The effects on 4-aminoantifolates on 5-formyltetrahydrofolate metabolism in L1210 cells. A biochemical basis of the selectivity of leucovorin rescue

This report describes studies designed to evaluate possible inhibitory effects of diaminoantifolates on folate-dependent biosynthetic enzymes in intact L1210 leukemia cells. A novel approach is described which involves an assessment of the metabolism of and biosynthetic flux of the one-carbon moiety from (6S)5-formyltetrahydrofolate in folate-depleted cells. Pretreatment with methotrexate (10 microM), resulting in the formation of methotrexate polyglutamates, or continuous incubation with trimetrexate (1 microM) inhibited growth of folate-depleted L1210 cells in the presence of folic acid or 5-formyltetrahydrolate. In both control and drug-treated cells, double-labeled (6S)-5-[14C]formyl[3H]tetrahydrofolate was rapidly metabolized with the loss of the [14C]formyl group. Under all conditions, the predominant metabolite was 10-formyl[3H]tetrahydrofolate, detectable both intracellularly and extracellularly. In drug-treated cells, there was a remarkably small decrease in the level of 10-formyl[3H]tetrahydrofolate (approximately 30%) and a 10-fold rise in the level of [3H]dihydrofolate to less than 20% of the total folate pool. The incorporation of [14C]formyl group from 5-[14C]formyltetrahydrofolate into thymidylate, serine, and methionine was unaffected by the presence of 1 microM trimetrexate, consistent with the generation of sufficient 5,10-[14C]methylenetetrahydrofolate to drive these reactions. Similarly, the presence of methotrexate polyglutamates had no effect at the level of amino acid synthesis; however, carbon transfer into thymidylate was markedly inhibited. Even though 10-formyltetrahydrofolate was readily formed from 5-formyltetrahydrofolate in this model, the net incorporation of 14C from 5-[14C]formyltetrahydrofolate into purine nucleotides was inhibited by both methotrexate and trimetrexate treatments. Similar findings were obtained when [14C]glycine incorporation into purine nucleotides was monitored in cells incubated with unlabeled 5-formyltetrahydrofolate. Finally, in antifolate-treated cells incubated with unlabeled 5-formyl-tetrahydrofolate, transfer of 14C from [14C]formate or [14C]serine into biosynthetic products or incorporation of [3H]deoxyuridine into nucleic acids was potently inhibited. These results suggest that insufficient levels of tetrahydrofolate and 5, 10-methylenetetrahydrofolate were formed to drive these reactions despite the presence of high levels of 10-formyltetrahydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)     

A sensitive radioenzymatic assay for (S)-5-formyltetrahydrofolate.

A highly sensitive, radioenzymatic method has been developed for the specific and quantitative estimation of (S)-5-formyltetrahydrofolate. This method is based on enzymatic cycling of the 5-formyl derivative to methylenetetrahydrofolate followed by entrapment into a stable ternary complex with thymidylate synthase and tritiated fluorodeoxyuridylate. Determination of bound radiolabeled ligand permits estimation of the original folate. The initial cycling step is catalyzed by the enzyme, methenyltetrahydrofolate synthetase, which is specific for the (S)-diastereomer of 5-formyltetrahydrofolate and generates a product which can be further cycled to tetrahydrofolate using either 10-formyltetrahydrofolate deacylase or glycinamide ribonucleotide transformylase. Tetrahydrofolate is ultimately converted to the entrapable methylene derivative in the presence of excess formaldehyde. Using this assay recovery of reference (S)-5-formyltetrahydrofolate was linear over the range 0.03-1.9 pmol with an average recovery of 83 +/- 2%. The method has been applied to estimation of plasma (S)-5-formyltetrahydrofolate from a volunteer who had been administered (R,S)-5-formyltetrahydrofolate. Where comparison was possible, estimation of plasma (S)-5-formyltetrahydrofolate by this one step ternary complex-based method yielded results that were very similar to those observed by Straw et al. (Cancer Res., 44, 3114, 1984) who used an HPLC-based method for separation of diastereomeric mixtures of reduced folates and microbiological growth dependence to determine (S)-5-formyltetrahydrofolate.     

