Dean R. Appling

Deletion of Mthfd1l causes embryonic lethality and neural tube and craniofacial defects in mice

Maternal supplementation with folic acid is known to reduce the incidence of neural tube defects (NTDs) by as much as 70%. Despite the strong clinical link between folate and NTDs, the biochemical mechanisms through which folic acid acts during neural tube development remain undefined. The Mthfd1l gene encodes a mitochondrial monofunctional 10-formyl-tetrahydrofolate synthetase, termed MTHFD1L. This gene is expressed in adults and at all stages of mammalian embryogenesis with localized regions of higher expression along the neural tube, developing brain, craniofacial structures, limb buds, and tail bud. In both embryos and adults, MTHFD1L catalyzes the last step in the flow of one-carbon units from mitochondria to cytoplasm, producing formate from 10-formyl-THF. To investigate the role of mitochondrial formate production during embryonic development, we have analyzed Mthfd1l knockout mice. All embryos lacking Mthfd1l exhibit aberrant neural tube closure including craniorachischisis and exencephaly and/or a wavy neural tube. This fully penetrant folate-pathway mouse model does not require feeding a folate-deficient diet to cause this phenotype. Maternal supplementation with sodium formate decreases the incidence of NTDs and partially rescues the growth defect in embryos lacking Mthfd1l. These results reveal the critical role of mitochondrially derived formate in mammalian development, providing a mechanistic link between folic acid and NTDs. In light of previous studies linking a common splice variant in the human MTHFD1L gene with increased risk for NTDs, this mouse model provides a powerful system to help elucidate the specific metabolic mechanisms that underlie folate-associated birth defects, including NTDs.

Regulation of S-adenosylmethionine levels in saccharomyces cerevisiae

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine biosynthesis. Methionine can be activated by ATP to give rise to the universal methyl donor, S-adenosylmethionine (AdoMet). Previously, a chimeric MTHFR (Chimera-1) comprised of the yeast Met13p N-terminal catalytic domain and the Arabidopsis thaliana MTHFR (AtMTHFR-1) C-terminal regulatory domain was constructed (Roje, S., Chan, S. Y., Kaplan, F., Raymond, R. K., Horne, D. W., Appling, D. R., and Hanson, A. D. (2002) J. Biol. Chem. 277, 4056–4061). Engineered yeast (SCY4) expressing Chimera-1 accumulated more than 100-fold more AdoMet and 7-fold more methionine than the wild type. Surprisingly, SCY4 showed no appreciable growth defect. The ability of yeast to hyperaccumulate AdoMet was investigated by studying the intracellular compartmentation of AdoMet as well as the mode of hyperaccumulation. Previous studies have established that AdoMet is distributed between the cytosol and the vacuole. A strain expressing Chimera-1 and lacking either vacuoles (vps33 mutant) or vacuolar polyphosphate (vtc1 mutant) was not viable when grown under conditions that favored AdoMet hyperaccumulation. The hyperaccumulation of AdoMet was a robust phenomenon when these cells were grown in medium containing glycine and formate but did not occur when these supplements were replaced by serine. The basis of the nutrient-dependent AdoMet hyperaccumulation effect is discussed in relation to homocysteine biosynthesis and sulfur metabolism.

Cloning and Characterization of Methenyltetrahydrofolate Synthetase from Saccharomyces cerevisiae

