Abstract
Tetrahydrofolates (THF) are a family of cofactors that function as one-carbon donors in folate-dependent one-carbon metabolism, a metabolic network required for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine in the cytoplasm. 5-FormylTHF is not a cofactor in one-carbon metabolism, but serves as a storage form of THF cofactors. 5-formylTHF is mobilized back into the THF cofactor pool by methenyltetrahydrofolate synthetase (MTHFS), which catalyzes the irreversible and ATP-dependent conversion 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Mthfs is not an essential gene in Arabidopsis, but MTHFS expression is elevated in animal tumors, enhances de novo purine synthesis, confers partial resistance to antifolate purine synthesis inhibitors and increases rates of folate catabolism in mammalian cell cultures. However, the mechanisms underlying these effects of MTHFS expression have yet to be established. The purpose of this study was to investigate the role and essentiality of MTHFS in mice. Mthfs was disrupted through the insertion of a gene trap vector between exons 1 and 2. Mthfsgt/+ mice were fertile and viable. No Mthfsgt/gt embryos were recovered from Mthfsgt/+ intercrosses, indicating Mthfs is an essential gene in mice. Tissue MTHFS protein levels are decreased in both Mthfsgt/+ and Mthfs+/+ mice placed on a folate and choline deficient diet, and mouse embryonic fibroblasts from Mthfsgt/+ embryos exhibit decreased capacity for de novo purine synthesis without impairment in de novo thymidylate synthesis. MTHFS was shown to co-localize with two enzymes of the de novo purine synthesis pathway in HeLa cells in a cell cycle-dependent manner, and to be modified by the small ubiquitin-like modifier (SUMO) protein. Mutation of the consensus SUMO modification sites on MTHFS eliminated co-localization of MTHFS with the de novo purine biosynthesis pathway under purine-deficient conditions. The results from this study indicate that MTHFS enhances purine biosynthesis by delivering 10-formylTHF to the purinosome in a SUMO-dependent fashion.
Highlights
Tetrahydrofolates (THF) are a family of cofactors that carry and chemically activate single carbons on N5, N10, or bridged between N5 and N10 (Shane, 1995)
The results demonstrate that Mthfs is an essential gene in mice and that reduced methenyltetrahydrofolate synthetase (MTHFS) expression in mouse embryonic fibroblast (MEF) cells is associated with decreased capacity for de novo purine synthesis in these cells
No Mthfsgt/gt embryos were observed at day 9.5 dpc in seven litters containing 42 fetuses (Table 1), indicating that embryos homozygous for the Mthfsgt allele did not survive early embryogenesis and that Mthfs is an essential gene in C57Bl/6 mice
Summary
Tetrahydrofolates (THF) are a family of cofactors that carry and chemically activate single carbons on N5, N10, or bridged between N5 and N10 (Shane, 1995). THF cofactors contain a glutamate polypeptide that contains 2–9 glutamate residues linked through γ-peptide linkages (Shane, 1995). The glutamate residues serve both to retain the co-factors within the cell and increase the affinity of folate-utilizing enzymes. THF cofactors donate or accept one-carbons in a network of interconnected pathways referred to as folate-mediated one-carbon metabolism (OCM). Folate-mediated OCM is required for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine (Figure 1). Impairments in OCM can result from primary folate deficiency, polymorphisms in genes that encode folatedependent enzymes, increased rates of folate degradation, and secondary nutrient deficiencies including vitamin B6 or vitamin B12. Biomarkers for impaired OCM include elevations in tissue and serum homocysteine and S-adenosylhomocysteine (AdoHcy) levels (Lindenbaum and Allen, 1995; Selhub, 1999; Ueland et al, 2000; Yi et al, 2000), increased rates of uracil incorporation into DNA which increases the incidence of DNA strand breaks (Blount et al, 1997; Melnyk et al, 1999), and DNA hypomethylation (Yi et al, 2000; Friso et al, 2002)
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