NAD- and NADP-dependent mitochondrially targeted methylenetetrahydrofolate dehydrogenase-cyclohydrolases can rescue mthfd2 null fibroblasts

Mouse fibroblasts in which the mthfd2 gene encoding mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) was previously inactivated were infected with retroviral expression constructs of dehydrogenase/cyclohydrolase cDNA. Cellular fractionation confirmed that the expressed proteins were properly targeted to the mitochondria. Expression of the NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase enzyme in mitochondria corrected the glycine auxotrophy of the null mutant cells. A construct in which the cyclohydrolase activity of NMDMC was inactivated by point mutation also rescued the glycine auxotrophy, although poorly. This suggests that the cyclohydrolase activity is also required to ensure optimal production of 10-formyltetrahydrofolate. The expression of the NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase in the mitochondria also reversed the glycine requirement of the null cells demonstrating that the use of the NAD cofactor is not absolutely essential to maintain the flux of one-carbon metabolites. All rescued cells demonstrated a decrease in the ratio of incorporation of exogenous formate to serine in standardized radiolabeling studies. This ratio, which is approximately 2.5 for nmdmc(-/-) cells and 0.3 for the wild type cells under the conditions used, is a qualitative indicator of the ability of the mitochondria of the cells to generate formate.     

Hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at pH 2.5 to 4.5

At pH 4.0 to 4.5, 5,10-methenyltetrahydrofolate is hydrolyzed to only 5-formyltetrahydrofolate if reducing agents are present or iron-redox cycling is suppressed. At pH 4.0, the equilibrium position for this hydrolysis is approximately equal concentrations of both folates. If no reducing agents are used or iron-redox cycling is promoted, considerable amounts of 10-formyldihydrofolate are also formed. It is likely that 10-formyldihydrofolate has been misidentified as 5,10-hydroxymethylenetetrahydrofolate, which was reported to accumulate during the hydrolysis of 5, 10-methenyltetrahydrofolate to 5-formyltetrahydrofolate [Stover, P. and Schirch, V. (1992) Biochemistry 31, 2148-2155 and 2155-2164; (1990) J. Biol. Chem. 265, 14227-14233]. Since 5, 10-hydroxymethylenetetrahydrofolate is reported to be the viable in vivo substrate for serine hydroxymethyltransferase-catalyzed formation of 5-formyltetrahydrofolate, and 5, 10-hydroxymethylenetetrahydrofolate probably does not accumulate, the above folate metabolism is now doubtful. It is hypothesized that mildly acidic subcellular organelles provide an environment for the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate in vivo, and there is no requirement for enzyme catalysis. Finally, 10-formyltetrahydrofolate is susceptible to iron-catalyzed oxidation to 10-formyldihydrofolate at pH 4 to 4.5.     

C1-Tetrahydrofolate synthase from rabbit liver. Structural and kinetic properties of the enzyme and its two domains

