In eukaryotes, folate metabolism is compartmentalized between the cytoplasm and organelles. The folate pathways of mitochondria are adapted to serve the metabolism of the organism. In yeast, mitochondria support cytoplasmic purine synthesis through the generation of formate. This pathway is important but not essential for survival, consistent with the flexibility of yeast metabolism. In plants, the mitochondrial pathways support photorespiration by generating serine from glycine. This pathway is essential under photosynthetic conditions and the enzyme expression varies with photosynthetic activity. In mammals, the expression of the mitochondrial enzymes varies in tissues and during development. In embryos, mitochondria supply formate and glycine for purine synthesis, a process essential for survival; in adult tissues, flux through mitochondria can favor serine production. The differences in the folate pathways of mitochondria depending on species, tissues and developmental stages, profoundly alter the nature of their metabolic contribution.
Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceutical interest, as their function is crucial for amino acid and DNA biosynthesis. The crystal structure of the human cytosolic methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme has been determined previously with a bound NADP cofactor. While the substrate binding site was identified to be localized in a deep and rather hydrophobic cleft at the interface between two protein domains, the unambiguous assignment of catalytic residues was not possible. We succeeded in determining the crystal structures of three ternary DC301/NADP/inhibitor complexes. Investigation of these structures followed by site-directed mutagenesis studies allowed identification of the amino acids involved in catalysis by both enzyme activities. The inhibitors bind close to Lys56 and Tyr52, residues of a strictly conserved motif for active sites in dehydrogenases. While Lys56 is in a good position for chemical interaction with the substrate analogue, Tyr52 was found stacking against the inhibitors' aromatic rings and hence seems to be more important for proper positioning of the ligand than for catalysis. Also, Ser49 and/or Cys147 were found to possibly act as an activator for water in the cyclohydrolase step. These and the other residues (Gln100 and Asp125), with which contacts are made, are strictly conserved in THF dehydrogenases. On the basis of structural and mutagenesis data, we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase.
Methylenetetrahydrofolate dehydrogenase)methenyltetrahydrofolate cyclohydrolase)formyltetrahydrofolate synthetase (MTHFD1) is a trifunctional enzyme that interconverts tetrahydrofolate (THF) derivatives for nucleotide synthesis. A common variant in MTHFD1, p.Arg653Gln (c.1958G>A), may increase the risk for neural tube defects (NTD). To examine the biological impact of this variant on MTHFD1 function, we measured enzyme activity and stability in vitro and assessed substrate flux in transfected mammalian cells. The purified Arg653Gln enzyme has normal substrate affinity but a 36% reduction in half)life at 42 degrees C. Thermolability is reduced by magnesium adenosine triphosphate and eliminated by the substrate analog folate pentaglutamate, suggesting that folate status may modulate impact of the variant. The mutation reduces the metabolic activity of MTHFD1 within cells: formate incorporation into DNA in murine Mthfd1 knockout cells transfected with Arg653Gln is reduced by 26%+/-7.7% (P<0.05), compared to cells transfected with wild)type protein, indicating a disruption of de novo purine synthesis. We assessed the impact of the variant on risk for congenital heart defects (CHD) in a cohort of Quebec children (158 cases, 110 controls) and mothers of children with heart defects (199 cases, 105 controls). The 653QQ genotype in children is associated with increased risk for heart defects (odds ratio [OR], 2.11; 95% confidence interval [CI], 1.01-4.42), particularly Tetralogy of Fallot (OR, 3.60; 95% CI, 1.38-9.42) and aortic stenosis (OR, 3.13; 95% CI, 1.13-8.66). There was no effect of maternal genotype. Our results indicate that the Arg653Gln polymorphism decreases enzyme stability and increases risk for CHD. Further evaluation of this polymorphism in folate)related disorders and its potential interaction with folate status is warranted.
