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.     

Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme

C1-tetrahydrofolate (THF) synthase is a trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The current model divides this protein into two functionally independent domains with dehydrogenase/cyclohydrolase activities sharing an overlapping active site on the N-terminal domain and synthetase activity associated with the C-terminal domain. Previous chemical modification studies on C1-THF synthase from the yeast Saccharomyces cerevisiae indicated at least two cysteinyl residues involved in the dehydrogenase/cyclohydrolase reactions [Appling, D. R., & Rabinowitz, J. C. (1985) Biochemistry 24, 3540-3547]. In the present work, site-directed mutagenesis of the S. cerevisiae ADE3 gene, which encodes C1-THF synthase, was used to individually change each cysteine contained within the dehydrogenase/cyclohydrolase domain (Cys-11, Cys-144, and Cys-257) to serine. The resulting proteins were overexpressed in yeast and purified for kinetic analysis. Site-specific mutations in the dehydrogenase/cyclohydrolase domain did not affect synthetase activity, consistent with the proposed domain structure. The C144S and C257S mutations result in 7- and 2-fold increases, respectively, in the dehydrogenase Km for NADP+. C144S lowers the dehydrogenase maximal velocity roughly 50% while C257S has a maximal velocity similar to that of the wild type. Cyclohydrolase catalytic activity is reduced 20-fold by the C144S mutation but increased 2-fold by the C257S mutation. Conversion of Cys-11 to serine has a negligible effect on dehydrogenase/cyclohydrolase activity. A double mutant, C144S/C257S, results in catalytic properties roughly multiplicative of the individual mutations.(ABSTRACT TRUNCATED AT 250 WORDS)     

A general method for generation and analysis of defined mutations in enzymes involved in a tetrahydrofolate-interconversion pathway

In eukaryotes, 10-formyltetrahydrofolate (THF) synthetase, 5,10-methenyl-THF cyclohydrolase and 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. These reactions are generally catalyzed by three separate monofunctional enzymes in prokaryotic cells. In this report a general method for the generation, detection and analysis of specific mutations affecting the catalytic activity of any of the reactions catalyzed by C1-THF synthase or its monofunctional counterparts is described. The method relies on plasmid-borne expression of genes in strains of the yeast Saccharomyces cerevisiae that are missing one or more of the activities of C1-THF synthase. Specific segments of the gene are subjected in vitro to random mutagenesis, the mutant genes expressed in yeast and screened by phenotype for inactivating mutations. Plasmids encoding mutant enzymes are recovered for sequence analysis. One-step purification of C1-THF synthase from the yeast expression system is demonstrated. The feasibility and versatility of the method is shown with the yeast ADE3 gene encoding the cytoplasmic C1-THF synthase and the gene encoding the monofunctional 10-formyl-THF synthetase from Clostridium acidiurici.     

Mammalian MTHFD2L encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues

Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH(2)-THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH(2)-THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP(+)-dependent CH(2)-THF dehydrogenase activity when expressed in yeast. The enzyme is localized to mitochondria when expressed in CHO cells and behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. The MTHFD2L gene is subject to alternative splicing and is expressed in adult tissues in humans and rodents. This CH(2)-THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH(2)-THF to formate in adult mammalian mitochondria.     

13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has both cytoplasmic and mitochondrial C1-tetrahydrofolate (THF) synthases. These trifunctional isozymes are central to single-carbon metabolism and are responsible for interconversion of the THF derivatives in the respective compartments. In the present work, we have used 13C NMR to study folate-mediated single-carbon metabolism in these two compartments, using glycine and serine synthesis as metabolic endpoints. The availability of yeast strains carrying deletions of cytoplasmic and/or mitochondrial C1-THF synthase allows a dissection of the role each compartment plays in this metabolism. When yeast are incubated with [13C]formate, 13C NMR spectra establish that production of [3-13C]serine is dependent on C1-THF synthase and occurs primarily in the cytosol. However, in a strain lacking cytoplasmic C1-THF synthase but possessing the mitochondrial isozyme, [13C]formate can be metabolized to [2-13C]glycine and [3-13C]serine. This provides in vivo evidence for the mitochondrial assimilation of formate, activation and conversion to [13C]CH2-THF via mitochondrial C1-THF synthase, and subsequent glycine synthesis via reversal of the glycine cleavage system. Additional supporting evidence of reversibility of GCV in vivo is the production of [2-13C]glycine and [2,3-13C]serine in yeast strains grown with [3-13C]serine. This metabolism is independent of C1-THF synthase since these products were observed in strains lacking both the cytoplasmic and mitochondrial isozymes. These results suggest that when formate is the one-carbon donor, assimilation is primarily cytoplasmic, whereas when serine serves as one-carbon donor, considerable metabolism occurs via mitochondrial pathways.     