C1-Tetrahydrofolate synthase is a multifunctional enzyme which catalyzes three reactions in 1-carbon metabolism: 10-formyltetrahydrofolate synthetase; 5,10-methenyltetrahydrofolate cyclohydrolase; 5,10-methylenetetrahydrofolate dehydrogenase. A rapid 1-day purification procedure has been developed which gives 40 mg of pure enzyme from 10 rabbit livers. The 10-formyltetrahydrofolate synthetase activity of this trifunctional enzyme has a specific activity that is 4-fold higher than the enzyme previously purified from rabbit liver. Conditions have been developed for the rapid isolation of a tryptic fragment of the enzyme which contains the methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities. This fragment is a monomer exhibiting a subunit and native molecular weight of 36,000 in most buffers. However, in phosphate buffers the native molecular weight suggests that the fragment is a dimer. Conditions are also given whereby chymotryptic digestion allows the simultaneous isolation from the native enzyme of a large fragment containing the 10-formyltetrahydrofolate synthetase activity and a smaller fragment containing the dehydrogenase and cyclohydrolase activities. The large fragment is a dimer with a subunit molecular weight of 66,000. The small fragment retains all of the dehydrogenase and cyclohydrolase activities of the native enzyme. The large fragment is unstable but retains most of the 10-formyltetrahydrofolate synthetase activity. Km values of substrates for the two fragments are the same as the values for the native enzyme. The 10-formyltetrahydrofolate synthetase activity of the native enzyme requires ammonium or potassium ions for expression of full catalytic activity. The effect of these two ions on the catalytic activity of the large chymotryptic fragment is the same as with the native enzyme. We have shown by differential scanning calorimetry that the native enzyme contains two protein domains which show thermal transitions at 47 and 60 degrees C. Evidence is presented that the two domains are related to the two protein fragments generated by proteolysis of the native enzyme. The larger of the two domains contains the active site for the 10-formyltetrahydrofolate synthetase activity while the smaller domain contains the active site which catalyzes the dehydrogenase and cyclohydrolase reactions. Replacement of sodium ion buffers with either ammonium or potassium ions results in an increase in stability of the large domain of the native enzyme. This change in stability is not accompanied by a change in the quaternary structure of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resolution of rat liver 10-formyltetrahydrofolate dehydrogenase/hydrolase activities

10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) catalyzes the NADP-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Previous studies of 10-formyltetrahydrofolate dehydrogenase purified from rat or pig liver homogenized in phosphate buffers indicated the presence of copurifying 10-formyltetrahydrofolate hydrolase activity, which catalyzes conversion of 10-formyltetrahydrofolate to tetrahydrofolate and formate. We find that the supernatant from rat liver homogenized in mannitol/sucrose/EDTA medium contains essentially all of the total cellular 10-formyltetrahydrofolate dehydrogenase activity, but no measurable hydrolase activity. Treating mannitol/sucrose/EDTA-washed mitochondria with Triton X-100 (0.5%) releases hydrolase activity in soluble form. 10-Formyltetrahydrofolate dehydrogenase purified from the mannitol/sucrose/EDTA supernatant has no 10-formyltetrahydrofolate hydrolase activity. Results of kinetic experiments using the hydrolase-free dehydrogenase give a complex rate equation with respect to (6R,S)-10-formyltetrahydrofolate. Double-reciprocal plots fit a 2/1 hyperbolic function with apparent Km values of 3.9 and 68 microM. Our results indicate that 10-formyltetrahydrofolate hydrolase and dehydrogenase are not alternate catalytic activities of a single protein, but represent two closely related and separately compartmentalized hepatic enzymes.     

Identification of 10-formyltetrahydrofolate dehydrogenase-hydrolase as a major folate binding protein in liver cytosol

10-Formyltetrahydrofolate dehydrogenase (10-formyltetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.6) purified from pig liver contained bound tetrahydropteroylhexa-gamma-glutamate, a potent product inhibitor. Dehydrogenase purified from rat liver had chromatographic properties indistinguishable from those of a previously described major cytosolic folate binding protein of unknown function (Zamierowski, M.M. and Wagner, C. (1977) J. Biol. Chem. 252, 933-938; Cook, R.J. and Wagner, C. (1982) Biochemistry 21, 4427-4434). The dehydrogenase catalyzes the oxidative deformylation of 10-formyltetrahydrofolate to carbon dioxide and tetrahydrofolate. The tight binding of product to the enzyme suggests that oxidation of one-carbon moieties is regulated by the ratio of formyltetrahydrofolate to tetrahydrofolate in liver.     

Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase.

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7--0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.     