Folate-dependent enzymes are compartmentalized between the cytoplasm and mitochondria of eukaryotes. The role of mitochondrial folate-dependent metabolism and the extent of its contribution to cytoplasmic processes are areas of active investigation. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) catalyzes the interconversion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate in mitochondria of mammalian cells, but its metabolic role is not yet clear. Its expression in embryonic tissues but not in most adult tissues as well as its stringent transcriptional regulation led us to postulate that it may play a role in embryonic development. To investigate the metabolic role of NMDMC, we used a knockout approach to delete the nmdmc gene in mice. Heterozygous mice appear healthy, but homozygous NMDMC knockout mice die in utero. At embryonic day 12.5 (E12.5), homozygous null embryos exhibit no obvious developmental defects but are smaller and pale and die soon thereafter. Mutant fetal livers contain fewer nucleated cells and lack the characteristic redness of wild-type or heterozygous livers. The frequencies of CFU-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) from fetal livers of E12.5 null mutants were not reduced compared with those of wild-type or heterozygous embryos. It has been assumed that initiation of protein synthesis in mitochondria requires a formylated methionyl-tRNA fmet . One role postulated for NMDMC is to provide 10-formyltetrahydrofolate as a formyl group donor for the synthesis of this formylmethionyl-tRNA fmet . To determine if the loss of NMDMC impairs protein synthesis and thus could be a cause of embryonic lethality, mitochondrial translation products were examined in cells in culture. Mitochondrial protein synthesis was unaffected in NMDMC-null mutant cell lines compared with the wild type. These results show that NMDMC is not required to support initiation of protein synthesis in mitochondria in isolated cells but instead demonstrate an essential role for mitochondrial folate metabolism during embryonic development.Folic acid is essential for the synthesis of purines and thymidylate and is also required for the remethylation of homocysteine to methionine to support the many cellular methylation reactions that use [S]adenosylmethionine. Because of its numerous roles, it is not surprising that the status of folic acid has been linked with a number of conditions that include megaloblastic anemia, cardiovascular disease, and birth defects, as well as with several types of cancer (for a review, see reference 13). The metabolically active form, tetrahydrofolate (THF), functions to transfer one-carbon units that exist in different oxidation states (14). These one-carbon folates are interconverted by enzymes that are present in cytosolic and mitochondrial compartments in mammalian cells (Fig. 1). Recently, there has been a considerable interest in understanding the metabolic advantages for this compartmentalization, support...
Analysis of the structure suggests strongly that folate-binding sites for both activities are within the cleft, providing direct support for the proposed overlapping site model. The orientation of the nicotinamide ring suggests that in the dehydrogenase-catalyzed reaction hydride transfer occurs to the pro-R side of the ring. The identity of the cyclohydrolase active site is not obvious. We propose that a conserved motif-Tyr52-X-X-X-Lys56- and/or a Ser49-Gln100-Pro102 triplet have a role in this activity.
Primary fibroblasts established from embryos of NAD-dependent mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) knockout mice were spontaneously immortalized or transformed with SV40 Large T antigen. Mitotracker Red CMXRos staining of the cells indicates the presence of intact mitochondria with a membrane potential. The nmdmc(-/-) cells are auxotrophic for glycine, demonstrating that NMDMC is the only methylenetetrahydrofolate dehydrogenase normally expressed in the mitochondria of these cell lines. Growth of null mutant but not wild type cells on complete medium with dialyzed serum is stimulated about 2-fold by added formate or hypoxanthine. Radiolabeling experiments demonstrated a 3-10 x enhanced incorporation of radioactivity into DNA from formate relative to serine by nmdmc(-/-) cells. The generation of one-carbon units by mitochondria in nmdmc(-/-) cells is completely blocked, and the cytoplasmic folate pathways alone are insufficient for optimal purine synthesis. The results demonstrate a metabolic role for NMDMC in supporting purine biosynthesis. Despite the recognition of these metabolic defects in the mutant cell lines, the phenotype of nmdmc(-/-) embryos that begin to die at E13.5 is not improved when pregnant dams are given a glycine-rich diet or daily injections of sodium formate.
Genetic variants in one-carbon folate metabolism have been identified as risk factors for disease because they may impair the production or use of one-carbon folates required for nucleotide synthesis and methylation. p.R653Q (1958G>A) is a single-nucleotide polymorphism (SNP) in the 10-formyltetrahydrofolate (formylTHF) synthetase domain of the trifunctional enzyme MTHFD1; this domain produces the formylTHF which is required for the de novo synthesis of purines. Approximately 20% of Caucasians are homozygous for the Q allele. MTHFD1 p.R653Q has been proposed as a risk factor for neural tube defects (NTDs), congenital heart defects (CHDs) and pregnancy losses. We have generated a novel mouse model in which the MTHFD1 synthetase activity is inactivated without affecting protein expression or the other activities of this enzyme. Complete loss of synthetase activity (Mthfd1S(-/-)) is incompatible with life; embryos die shortly after 10.5 days gestation, and are developmentally delayed or abnormal. The proportion of 10-formylTHF in the plasma and liver of Mthfd1S(+/-) mice is reduced (P < 0.05), and de novo purine synthesis is impaired in Mthfd1S(+/-) mouse embryonic fibroblasts (MEFs, P < 0.005). Female Mthfd1S(+/-) mice had decreased neutrophil counts (P < 0.05) during pregnancy and increased incidence of developmental defects in embryos (P = 0.052). These findings suggest that synthetase deficiency may lead to pregnancy complications through decreased purine synthesis and reduced cellular proliferation. Additional investigation of the impact of synthetase polymorphisms on human pregnancy is warranted.
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