NAD- and NADP-dependent mitochondrially targeted methylenetetrahydrofolate dehydrogenase-cyclohydrolases can rescue mthfd2 null fibroblasts.

Mouse fibroblasts in which the mthfd2 gene encoding mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) was previously inactivated were infected with retroviral expression constructs of dehydrogenase/cyclohydrolase cDNA. Cellular fractionation confirmed that the expressed proteins were properly targeted to the mitochondria. Expression of the NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase enzyme in mitochondria corrected the glycine auxotrophy of the null mutant cells. A construct in which the cyclohydrolase activity of NMDMC was inactivated by point mutation also rescued the glycine auxotrophy, although poorly. This suggests that the cyclohydrolase activity is also required to ensure optimal production of 10-formyltetrahydrofolate. The expression of the NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase in the mitochondria also reversed the glycine requirement of the null cells demonstrating that the use of the NAD cofactor is not absolutely essential to maintain the flux of one-carbon metabolites. All rescued cells demonstrated a decrease in the ratio of incorporation of exogenous formate to serine in standardized radiolabeling studies. This ratio, which is approximately 2.5 for nmdmc(-/-) cells and 0.3 for the wild type cells under the conditions used, is a qualitative indicator of the ability of the mitochondria of the cells to generate formate.

Disruption of the mthfd1 gene reveals a monofunctional 10-formyltetrahydrofolate synthetase in mammalian mitochondria.

The Mthfd1 gene encoding the cytoplasmic methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase enzyme (DCS) was inactivated in embryonic stem cells. The null embryonic stem cells were used to generate spontaneously immortalized fibroblast cell lines that exhibit the expected purine auxotrophy. Elimination of these cytoplasmic activities allowed for the accurate assessment of similar activities encoded by other genes in these cells. A low level of 10-formyltetrahydrofolate synthetase was detected and was shown to be localized to mitochondria. However, NADP-dependent methylenetetrahydrofolate dehydrogenase activity was not detected. Northern blot analysis suggests that a recently identified mitochondrial DCS (Prasannan, P., Pike, S., Peng, K., Shane, B., and Appling, D. R. (2003) J. Biol. Chem. 278, 43178-43187) is responsible for the synthetase activity. The lack of NADP-dependent dehydrogenase activity suggests that this RNA may encode a monofunctional synthetase. Moreover, examination of the primary structure of this novel protein revealed mutations in key residues required for dehydrogenase and cyclohydrolase activities. This monofunctional synthetase completes the pathway for the production of formate from formyltetrahydrofolate in the mitochondria in our model of mammalian one-carbon folate metabolism in embryonic and transformed cells.

Mammalian mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase derived from a trifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase.

We have isolated the cDNA and the gene encoding the murine cytoplasmic methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (DCS). Comparison of these sequences with the 3'-untranslated region of the mitochondrial NAD(+)-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (mt-DC) revealed areas of significant homology. Both exon and intron sequences of the synthetase domain of DCS are homologous to sequences in the untranslated region of mt-DC. A similar comparison between the mt-DC and the DCS sequences of humans as well as Drosophila supports the conclusion that in higher eukaryotes the bifunctional mt-DC replaced a trifunctional precursor through inactivation of the synthetase domain. The mt-DC should be considered in models of one-carbon folate fluxes in mammals.