Bioactivity of orally administered unnatural isomers, [6R]-5-formyltetrahydrofolate and [6S]-5,10-methenyltetrahydrofolate, in humans

It has been assumed that humans cannot utilize 5,6,7,8-tetrahydrofolates with the unnatural configuration at carbon 6, since these folates are enzymatically and microbiologically inactive. We hypothesized that orally administered unnatural [6R]-5-formyltetrahydrofolate or [6S]-5,10-methenyltetrahydrofolate is bioactive in humans. Subjects were given independent oral doses of these unnatural folates and of a natural [6S]-5-formyltetrahydrofolate. Plasma, before and after the dose for 4 h, and 2 h urine were collected. Areas under the curve for the change in plasma folate concentrations were measured microbiologically and urinary folates were measured using HPLC. Based on findings of plasma and urinary folates, the unnatural folates were estimated to be 14-50% active as compared to [6S]-5-formyltetrahydrofolate. The major plasma and urinary folate was [6S]-5-methyltetrahydrofolate in all experiments. In urine, a [6S]-5-formyltetrahydrofolate peak was observed only after a [6S]-5-HCO-H4folate dose and peaks of unnatural [6S]-10-formyltetrahydrofolate and 5-formyltetrahydrofolate were identified after a [6R]-5-formyltetrahydrofolate dose. A possible pathway that explains our findings is discussed. This pathway includes the oxidation of the unnatural [6S]-10-formyltetrahydrofolate to 10-formyl-7,8-dihydrofolate which can be further metabolized by 5-amino-4-imidazolecarboxamide-ribotide transformylase producing dihydrofolate. Dihydrofolate can then be metabolized to [6S]-5-methyltetrahydrofolate by well-established metabolism.     

Structure of a murine cytoplasmic serine hydroxymethyltransferase quinonoid ternary complex: evidence for asymmetric obligate dimers

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.     

Knocking down 10-formyltetrahydrofolate dehydrogenase increased oxidative stress and impeded zebrafish embryogenesis by obstructing morphogenetic movement

Background

Folate is an essential nutrient for cell survival and embryogenesis. 10-Formyltetrahydrofolate dehydrogenase (FDH) is the most abundant folate enzyme in folate-mediated one-carbon metabolism. 10-Formyltetrahydrofolate dehydrogenase converts 10-formyltetrahydrofolate to tetrahydrofolate and CO₂, the only pathway responsible for formate oxidation in methanol intoxication. 10-Formyltetrahydrofolate dehydrogenase has been considered a potential chemotherapeutic target because it was down-regulated in cancer cells. However, the normal physiological significance of 10-Formyltetrahydrofolate dehydrogenase is not completely understood, hampering the development of therapeutic drug/regimen targeting 10-Formyltetrahydrofolate dehydrogenase.

Methods

10-Formyltetrahydrofolate dehydrogenase expression in zebrafish embryos was knocked-down using morpholino oligonucleotides. The morphological and biochemical characteristics of fdh morphants were examined using specific dye staining and whole-mount in-situ hybridization. Embryonic folate contents were determined by HPLC.

Results

The expression of 10-formyltetrahydrofolate dehydrogenase was consistent in whole embryos during early embryogenesis and became tissue-specific in later stages. Knocking-down fdh impeded morphogenetic movement and caused incorrect cardiac positioning, defective hematopoiesis, notochordmalformation and ultimate death of morphants. Obstructed F-actin polymerization and delayed epiboly were observed in fdh morphants. These abnormalities were reversed either by adding tetrahydrofolate or antioxidant or by co-injecting the mRNA encoding 10-formyltetrahydrofolate dehydrogenase N-terminal domain, supporting the anti-oxidative activity of 10-formyltetrahydrofolate dehydrogenase and the in vivo function of tetrahydrofolate conservation for 10-formyltetrahydrofolate dehydrogenase N-terminal domain.

Conclusions

10-Formyltetrahydrofolate dehydrogenase functioned in conserving the unstable tetrahydrofolate and contributing to the intracellular anti-oxidative capacity of embryos, which was crucial in promoting proper cell migration during embryogenesis.

