Disruption of the Mthfd1 Gene Reveals a Monofunctional 10-Formyltetrahydrofolate Synthetase in Mammalian Mitochondria

Abstract

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 MTHFD2L Encodes a Mitochondrial Methylenetetrahydrofolate Dehydrogenase Isozyme Expressed in Adult Tissues

“…Importantly, the predicted MTHFD2L proteins possess four amino acids (Lys-93, Arg-206, Gly-211, and Arg-238 in Fig. 2) shown to be critical for dehydrogenase and/or cyclohydrolase activity (24). These four residues have been substituted in all MTHFD1L-encoded monofunctional mitochondrial C 1 -THF synthases (33).…”

Magnesium and Phosphate Ions Enable NAD Binding to Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase

“…This change in cofactor specificity is important because the use of NAD rather than NADP in mitochondria shifts the equilibrium of the reaction to favor the production of formyl-THF (5). The increased production of formyl-THF is required to meet the demand for glycine and purines during embryogenesis (1,2,6). However, it is not known how this cofactor specificity change was accomplished.…”

“…The mitochondrial formyl-THF is converted to formate by a monofunctional formyl-THF synthetase and is released to the cytoplasm for reconversion into formyl-THF to support purine biosynthesis (1). NMDMC can use both NAD and NADP as a cofactor in its dehydrogenase activity, although the maximal activity with NADP is only about twenty percent of that with NAD (3).…”

Polymorphisms of MTHFD, Plasma Homocysteine Levels, and Risk of Gastric Cancer in a High-Risk Chinese Population

“…Examination of the primary structure of this protein revealed mutations in key residues required for dehydrogenase and cyclohydrolase activities (22). This monofunctional synthetase completes the pathway for the production of formate from formyltetrahydrofolate in the mitochondria in the model of mammalian one-carbon folate metabolism in embryonic and transformed cells (22).…”

“…Examination of the primary structure of this protein revealed mutations in key residues required for dehydrogenase and cyclohydrolase activities (22). This monofunctional synthetase completes the pathway for the production of formate from formyltetrahydrofolate in the mitochondria in the model of mammalian one-carbon folate metabolism in embryonic and transformed cells (22). It has been reported that MTHFD single nucleotide polymorphisms were associated with serum folic acid and homocysteine levels in congenital heart disease families (23,24), indicating its important roles in folate and homocysteine metabolism pathway.…”