Disruption of the Mthfd1 Gene Reveals a Monofunctional 10-Formyltetrahydrofolate Synthetase in Mammalian Mitochondria
| Autor: | Harshila Patel, Robert E. MacKenzie, Karen E. Christensen, Narciso R. Mejia, Uros Kuzmanov |
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| Rok vydání: | 2005 |
| Předmět: |
Heterozygote
DNA Complementary Time Factors Genotype Auxotrophy Molecular Sequence Data Methenyltetrahydrofolate Cyclohydrolase Mice Transgenic Dehydrogenase Mitochondrion Biology Models Biological Biochemistry Cell Line Formate-Tetrahydrofolate Ligase Mice chemistry.chemical_compound Animals Humans Amino Acid Sequence Northern blot Molecular Biology Gene Cell Line Transformed Methylenetetrahydrofolate Dehydrogenase (NADP) Mice Knockout Models Genetic Sequence Homology Amino Acid 10-Formyltetrahydrofolate Stem Cells Homozygote Exons Cell Biology Fibroblasts Blotting Northern Embryo Mammalian Mitochondria Databases as Topic chemistry Cell culture Methylenetetrahydrofolate dehydrogenase Mutation RNA Software |
| Zdroj: | Journal of Biological Chemistry. 280:7597-7602 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m409380200 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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