General significance

These newly reported tetrahydrofolate conserving and anti-oxidative activities of 10-formyltetrahydrofolate dehydrogenase shall be important for unraveling 10-formyltetrahydrofolate dehydrogenase biological significance and the drug development targeting 10-formyltetrahydrofolate dehydrogenase.     

The regulation of folate and methionine metabolism.

1. The isolated perfused rat liver and suspensions of isolated rat hepatocytes fail to form glucose from histidine, in contrast with the liver in vivo. Both rat liver preparations readily metabolize histidine. The main end product is N-formiminoglutamate. In this respect the liver preparations behave like the liver of cobalamin- or folate-deficient mammals. 2. Additions of L-methionine in physiological concentrations (or of ethionine [2-amino-4-(ethylthio)butyric acid]) promotes the degradation of formiminoglutamate, as is already known to be the case in cobalamin of folate deficiency. Added methionine also promotes glucose formation from histidine. 3. Addition of methionine accelerates the oxidation of formate to bicarbonate by hepatocytes. 4. A feature common to cobalamin-deficient liver and the isolated liver preparations is taken to be a low tissue methionine concentration, to be expected in cobalamin deficiency through a decreased synthesis of methionine and caused in liver preparations by a washing out of amino acids during the handling of the tissue. 5. The available evidence is in accordance with the assumption that methionine does not directly increase the catalytic capacity of formyltetrahydrofolate dehydrogenase; rather, that an increased methionine concentration raises the concentration of S-adenosylmethionine, thus leading to the inhibition of methylenetetrahydrofolate reductase activity [Kutzbach & Stokstad (1967) Biochim. Biophys. Acta 139, 217-220; Kutzbach & Stokstad (1971) Methods Enzymol. 18B, 793-798], that this inhibition causes an increase in the concentration of methylenetetrahydrofolate and the C1 tetrahydrofolate derivatives in equilibrium with methylenetetrahydrofolate, including 10-formyltetrahydrofolate; that the increased concentration of the latter accelerates the formyltetrahydrofolate dehydrogenase reaction, because the normal concentration of the substrate is far below the Km value of the enzyme for the substrate. 6. The findings are relevant to the understanding of the regulation of both folate and methionine metabolism. When the methionine concentration is low, C1 units are preserved by the decreased activity of formyltetrahydrofolate dehydrogenase and are utilized for the synthesis of methionine, purines and pyrimidines. On the other hand when the concentration of methionine, and hence adenosylmethionine, is high and there is a surplus of C1 units as a result of excess of dietary supply, formyltetrahydrofolate dehydrogenase disposes of the excess. When ample dietary supply causes an excess of methionine, which has to be disposed of by degradation, the increased activity of formyltetrahydrofolate dehydrogenase decreases the supply of methyltetrahydrofolate. Thus homocysteine, instead of being remethylated, enters the pathway of degradation via cystathionine. 7. The findings throw light on the biochemical abnormalities associated with cobalamin deficiency (megaloblastic anaemia), especially on the 'methylfolate-trap hypothesis'. This is discussed. 8...     

N10-Formyltetrahydrofolate is the formyl donor for glycinamide ribotide transformylase in Escherichia coli.

Glycinamide ribotide transformylase from Escherichia coli was obtained free of N5,N10-methenyltetrahydrofolate cyclohydrolase activity by DEAE-cellulose chromatography. In reaction mixtures containing this enzyme preparation in potassium maleate buffer, pH 7.2, no detectable interconversion of N5,N10-methenyltetrahydrofolate occurred. Upon addition of glycinamide ribotide, N-formylglycinamide ribotide was formed when N10-formyltetrahydrofolate was present; no formylation occurred in the presence of N5,N10-methenyltetrahydrofolate. A method for the synthesis and purification of glycinamide ribotide is presented.     

