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.
Christensen
Karen E
KE
Department of Biochemistry, McGill University, Montréal, Québec H3G 1Y6, Canada.
Patel
Harshila
H
Kuzmanov
Uros
U
Mejia
Narciso R
NR
MacKenzie
Robert E
RE
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D002461:Cell Line, Transformed; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D004622:Embryo, Mammalian / Q000166:cytology*; D005091:Exons; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005574:Formate-Tetrahydrofolate Ligase / Q000235:genetics* / Q000502:physiology*; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D045064:Methenyltetrahydrofolate Cyclohydrolase / Q000737:chemistry; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D012313:RNA / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012984:Software; D013234:Stem Cells / Q000166:cytology*; D013997:Time Factors
D018076:DNA, Complementary; D012313:RNA; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D045064:Methenyltetrahydrofolate Cyclohydrolase; D005574:Formate-Tetrahydrofolate Ligase