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: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The use and metabolism of folates by leishmanias have been studied by assessing the growth of promastigotes in defined media with different folates and the cell content of folate-metabolising enzymes. The folates present in Leishmania mexicana mexicana have been determined using HPLC. Folic acid, 5-formyltetrahydrofolate (THF) and 5-methyl-THF each supported growth of L. m. mexicana promastigotes in defined medium, whereas the parasites did not survive in the absence of folates; p-aminobenzoic acid could not replace the folate requirement. The only folate present at detectable levels in L. m. mexicana promastigotes was 5-methyl-THF. Dihydrofolate reductase (EC 1.5.1.3), methylene-THF reductase (EC 1.1.1.68), serine hydroxymethyltransferase (EC 2.1.2.1) and thymidylate synthetase (EC 2.1.1.45) were all detected in extracts of promastigotes of L. m. mexicana, L. donovani and L. major. Some of these activities were also found in extracts of amastigotes of the former two species. The enzymes of L. m. mexicana have been partially characterised. Methylene-THF reductase may be involved in the conversion in vivo of 5-methyl-THF to 5,10-methylene-THF.
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PMID:Folate utilisation by Leishmania species and the identification of intracellular derivatives and folate-metabolising enzymes. 357 56

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

10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate to tetrahydrofolate (THF). Expression of the enzyme in FDH-deficient cancer cells induces cytotoxicity that can be reversed by supplementation with high concentrations of a reduced folate, 5-formyl-THF (leucovorin). In contrast, non-tumor cells are resistant to FDH. The present study was undertaken to investigate mechanisms that could protect cells against FDH suppressor effects. Using 10 microM leucovorin supplementation of FDH-sensitive A549 cells transfected for FDH expression, we selected clones that have acquired resistance against FDH. Resistant cells expressed high levels of FDH and were capable of growing after withdrawal of leucovorin. These cells, however, have increased doubling time due to prolonged S phase. They also have significantly increased levels of total folate pool and THF/5,10-methylene-THF pool while the level of 10-formyl-THF was two-fold lower than in parental FDH-sensitive cells. We have shown that the FDH-catalyzed reaction proceeds at about a three-fold slower rate at the ratio of 10-formyl-THF/THF corresponding to the resistant cells than at the ratio corresponding to parental sensitive cells, due to product inhibition (KI is 2.35 microM). FDH-resistant cells have strongly up-regulated dihydrofolate reductase (DHFR) that is proposed to be a mechanism for the alteration of folate pools and a key component of the acquired resistance. Elevation of DHFR in A549 cells by transient transfection decreased sensitivity to FDH toxicity and allowed selection of FDH-resistant clones. DHFR-induced repression of FDH catalysis could be an S phase-related metabolic adjustment that provides protection against FDH suppressor effects.
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PMID:Leucovorin-induced resistance against FDH growth suppressor effects occurs through DHFR up-regulation. 1671 99

Serine hydroxymethyltransferases (SHMTs) reversibly transform serine into glycine in a reaction accompanied with conversion of tetrahydrofolate (THF) into 5,10-methylene-THF (5,10-meTHF). In vivo, 5,10-meTHF is the main carrier of one-carbon (1C) units, which are utilized for nucleotide biosynthesis and other processes crucial for every living cell, but hyperactivated in overproliferating cells (e.g. cancer tissues). SHMTs are emerging as a promising target for development of new drugs because it appears possible to inhibit growth of cancer cells by cutting off the supply of 5,10-meTHF. Methotrexate (MTX) and pemetrexed (PTX) are two examples of antifolates that have cured many patients over the years but target different enzymes from the folate cycle (mainly dihydrofolate reductase and thymidylate synthase, respectively). Here we show crystal structures of MTX and PTX bound to plant SHMT isozymes from cytosol and mitochondria-human isozymes exist in the same subcellular compartments. We verify inhibition of the studied isozymes by a thorough kinetic analysis. We propose to further exploit antifolate scaffold in development of SHMT inhibitors because it seems likely that especially polyglutamylated PTX inhibits SHMTs in vivo. Structure-based optimization is expected to yield novel antifolates that could potentially be used as chemotherapeutics.
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PMID:Structural basis of methotrexate and pemetrexed action on serine hydroxymethyltransferases revealed using plant models. 3187 25

One-carbon metabolism produces methionine and N¹⁰-formyl-tetrahydrofolate (N¹⁰-fTHF) required for aminoacylation and formylation of initiator tRNA (i-tRNA), respectively. In Escherichia coli, N¹⁰-fTHF is made from 5, 10-methylene-THF by a two-step reaction using 5,10-methylene-THF dehydrogenase/cyclohydrolase (FolD). The i-tRNAs from all domains of life possess a highly conserved sequence of three consecutive G-C base pairs (3GC pairs) in their anticodon stem. A 3GC mutant i-tRNA (wherein the 3GC pairs are mutated to those found in elongator tRNA^Met) is incompetent in initiation in E. coli (even though it is efficiently aminoacylated and formylated). Here, we show that E. coli strains having mutations in FolD (G122D or C58Y or P140L) allow a plasmid encoded 3GC mutant i-tRNA to participate in initiation. In vitro, the FolD mutants are highly compromised in their dehydrogenase/cyclohydrolase activities leading to reduced production of N¹⁰-fTHF and decreased rates of i-tRNA formylation. The perturbation of one-carbon metabolism by trimethoprim (inhibitor of dihydrofolate reductase) phenocopies FolD deficiency and allows initiation with the 3GC mutant i-tRNA. This study reveals an important crosstalk between one-carbon metabolism and the fidelity of translation initiation via formylation of i-tRNA, and suggests that augmentation of the age old sulfa drugs with FolD inhibitors could be an important antibacterial strategy.
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PMID:Metabolic Flux of N¹⁰-Formyltetrahydrofolate Plays a Critical Role in the Fidelity of Translation Initiation in Escherichia coli. 3279 32

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