Regulation of expression of the ADE3 gene for yeast C1-tetrahydrofolate synthase, a trifunctional enzyme involved in one-carbon metabolism

C1-THF (5,6,7,8-tetrahydrofolate) synthase is a trifunctional protein catalyzing the sequential reactions specified by the enzymes 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5). These three activities supply the activated one-carbon units required for the biosynthesis of purines, thymidylate, the amino acids histidine and methionine, the vitamin pantothenic acid, and the formyl group of mitochondrial fMet-tRNAfMet. Extracts of Saccharomyces cerevisiae whose growth is dependent on the three activities of C1-THF synthase contain 2-3 times the level of enzyme activity of extracts from cells grown under conditions where they are independent of this enzyme. Repression of C1-THF synthase activity requires the simultaneous presence of adenine, histidine, methionine, and pantothenic acid. Starvation of the cells for any one of these nutrients leads to derepression of the enzyme. Drug-induced folate starvation also leads to derepression of enzyme activity. The response to changing nutritional conditions occurs within 1 h and is due to changes in the steady-state concentration of C1-THF synthase enzyme, rather than to activation or deactivation of a pre-existing pool of enzyme. Determination of the amount of C1-THF synthase mRNA under the various growth conditions by an in vitro translation/immunoprecipitation assay indicates that regulation of the enzyme occurs predominantly at a pretranslational level since steady-state levels of C1-THF synthase mRNA are 2-3-fold higher in derepressed cells than in repressed cells.     

Isolation, characterization, and structural organization of 10-formyltetrahydrofolate synthetase from spinach leaves

One-carbon metabolism mediated by folate coenzymes plays an essential role in several major cellular processes. In the prokaryotes studied, three folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) generally exist as monofunctional or bifunctional proteins, whereas in eukaryotes the three activities are present on one polypeptide. The structural organization of these enzymes in plants had not previously been examined. We have purified the 10-formyltetrahydrofolate synthetase activity from spinach leaves to homogeneity and raised antibodies to it. The protein was a dimer with a subunit molecular weight of Mr = 67,000. The Km values for the three substrates, (6R)-tetrahydrofolate, ATP, and formate were 0.94, 0.043, and 21.9 mM, respectively. The enzyme required both monovalent and divalent cations for maximum activity. The 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities of spinach coeluted separately from the 10-formyltetrahydrofolate synthetase activity on a Matrex Green-A column. On the same column, the activities of the yeast trifunctional C1-tetrahydrofolate synthase coeluted. In addition, antibodies raised to the purified spinach protein immunoinactivated and immunoprecipitated only the 10-formyltetrahydrofolate synthetase activity in a crude extract of spinach leaves. These results suggest that unlike the trifunctional form of C1-tetrahydrofolate synthase in the other eukaryotes examined, 10-formyltetrahydrofolate synthetase in spinach leaves is monofunctional and 5,10-methyl-enetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase appear to be bifunctional. Although structurally dissimilar to the other eukaryotic trifunctional enzymes, the 35 amino-terminal residues of spinach 10-formyltetrahydrofolate synthetase showed 35% identity with six other tetrahydrofolate synthetases.     

13C nuclear magnetic resonance detection of interactions of serine hydroxymethyltransferase with C1-tetrahydrofolate synthase and glycine decarboxylase complex activities in Arabidopsis.

In C3 plants, serine synthesis is associated with photorespiratory glycine metabolism involving the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (GDC) and serine hydroxymethyl transferase (SHMT). Alternatively, THF-dependent serine synthesis can occur via the C1-THF synthase/SHMT pathway. We used 13C nuclear magnetic resonance to examine serine biosynthesis by these two pathways in Arabidopsis thaliana (L.) Heynh. Columbia wild type. We confirmed the tight coupling of the GDC/ SHMT system and observed directly in a higher plant the flux of formate through the C1-THF synthase/SHMT system. The accumulation of 13C-enriched serine over 24 h from the GDC/SHMT activities was 4-fold greater than that from C1-THF synthase/SHMT activities. Our experiments strongly suggest that the two pathways operate independently in Arabidopsis. Plants exposed to methotrexate and sulfanilamide, powerful inhibitors of THF biosynthesis, reduced serine synthesis by both pathways. The results suggest that continuous supply of THF is essential to maintain high rates of serine metabolism. Nuclear magnetic resonance is a powerful tool for the examination of THF-mediated metabolism in its natural cellular environment.     

