cDNA library, purified to homogeneity, and crystallized. The crystals belong to space group I222, with unit cell dimensions of a = 64.5 A, b = 84.9 A, c = 146.1 A. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme monomer, and a specific volume of the unit cell of 3.2 A3/Da. The crystals diffract to at least 2.8 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. A 2.56 A resolution native data set has been collected at beamline X12-C at the NSLS.
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PMID:Purification, crystallization, and preliminary x-ray studies of a bifunctional 5,10-methenyl/methylene-tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli. 906 97

Deficient activity of an enzyme can result from a defect in the conversion of the vitamin to a co-enzyme as well from an abnormal apo-enzyme or disturbed binding of coenzyme to enzyme. Conversion of dietary vitamin to intracellular active co-enzyme can be complex and require many physiological and biochemical processes including stomach release of bound vitamin, intestinal uptake, carriers/transport, blood transport, cellular uptake, intracellular release and intracellular compartmentalisation. Disorders of malabsorption (food cobalamin malabsorption, intrinsic factor deficiency and abnormal enterocyte cobalamin processing) and transport proteins (transcobalamin II deficiency, R-binder deficiency) mostly lead to disturbed function of the two cobalamin requiring enzymes, methylmalonyl CoA mutase and methionine synthase. Defects of early steps of intracellular cobalamin (cblF, cbl C/D) result in marked deficiencies of both cobalamin co-enzymes and homocystinuria combined with methylmalonic aciduria. Defective synthesis of adenosyl cobalamin in the cbl A/B defects leads to methylmalonyl CoA mutase. Isolated methionine synthase deficiency is also classified as a cobalamin disorder due to its associated deficient formation of methylcobalamin. Folate disorders include methylene-tetrahydrofolate reductase deficiency and glutamate formimino-transferase deficiency. In addition a hereditary disorder of intestinal folate transport has been described. Less well established are disorders of dihydrofolate reductase, methenyl-tetrahydrofolate cyclohydrolase, and defects of cellular folate uptake.
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PMID:Genetic defects of folate and cobalamin metabolism. 958 28

Recently it was found that Methylobacterium extorquens AM1 contains both tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F) as carriers of C1 units. In this paper we report that the aerobic methylotroph contains a methenyl H4MPT cyclohydrolase (0.9 U x mg-1 cell extract protein) and a methenyl H4F cyclohydrolase (0.23 U x mg-1). Both enzymes, which were specific for their substrates, were purified and characterized and the encoding genes identified via the N-terminal amino acid sequence. The purified methenyl H4MPT cyclohydrolase with a specific activity of 630 U x mg-1 (Vmax = 1500 U x mg-1; Km = 30 microm) was found to be composed of two identical subunits of molecular mass 33 kDa. Its sequence was approximately 40% identical to that of methenyl H4MPT cyclohydrolases from methanogenic archaea. The methenyl H4F cyclohydrolase with a specific activity of 100 U x mg-1 (Vmax = 330 U x mg-1; Km = 80 microm) was found to be composed of two identical subunits of molecular mass 22 kDa. Its sequence was not similar to that of methenyl H4MPT cyclohydrolases or to that of other methenyl H4F cyclohydrolases. Based on the specific activities in cell extract and from the growth properties of insertion mutants it is suggested that the methenyl H4MPT cyclohydrolase might have a catabolic, and the methenyl-H4F cyclohydrolase an anabolic function in the C1-unit metabolism of M. extorquens AM1.
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PMID:A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1. 1021 59

Formiminotransferase-cyclodeaminase (E.C. 2.1.2.5-E.C. 4.3.1.4) is a bifunctional enzyme involved in the histidine-degradation pathway which exhibits specificity for polyglutamylated folate substrates. The first function of the enzyme transfers the formimino group of formiminoglutamate to the N5 position of tetrahydrofolate, while the second function catalyses the cyclodeamination of the formimino group, yielding N5,10-methenyl-tetrahydrofolate, with efficient channeling of the intermediate between these activities. Initial studies have shown that the enzyme consists of eight identical subunits of 62 kDa each, arranged as a circular tetramer of dimers. It is this formation which results in two different dimeric interfaces, which are necessary for the two different activities. The identical subunits have been shown to consist of two domains, each of which can be obtained as dimers. The formiminotransferase domain has been crystallized in the presence of the substrate analogue folinic acid. The crystals belong to space group P212121, with unit-cell dimensions a = 64.4, b = 103.7, c = 122.3 A. Both a native data set and a mercurial derivative data set have been collected to 2.8 A resolution.
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PMID:Crystallization and preliminary X-ray analysis of the formiminotransferase domain from the bifunctional enzyme formiminotransferase-cyclodeaminase. 1032 87

Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65. The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5, 10-methenyltetrahydrofolate were not significantly changed. However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.
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PMID:Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase. 1085 98


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