Inhibition of 5,10-methenyltetrahydrofolate synthetase.

Cornell University, Graduate Field of Biochemistry, Molecular and Cell Biology, Ithaca, NY 14853, USA.
Archives of Biochemistry and Biophysics (Impact Factor: 3.37). 03/2007; 458(2):194-201. DOI: 10.1016/
Source: PubMed

ABSTRACT The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-Formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (K(i)=0.7 microM), whereas 5-formyl,10-methyltetrahydrofolate was a weak inhibitor (K(i)=10 microM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-Formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.

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    ABSTRACT: Tetrahydrofolate (THF) polyglutamates are a family of cofactors that carry and chemically activate one-carbon units for biosynthesis. THF-mediated one-carbon metabolism is a metabolic network of interdependent biosynthetic pathways that is compartmentalized in the cytoplasm, mitochondria, and nucleus. One-carbon metabolism in the cytoplasm is required for the synthesis of purines and thymidylate and the remethylation of homocysteine to methionine. One-carbon metabolism in the mitochondria is required for the synthesis of formylated methionyl-tRNA; the catabolism of choline, purines, and histidine; and the interconversion of serine and glycine. Mitochondria are also the primary source of one-carbon units for cytoplasmic metabolism. Increasing evidence indicates that folate-dependent de novo thymidylate biosynthesis occurs in the nucleus of certain cell types. Disruption of folate-mediated one-carbon metabolism is associated with many pathologies and developmental anomalies, yet the biochemical mechanisms and causal metabolic pathways responsible for the initiation and/or progression of folate-associated pathologies have yet to be established. This chapter focuses on our current understanding of mammalian folate-mediated one-carbon metabolism, its cellular compartmentation, and knowledge gaps that limit our understanding of one-carbon metabolism and its regulation.
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    ABSTRACT: 5-Formyltetrahydrofolate (5-CHO-THF) is formed by a side reaction of serine hydroxymethyltransferase. Unlike other folates, it is not a one-carbon donor but a potent inhibitor of folate enzymes and must therefore be metabolized. Only 5-CHO-THF cycloligase (5-FCL) is generally considered to do this. However, comparative genomic analysis indicated (i) that certain prokaryotes lack 5-FCL, implying that they have an alternative 5-CHO-THF-metabolizing enzyme, and (ii) that the histidine breakdown enzyme glutamate formiminotransferase (FT) might moonlight in this role. A functional complementation assay for 5-CHO-THF metabolism was developed in Escherichia coli, based on deleting the gene encoding 5-FCL (ygfA). The deletion mutant accumulated 5-CHO-THF and, with glycine as sole nitrogen source, showed a growth defect; both phenotypes were complemented by bacterial or archaeal genes encoding FT. Furthermore, utilization of supplied 5-CHO-THF by Streptococcus pyogenes was shown to require expression of the native FT. Recombinant bacterial and archaeal FTs catalyzed formyl transfer from 5-CHO-THF to glutamate, with k(cat) values of 0.1-1.2 min(-1) and K(m) values for 5-CHO-THF and glutamate of 0.4-5 μM and 0.03-1 mM, respectively. Although the formyltransferase activities of these proteins were far lower than their formiminotransferase activities, the K(m) values for both substrates relative to their intracellular levels in prokaryotes are consistent with significant in vivo flux through the formyltransferase reaction. Collectively, these data indicate that FTs functionally replace 5-FCL in certain prokaryotes.
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