Purification and characterization of a mitochondrial isozyme of C1-tetrahydrofolate synthase from Saccharomyces cerevisiae

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is encoded by the ADE3 locus, yet ade3 mutants have low but detectable levels of these enzyme activities. Synthetase, cyclohydrolase, and dehydrogenase activities in an ade3 deletion strain co-purify 4,000-fold to yield a single protein species as seen on sodium dodecyl sulfate-polyacrylamide gels. The native molecular weight of the isozyme (Mr = 200,000 by gel exclusion chromatography) and the size of its subunits (Mr = 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are similar to those of C1-tetrahydrofolate synthase. Cell fractionation experiments show that the isozyme, but not C1-tetrahydrofolate synthase, is localized in the mitochondria. Genetic studies indicate that the isozyme is encoded in the nuclear genome. Peptide mapping experiments show that C1-tetrahydrofolate synthase and the isozyme are not structurally identical. However, immunotitration experiments and amino acid sequence analysis suggest that C1-tetrahydrofolate synthase and the isozyme are structurally related. We propose to call the isozyme "mitochondrial C1-tetrahydrofolate synthase."     

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.     

Methenyltetrahydrofolate synthetase prevents the inhibition of phosphoribosyl 5-aminoimidazole 4-carboxamide ribonucleotide formyltransferase by 5-formyltetrahydrofolate polyglutamates

Methenyltetrahydrofolate synthetase (EC 6.3.3.2) catalyzes the irreversible ATP and Mg2+-dependent transformation of 5-formyltetrahydrofolate (N5-HCO-H4-pteroylglutamic acid (PteGlu] to 5,10-methenyltetrahydrofolate. The physiological function of this reaction remains unknown even though it is potentially involved in the intracellular metabolism of the large doses of N5-HCO-H4-PteGlu (leucovorin) administered to cancer patients. We have tried to elucidate methenyltetrahydrofolate synthetase's physiological role by examining the consequences of its inhibition in MCF-7 human breast cancer cells by the folate analog 5-formyltetrahydrohomofolate (fTHHF), a potent competitive inhibitor with a Ki of 1.4 microM. fTHHF inhibited MCF-7 cell growth with an IC50 of 2.0 microM during 72-h exposures, and this effect was fully reversible by hypoxanthine but not thymidine, indicating specific inhibition of de novo purine synthesis. A correlation was observed between increases in intracellular N5-HCO-H4-PteGlu concentrations following fTHHF and cell growth inhibition. De novo purine synthesis was inhibited at the second folate-dependent enzyme, phosphoribosyl aminoimidazole-carboxamide formyltransferase (AICAR transferase; EC 2.1.2.3), as determined by aminoimidazole carboxamide rescue and azaserine inhibition studies. N5-HCO-H4-PteGlu pentaglutamate was a potent inhibitor of purified MCF-7 cell AICAR transferase with a Ki of 3.0 microM while the monoglutamate was not an inhibitor up to 10 microM and fTHHF was only weakly inhibitory with a Ki of 16 microM. These findings suggest that methenyltetrahydrofolate synthetase activity is needed to prevent de novo purine synthesis inhibition by N5-HCO-H4-PteGlu polyglutamates.     

Studies on the polyglutamate specificity of thymidylate synthase from fetal pig liver