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.     

Isolation and sequencing of the cDNA coding for spinach 10-formyltetrahydrofolate synthetase. Comparisons with the yeast, mammalian, and bacterial proteins.

The one-carbon metabolism 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) can be found on a single trifunctional protein in the eukaryotes examined. The one exception is in spinach leaves where 10-formyltetrahydrofolate synthetase is monofunctional (Nour, J. M., and Rabinowitz, J. C. (1991) J. Biol. Chem. 266, 18363-18369). In the prokaryotes examined, 10-formyltetrahydrofolate synthetase is either absent or is monofunctional. A cDNA clone encoding spinach leaf 10-formyltetrahydrofolate synthetase was isolated through the use of antibodies to the purified enzyme. This clone had an open reading frame of 1914 base pairs and encoded for a protein containing 636 amino acids with a calculated M(r) of 67,727. The percentage identity between spinach 10-formyltetrahydrofolate synthetase and the synthetase domains in the four trifunctional eukaryotic enzymes and the two monofunctional prokaryotic enzymes that have been cloned and sequenced was: 64.9% human, 63.8% rat, 55.6% yeast cytoplasm, 53.8% yeast mitochondria, 47.8% Clostridium acidi-urici, and 47.9% Clostridium thermoaceticum. Clearly the spinach monofunctional protein had greatest homology with the mammalian proteins. The spinach protein is longer than the two other monofunctional prokaryotic proteins. Possible reasons for this are presented. The codon usage and the putative translation initiation sites are examined and compared with other spinach proteins.     

Isolation and characterization of cDNA clones for rat liver 10-formyltetrahydrofolate dehydrogenase.

We have isolated and characterized cDNA clones encoding rat liver cytosol 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6). An open reading frame of 2706 base pairs encodes for 902 amino acids of Mr 99,015. The deduced amino acid sequence contains exact matches to the NH2-terminal sequence (28 residues) and the sequences of five peptides derived from cyanogen bromide cleavage of the purified protein. The amino acid sequence of 10-formyltetrahydrofolate dehydrogenase has three putative domains. The NH2-terminal sequence (residues 1-203) is 24-30% identical to phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) from Bacillus subtilis (30%), Escherichia coli (24%), Drosophila melanogaster (24%), and human hepatoma HepG2 (27%). Residues 204-416 show no extensive homology to any known protein sequence. Sequence 417-900 is 46% (mean) identical to the sequences of a series of aldehyde dehydrogenase (NADP+) (EC 1.2.1.3). Intact 10-formyltetrahydrofolate dehydrogenase exhibits NADP-dependent aldehyde dehydrogenase activity. The sequence identity to phosphoribosylglycinamide formyltransferase is discussed, and a binding region for 10-formyltetrahydrofolate is proposed.     

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.     

Synthesis and physicochemical characterization of the one-carbon carrier 10-formyltetrahydrofolate; a reference standard for metabolomics

Introduction

Metabolomics analysis depends on the identification and validation of specific metabolites. This task is significantly hampered by the absence of well-characterized reference standards. The one-carbon carrier 10-formyltetrahydrofolate acts as a donor of formyl groups in anabolism, where it is a substrate in formyltransferase reactions in purine biosynthesis. It has been reported as an unstable substance and is currently unavailable as a reference standard for metabolomics analysis.

Objectives

The current study was undertaken to provide the metabolomics community thoroughly characterized 10-formyltetrahydrofolate along with analytical methodology and guidelines for its storage and handling.

Methods

Anaerobic base treatment of 5,10-methenyltetrahydrofolate chloride in the presence of antioxidant was utilized to prepare 10-formyltetrahydrofolate.

Results

Pure 10-formyltetrahydrofolate has been prepared and physicochemically characterized. Conditions toward maintaining the stability of a solution of the dipotassium salt of 10-formyltetrahydrofolate have been determined.