The folate derivative 5-formyltetrahydrofolate (folinic acid; 5-CHO-THF) was discovered over 40 years ago, but its role in metabolism remains poorly understood. Only one enzyme is known that utilizes 5-CHO-THF as a substrate: 5,10-methenyltetrahydrofolate synthetase (MTHFS). A BLAST search of the yeast genome using the human MTHFS sequence revealed a 211-amino acid open reading frame (YER183c) with significant homology. The yeast enzyme was expressed inEscherichia coli, and the purified recombinant enzyme exhibited kinetics similar to previously purified MTHFS. No new phenotype was observed in strains disrupted at MTHFS or in strains additionally disrupted at the genes encoding one or both serine hydroxymethyltransferases (SHMT) or at the genes encoding one or both methylenetetrahydrofolate reductases. However, when the MTHFS gene was disrupted in a strain lacking the de novo folate biosynthesis pathway, folinic acid (5-CHO-THF) could no longer support the folate requirement. We have thus named the yeast gene encoding methenyltetrahydrofolate synthetase FAU1(folinic acid utilization). Disruption of the FAU1 gene in a strain lacking both 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase isozymes (ADE16 and ADE17) resulted in a growth deficiency that was alleviated by methionine. Genetic analysis suggested that intracellular accumulation of the purine intermediate AICAR interferes with a step in methionine biosynthesis. Intracellular levels of 5-CHO-THF were determined in yeast disrupted atFAU1 and other genes encoding folate-dependent enzymes. In fau1 disruptants, 5-CHO-THF was elevated 4-fold over wild-type yeast. In yeast lacking MTHFS along with both AICAR transformylases, 5-CHO-THF was elevated 12-fold over wild type. 5-CHO-THF was undetectable in strains lacking SHMT activity, confirming SHMT as the in vivo source of 5-CHO-THF. Taken together, these results indicate that S. cerevisiae harbors a single, nonessential, MTHFS activity. Growth phenotypes of multiply disrupted strains are consistent with a regulatory role for 5-CHO-THF in one-carbon metabolism and additionally suggest a metabolic interaction between the purine and methionine pathways.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme.

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Characterization of two 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase isozymes from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ADE16and ADE17 genes encode 5-aminoimidazole-4-carboxamide ribonucleotide transformylase isozymes that catalyze the penultimate step of the de novo purine biosynthesis pathway. Disruption of these two chromosomal genes results in adenine auxotrophy, whereas expression of either gene alone is sufficient to support growth without adenine. In this work, we show that anade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy ofade3 mutant yeast strains. We also report the purification and characterization of the ADE16 and ADE17gene products (Ade16p and Ade17p). Like their counterparts in other organisms, the yeast isozymes are bifunctional, containing both 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and inosine monophosphate cyclohydrolase activities, and exist as homodimers based on cross-linking studies. Both isozymes are localized to the cytosol, as shown by subcellular fractionation experiments and immunofluorescent staining. Epitope-tagged constructs were used to study expression of the two isozymes. The expression of Ade17p is repressed by the addition of adenine to the media, whereas Ade16p expression is not affected by adenine. Ade16p was observed to be more abundant in cells grown on nonfermentable carbon sources than in glucose-grown cells, suggesting a role for this isozyme in respiration or sporulation.

Human mitochondrial C1-tetrahydrofolate synthase: submitochondrial localization of the full-length enzyme and characterization of a short isoform.

Mammalian mitochondrial C(1)-tetrahydrofolate (THF) synthase (MTHFDIL gene product) is a monofunctional 10-formyl-THF synthetase, lacking the 5,10-methylene-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities typically found in the trifunctional cytoplasmic proteins. Here, we report the submitochondrial localization of epitope-tagged human mitochondrial C(1)-THF synthase expressed in Chinese hamster ovary cells. Mitochondrial fractionation experiments show that human mitochondrial C(1)-THF synthase behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. Inner mitochondrial membrane association was also observed for the endogenous mitochondrial C(1)-THF synthase in adult rat spleen. We also purified and characterized the recombinant protein product (short isoform) of the alternatively spliced short transcript of the mitochondrial isozyme. Methylene-THF dehydrogenase assays confirmed that the short isoform is not enzymatically active. The purified short isoform was used in the production of polyclonal antibodies specific for the mitochondrial isozyme. These antibodies detected endogenous full-length mitochondrial C(1)-THF synthase in mitochondria from adult rat spleen and human placenta, confirming the expression of the mitochondrial isozyme in adult mammalian tissues.

Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues

Background: Mitochondria produce one-carbon units for cytoplasmic nucleotide and methyl group synthesis. Results: MTHFD2L uses both NAD+ and NADP+ and is expressed in embryonic tissues during neural tube closure. Conclusion: This cofactor specificity allows for rapid response to changing metabolic conditions. Significance: These findings help explain why mammals possess two distinct mitochondrial isozymes that switch expression during neural tube closure. Mammalian mitochondria are able to produce formate from one-carbon donors such as serine, glycine, and sarcosine. This pathway relies on the mitochondrial pool of tetrahydrofolate (THF) and several folate-interconverting enzymes in the mitochondrial matrix. We recently identified MTHFD2L as the enzyme that catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria. We show here that the MTHFD2L enzyme is bifunctional, possessing both CH2-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities. The dehydrogenase activity can use either NAD+ or NADP+ but requires both phosphate and Mg2+ when using NAD+. The NADP+-dependent dehydrogenase activity is inhibited by inorganic phosphate. MTHFD2L uses the mono- and polyglutamylated forms of CH2-THF with similar catalytic efficiencies. Expression of the MTHFD2L transcript is low in early mouse embryos but begins to increase at embryonic day 10.5 and remains elevated through birth. In adults, MTHFD2L is expressed in all tissues examined, with the highest levels observed in brain and lung.

Purification and characterization of yeast mitochondrial initiation factor 2.

Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E. coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions. In addition, the isolated ymIF2 was compared to E. coli IF2 in four other assays commonly used to characterize this initiation factor. Formylated and nonformylated Met-tRNA(f)(Met) were bound to E. coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA. The GTPase activity of ymIF2 was found to be dependent on the presence of E. coli ribosomes. The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E. coli IF2 against nonenzymatic deaminoacylation. In contrast to E. coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay. In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.

Purification, immunoassay, and tissue distribution of rat C1-tetrahydrofolate synthase☆

C1-tetrahydrofolate synthase (C1-THF synthase), a eukaryotic trifunctional enzyme, catalyzes three sequential folate-mediated one-carbon interconversions. These three reactions supply the activated one-carbon units required in the metabolism of purines, thymidylate, and several amino acids. In order to study the regulation of C1-THF synthase expression in mammals, we have purified the enzyme to homogeneity from rat liver, raised polyclonal antisera to it in rabbits, and developed a sensitive solid-phase immunoassay for the enzyme. The enzyme was purified approximately 600-fold to a specific activity of 24.6 U/mg protein based on 10-formyl-THF synthetase activity. Western blot analysis indicated that the antisera is specific for one protein in crude liver extracts which comigrates with purified C1-THF synthase. Using the solid-phase immunoassay, as little as 200 pg of immunoreacting protein can be detected in tissue homogenates. Several rat tissues were examined for the three C1-THF synthase enzymatic activities and immunoreactive protein. The results indicated that the level of C1-THF synthase is regulated in a tissue-specific manner. Enzyme assays revealed that certain tissues differ by more than 100-fold in enzyme activity, with liver and kidney containing the highest levels, and lung and muscle the lowest. However, immunoassay of these same tissues indicated only a 10-fold difference in C1-THF synthase concentration. This apparent masking of enzyme activity was observed in all tissues, but to varying degrees. These results emphasize the advantages of an immunoassay in studying the regulation of C1-THF synthase.

Mitochondrial one-carbon metabolism and neural tube defects

BACKGROUND Neural tube defects (NTDs) are one of the most common birth defects in humans. Maternal intake of folic acid was linked to prevention of NTDs in the 1970s. This realization led to the establishment of mandatory and/or voluntary food folic acid fortification programs in many countries that have reduced the incidence of NTDs by up to 70% in humans. Despite 40 years of intensive research, the biochemical mechanisms underlying the protective effects of folic acid remain unknown. RESULTS Recent research reveals a role for mitochondrial folate-dependent one-carbon metabolism in neural tube closure. CONCLUSION In this article, we review the evidence linking NTDs to aberrant mitochondrial one-carbon metabolism in humans and mouse models. The potential of formate, a product of mitochondrial one-carbon metabolism, to prevent NTDs is also discussed.