Thymidylate synthase has been purified 1700-fold from fetal pig livers by using chromatography on Affigel-Blue, DEAE-52, and hydroxylapatite. Steady-state kinetic measurements indicate that catalysis proceeds via an ordered sequential mechanism. When 5,10-methylenetetrahydro-pteroylmonoglutamate (CH2-H4PteGlu1) is used as the substrate, dUMP is bound prior to CH2-H4PTeGlu1, and 7,8-dihydropteroylmonoglutamate (H2PteGlu1) is released prior to dTMP. Pteroylpolyglutamates (PteGlun) are inhibitors of thymidylate synthase activity and are competitive with respect to CH2-H4PteGlu1 and uncompetitive with respect to dUMP. Inhibition constants (Ki values), which correspond to dissociation constants for the dissociation of PteGlun from the enzyme-dUMP-PteGlun ternary complex, have been determined for PteGlun derivatives with one to seven glutamyl residues: PteGlu1, 10 microM; PteGlu2, 0.3 microM; PteGlu3, 0.2 microM; PteGlu4, 0.06 microM; PteGlu5, 0.10 microM; PteGlu6, 0.12 microM; PteGlu7, 0.15 microM. Thus, thymidylate synthase from fetal pig liver preferentially binds pteroylpolyglutamates with four glutamyl residues, but derivatives with two to seven glutamyl residues all bind at least 30-fold more tightly than the monoglutamate. When CH2-H4PteGlu4 is used as the one carbon donor for thymidylate biosynthesis, the order of substrate binding and product release is reversed, with binding of CH2-H4PteGlu4 preceding that of dUMP and release of dTMP preceding release of H2PteGlu4. Vmax and Km values for dUMP and CH2-H4PteGlun show relatively little change as the polyglutamate chain length of the substrate is varied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a novel eukaryotic monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase

An NAD(+)-dependent 5,10-methylenetetrahydrofolate (THF) dehydrogenase has been purified to homogeneity from the yeast Saccharomyces cerevisiae. The purified enzyme exhibits a final specific activity of 5.4 units mg-1 and is represented by a single protein of apparent Mr = 33,000-38,000 as determined by sodium dodecyl sulfate gel electrophoresis. A native Mr = 64,000 was determined by gel filtration, suggesting a homodimer subunit structure. Cross-linking experiments with dimethyl suberimidate confirmed the dimeric structure. The enzyme is specific for NAD+ and is not dependent on Mg2+ for activity. The forward reaction initial velocity kinetics are consistent with a sequential reaction mechanism. With this model, Km values for NAD+ and (6R,S)-5,10-methylene-THF are 1.6 and 0.06 mM, respectively. In contrast to all other previously described eukaryotic 5,10-methylene-THF dehydrogenases, the purified enzyme is apparently monofunctional, with undetectable 5,10-methenyl-THF cyclohydrolase and 10-formyl-THF synthetase activities. Subcellular fractionation of yeast indicates the enzyme is cytoplasmic, with no NAD(+)-dependent 5,10-methylene-THF dehydrogenase detectable in mitochondria. The activity was found in all yeast strains examined, at all stages of growth from the lag phase through the stationary phase.     

Nitrous oxide exposure reduces hepatic C1-tetrahydrofolate synthase expression in rats

C1-tetrahydrofolate synthase (C1-THF synthase) is a trifunctional enzyme which catalyzes the interconversion of one-carbon units attached to the coenzyme THF. Nitrous oxide (N2O) inhalation is known to inactivate hepatic cobalamin-dependent methionine synthase leading to methionine deficiency and trapping of THF in the methyl-THF form. Liver tissue from rats exposed to N2O for 48 hours exhibited a coordinate decrease in all three activities of C1-THF synthase of approximately 25%. A corresponding 25% decrease in immunoreactive C1-THF synthase was also observed after 48 hours. Thus, the decrease in the concentration of C1-THF synthase accounted entirely for the decreases observed in the three activities. These results suggest that perturbations of hepatic THF pools by N2O affect the level of C1-THF synthase expression at a translational or pretranslational level.     