Conclusion

This study describes the facile preparation of pure (>90%) 10-formyltetrahydrofolate, its qualitative physicochemical characterization, as well as conditions to enable its use as a reference standard in physiologic samples.

     

Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate

The combined activities of rabbit liver cytosolic serine hydroxymethyltransferase and C1-tetrahydrofolate synthase convert tetrahydrofolate and formate to 5-formyltetrahydrofolate. In this reaction C1-tetrahydrofolate synthase converts tetrahydrofolate and formate to 5,10-methenyltetrahydrofolate, which is hydrolyzed to 5-formyltetrahydrofolate by a serine hydroxymethyltransferase-glycine complex. Serine hydroxymethyltransferase, in the presence of glycine, catalyzes the conversion of chemically synthesized 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate with biphasic kinetics. There is a rapid burst of product that has a half-life of formation of 0.4 s followed by a slower phase with a completion time of about 1 h. The substrate for the burst phase of the reaction was shown not to be 5,10-methenyltetrahydrofolate but rather a one-carbon derivative of tetrahydrofolate which exists in the presence of 5,10-methenyltetrahydrofolate. This derivative is stable at pH 7 and is not an intermediate in the hydrolysis of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate by C1-tetrahydrofolate synthase. Cytosolic serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate pentaglutamate to 5-formyltetrahydrofolate pentaglutamate 15-fold faster than the hydrolysis of the monoglutamate derivative. The pentaglutamate derivative of 5-formyltetrahydrofolate binds tightly to serine hydroxymethyltransferase and dissociates slowly with a half-life of 16 s. Both rabbit liver mitochondrial and Escherichia coli serine hydroxymethyltransferase catalyze the conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at rates similar to those observed for the cytosolic enzyme. Evidence that this reaction accounts for the in vivo presence of 5-formyltetrahydrofolate is suggested by the observation that mutant strains of E. coli, which lack serine hydroxymethyltransferase activity, do not contain 5-formyltetrahydrofolate, but both these cells, containing an overproducing plasmid of serine hydroxymethyltransferase, and wild-type cells do have measurable amounts of this form of the coenzyme.     

Susceptibility to intestinal tumorigenesis in folate-deficient mice may be influenced by variation in one-carbon metabolism and DNA repair

Low dietary folate is associated with increased risk of colorectal cancer. In earlier work, we showed that folate deficiency induced intestinal tumors in BALB/c but not C57Bl/6 mice through increased dUTP incorporation into DNA with consequent DNA damage. To determine whether strain differences between one-carbon metabolism and DNA repair pathways could contribute to increased tumorigenesis in BALB/c mice, we measured amino acids and folate in the normal intestinal tissue of both strains fed a control diet or a folate-deficient diet. We also determined the expression of critical folate-metabolizing enzymes and several DNA repair enzymes. BALB/c mice had lower intestinal serine (major cellular one-carbon donor), methionine and total folate than C57Bl/6 mice under both dietary conditions. BALB/c mice had higher messenger RNA and protein levels of three folate-interconverting enzymes: trifunctional methyleneTHF (5,10-methylenetetrahydrofolate) dehydrogenase–methenylTHF cyclohydrolase–formylTHF (10-formyltetrahydrofolate) synthetase 1, bifunctional methyleneTHF dehydrogenase–methenylTHF cyclohydrolase and methylenetetrahydrofolate reductase. This pattern of expression could limit the availability of methyleneTHF for conversion of dUMP to dTMP. BALB/c mice also had higher levels of uracil DNA glycosylase 2 protein without an increase in the rate-limiting DNA polymerase β enzyme, compared with C57Bl/6 mice. We conclude that BALB/c mice may be more prone to DNA damage through decreased amounts of one-carbon donors and the diversion of methyleneTHF away from the conversion of dUMP to dTMP. In addition, incomplete excision repair of uracil in DNA could lead to accumulation of toxic repair intermediates and promotion of tumorigenesis in this tumor-susceptible strain.