Investigating the regulation of one-carbon metabolism in Arabidopsis thaliana

Serine (Ser) biosynthesis in C(3) plants can occur via several pathways. One major route involves the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (GDC, EC 2.1.1.10) and serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) with glycine (Gly) as one-carbon (1-C) source. An alternative THF-dependent pathway involves the C1-THF synthase/SHMT activities with formate as 1-C source. Here, we have investigated aspects of the regulation of these two folate-mediated pathways in Arabidopsis thaliana (L.) Heynh. Columbia using two approaches. Firstly, transgenic plants overexpressing formate dehydrogenase (FDH, EC 1.2.1.2) were used to continue our previous studies on the function of FDH in formate metabolism. The formate pool size was approximately 73 nmol (g FW)(-1) in wild type (WT) Arabidopsis plants; three independent transgenic lines had similar-sized pools of formate. Transgenic plants produced more (13)CO(2) from supplied [(13)C]formate than did WT plants but were not significantly different from WT plants in their synthesis of Ser. We concluded that FDH has no direct role in the regulation of the above two pathways of Ser synthesis; the breakdown of formate to CO(2) by the FDH reaction is the primary and preferred fate of the organic acid in Arabidopsis. The ratio between the GDC/SHMT and C1-THF synthase/SHMT pathways of Ser synthesis from [alpha-(13)C]Gly and [(13)C]formate, respectively, in Arabidopsis shoots was 21 : 1; in roots, 9 : 1. In shoots, therefore, the pathway from formate plays only a small role in Ser synthesis; in the case of roots, results indicated that the 9 : 1 ratio was as a result of greater fluxes of (13)C through both pathways together with a relatively higher contribution from the C1-THF synthase/SHMT route than in shoots. We also examined the synthesis of Ser in a GDC-deficient mutant of Arabidopsis (glyD) where the GDC/SHMT pathway was impaired. Compared with WT, glyD plants accumulated 5-fold more Gly than WT after supplying [alpha-(13)C]Gly for 24 h; the accumulation of Ser from [alpha-(13)C]Gly was reduced by 25% in the same time period. On the other hand, the accumulation of Ser through the C1-THF synthase/SHMT pathway in glyD plants was 2.5-fold greater than that in WT plants. Our experiments confirmed that the GDC/SHMT and C1-THF synthase/SHMT pathways normally operate independently in Arabidopsis plants but that when the primary GDC/SHMT pathway is impaired the alternative C1-THF synthase/SHMT pathway can partially compensate for deficiencies in the synthesis of Ser.     

Mthfd1 Is an Essential Gene in Mice and Alters Biomarkers of Impaired One-carbon Metabolism

Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1^gt/+ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1^gt/+ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors.Folate-mediated one-carbon (1C)³ metabolism is compartmentalized in the cytoplasm, mitochondria, and nucleus of mammalian cells (1). In the cytoplasm, 1C metabolism functions to carry and chemically activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine (2) (see Fig. 1). Methionine can be adenosylated to form S-adenosylmethionine (AdoMet), the major cellular methyl group donor required for the methylation of DNA, RNA, histones, small molecules, and lipids. Nuclear 1C metabolism functions to synthesize thymidylate from dUMP and serine during S phase through the small ubiquitin-like modifier-dependent translocation of cytoplasmic serine hydroxymethyltransferase (cSHMT), dihydrofolate reductase, and thymidylate synthase into the nucleus (3).Open in a separate windowFIGURE 1.Folate-mediated one-carbon metabolism occurs in the mitochondria, nucleus, and cytoplasm. Mitochondrial-derived formate traverses to the cytoplasm where it is incorporated into the folate-activated one-carbon pool through the activity of FTHFS and utilized in the synthesis of purines, thymidylate, and the methylation of homocysteine to methionine. Methionine can be converted to a methyl donor through its adenosylation to AdoMet. Thymidylate biosynthesis occurs in the cytoplasm and nucleus. The one-carbon unit is labeled in bold. GCS, glycine cleavage system; mSHMT, mitochondrial serine hydroxymethyltransferase; mMTHFD, mitochondrial methylenetetrahydrofolate dehydrogenase; mMTHFC, mitochondrial methenyltetrahydrofolate cyclohydrolase; mFTHFS, mitochondrial formyltetrahydrofolate synthetase; MTHFD, methylenetetrahydrofolate dehydrogenase; MTHFC, methenyltetrahydrofolate cyclohydrolase; FTHFS, formyltetrahydrofolate synthetase; MTHFR, methylenetetrahydrofolate reductase; TS, thymidylate synthase; DHFR, dihydrofolate reductase; and cSHMT, cytoplasmic serine hydroxymethyltransferase.Serine, through its conversion to glycine by SHMT, is a primary source of 1Cs for nucleotide and methionine synthesis (4). SHMT generates 1Cs in the cytoplasm, mitochondria, and nucleus, although the generation of 1Cs through SHMT activity in the cytoplasm is not essential in mice, indicating the essentiality of mitochondria-derived 1Cs for cytoplasmic 1C metabolism (5). In mitochondria, the hydroxymethyl group of serine and the C2 carbon of glycine are transferred to tetrahydrofolate (THF) to generate 5,10-methylene-THF by the mitochondrial isozyme of SHMT and the glycine cleavage system, respectively (6). The 1C carried by methylene-THF is oxidized and hydrolyzed to generate formate by the NAD-dependent methylene-THF dehydrogenase (MTHFD) and methenyl-THF cyclohydrolase (MTHFC) activities encoded by a single gene, Mthfd2 (7), and 10-formyl-THF synthetase (FTHFS) activity, encoded by Mthfd1L (8) (see Fig. 1).In the cytoplasm, the product of the Mthfd1 gene, C1THF synthase, is a trifunctional enzyme that contains NADP-dependent MTHFD and MTHFC activities on the N-terminal domain of the protein, and FTHFS activity on the C-terminal domain (9). These three activities collectively catalyze the interconversion of THF, 10-formyl-THF, 5,10-methenyl-THF, and 5,10-methylene-THF (10) (Fig. 1). The ATP-dependent FTHFS activity of C1THF synthase condenses mitochondria-derived formate with THF to form 10-formyl-THF, which is required for the de novo synthesis of purines (9). The MTHFC and MTHFD activities convert 10-formyl-THF to methylene-THF (11). Methylene-THF is utilized in the de novo synthesis of thymidylate or, alternatively, can be irreversibly reduced by methylene-THF reductase to 5-methyl-THF, which is used in the remethylation of homocysteine to methionine (12).Impairments in 1C metabolism, due to insufficient folate cofactors and/or single nucleotide polymorphisms in genes that encode folate-dependent enzymes, are associated with numerous pathologies and developmental anomalies, including cancers, cardiovascular disease, and neural tube defects. The causal mechanisms underlying the folate-pathology relationship(s) remains to be established. However, a number of hypotheses have been proposed related to the role of 1C metabolism in genome stability and gene expression. Decreased thymidylate synthesis results in increased uracil misincorporation into DNA and decreased rates of cell division, causing double strand breaks in DNA and genomic instability (13). Decreased AdoMet synthesis alters methylation patterns in CpG islands in DNA and can result in histone hypomethylation, which can alter gene expression (2). Proliferating cells also require the de novo synthesis of purines to maintain rates of DNA synthesis (14).It has been shown that the gene product of Mthfd2, mitochondrial MTHFC/MTHFD is essential in mice, and Mthfd2 deficiency results in embryonic lethality (15). This protein is required for the generation of formate from serine in the mitochondria of embryonic cells. Here, we have investigated the essentiality of the Mthfd1 gene in mice and the effect of altered Mthfd1 gene expression on biomarkers of cytoplasmic 1C metabolism. Our data demonstrate that Mthfd1 is an essential gene in mice and that Mthfd1-deficient mice are a model for the study of folate-associated pathologies.     

Enzymatic mechanism for the hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate by serine hydroxymethyltransferase.

Serine hydroxymethyltransferase in the presence of glycine catalyzes the hydrolysis of (6R)-5,10-methenyltetrahydropteroylpolyglutamate to (6S)-5-formyltetrahydropteroylpolyglutamate. The enzyme also catalyzes the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from a compound in equilibrium with (6R)-5,10-methenyltetrahydropteroylpolyglutamate believed to be (6R,11R)-5,10-hydroxymethylenetetrahydropteroylpolyglutamate , a putative intermediate in the nonenzymatic hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate [Stover, P., & Schirch, V. (1992) Biochemistry (preceding paper in this issue)]. The enzymatic mechanism for the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from these substrates and the role of glycine in the reaction was addressed. Evidence suggests that (6R,11R)-5,10-hydroxymethylenetetrahydropteroyltetraglutamate++ + is a catalytically competent intermediate in the enzyme-catalyzed hydrolysis of (6R)-5,10-methenyltetrahydropteroyltetraglutamate. The enzyme displays a high Km of 40 microM for (6R)-5,10-methenyltetrahydropteroyltetraglutamate, while the Km for (6R,11R)-5,10-hydroxymethylenetetrahydropteroyltetraglutamate++ + is below 0.5 microM. The kcat values for both reactions are identical and equal to the rate of formation of an enzyme ternary complex absorbing at 502 nm which is formed from glycine and (6S)-5-formyltetrahydropteroylpolyglutamate. The hydrolysis reaction proceeds with exchange of the C11 formyl proton of (6R)-5,10-methenyltetrahydropteroyltetraglutamate, suggesting that the enzyme-catalyzed reaction occurs by the same C11 carbanion inversion mechanism as the nonenzymatic reaction. Isotope exchange experiments using [2-3H]glycine and differential scanning calorimetry data suggest both a catalytic and a conformational role for glycine in the enzymatic reaction. The results are discussed in terms of the similarity in mechanisms of the SHMT-catalyzed retroaldol cleavage of serine and hydrolysis of (6R)-5,10-methenyltetrahydropteroylpolyglutamates.