A novel mouse model for genetic variation in 10-formyltetrahydrofolate synthetase exhibits disturbed purine synthesis with impacts on pregnancy and embryonic development.

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.

Mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is essential for embryonic development.

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.

The 3-D structure of a folate-dependent dehydrogenase/cyclohydrolase bifunctional enzyme at 1.5 A resolution.

BACKGROUND: The interconversion of two major folate one-carbon donors occurs through the sequential activities of NAD(P)-dependent methylene[H4]folate dehydrogenase (D) and methenyl[H4]folate cyclohydrolase (C). These activities often coexist as part of a multifunctional enzyme and there are several lines of evidence suggesting that their substrates bind at overlapping sites. Little is known, however, about the nature of this site or the identity of the active-site residues for this enzyme family. RESULTS: We have determined, to 1.5 A resolution, the structure of a dimer of the D/C domain of the human trifunctional cytosolic enzyme with bound NADP cofactor, using the MAD technique. The D/C subunit is composed of two alpha/beta domains that assemble to form a wide cleft. The cleft walls are lined with highly conserved residues and NADP is bound along one wall. The NADP-binding domain has a Rossmann fold, characterized by a modified diphosphate-binding loop fingerprint-GXSXXXG. Dimerization occurs by antiparallel interaction of two NADP-binding domains. Superposition of the two subunits indicates domain motion occurs about a well-defined hinge region. CONCLUSIONS: 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.

The crystal structure of the formiminotransferase domain of formiminotransferase-cyclodeaminase: implications for substrate channeling in a bifunctional enzyme.

BACKGROUND: The bifunctional enzyme formiminotransferase-cyclodeaminase (FTCD) contains two active sites at different positions on the protein structure. The enzyme binds a gamma-linked polyglutamylated form of the tetrahydrofolate substrate and channels the product of the transferase reaction from the transferase active site to the cyclodeaminase active site. Structural studies of this bifunctional enzyme and its monofunctional domains will provide insight into the mechanism of substrate channeling and the two catalytic reactions. RESULTS: The crystal structure of the formiminotransferase (FT) domain of FTCD has been determined in the presence of a product analog, folinic acid. The overall structure shows that the FT domain comprises two subdomains that adopt a novel alpha/beta fold. Inspection of the folinic acid binding site reveals an electrostatic tunnel traversing the width of the molecule. The distribution of charged residues in the tunnel provides insight into the possible mode of substrate binding and channeling. The electron density reveals that the non-natural stereoisomer, (6R)-folinic acid, binds to the protein; this observation suggests a mechanism for product release. In addition, a single molecule of glycerol is bound to the enzyme and indicates a putative binding site for formiminoglutamate. CONCLUSIONS: The structure of the FT domain in the presence of folinic acid reveals a possible novel mechanism for substrate channeling. The position of the folinic acid and a bound glycerol molecule near to the sidechain of His82 suggests that this residue may act as the catalytic base required for the formiminotransferase mechanism.

Monofunctional domains of formiminotransferase-cyclodeaminase retain similar conformational stabilities outside the bifunctional octamer.

Each identical subunit of octameric formiminotransferase cyclodeaminase consists of a transferase and a deaminase domain connected by a short linker sequence. Both domains can be independently expressed in Escherichia coli as monofunctional dimers and show no indication of associating, suggesting that the linker mediates the only substantial interaction between the transferase and deaminase domains. To better understand the benefits arising from octamer formation, we have used equilibrium unfolding methods to examine the properties of the transferase and deaminase domains independently and within the octamer. Each isolated dimeric domain undergoes an apparent change in tertiary structure at low concentrations of urea (< 2 mol/l) which results in the concurrent loss of intrinsic fluorescence and catalytic activity. The full length octameric enzyme also undergoes inactivation and a loss of intrinsic fluorescence over this concentration range, without apparent change in secondary or quaternary structure. Between 2 and 2.5 M urea the isolated transferase and deaminase domains dissociate to monomers. However, only one of the subunit interfaces in the octamer is disrupted at this urea concentration and dissociation of the second interface occurs between 3.5 and 5 M urea. While each domain shows similar stability to denaturation within and outside of the octamer, one type of subunit interface achieves increased stability within the full length enzyme.

Primary structure of a folate-dependent trifunctional enzyme from Spodoptera frugiperda.

The dehydrogenase and synthetase activities of the NADP-dependent methylenetetra-hydrofolate dehydrogenase-cyclohydrolase-synthetase are undetectable in extracts of the Spodoptera frugiperda cell line, Sf9. However, a single cDNA encoding this protein was isolated from a library and sequenced. The deduced amino acid sequence codes for a protein of 933 amino acids in length that shows 59% identity to the human enzyme. The cDNA inserted in the yeast expression vector pVT102-U complements a purine auxotrophic yeast strain lacking this enzyme.

Methylenetetrahydrofolate dehydrogenase-cyclohydrolase from Photobacterium phosphoreum shares properties with a mammalian mitochondrial homologue.

The marine bioluminescent bacterium Photobacterium phosphoreum expresses a bifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase with dual cofactor specificity. An investigation of the kinetic parameters of the P. phosphoreum enzyme indicate that its utilization of dinucleotide cofactors shares similarities with the human mitochondrial dehydrogenase-cyclohydrolase. Both enzymes exhibit dual cofactor specificity and the NAD(+)-dependent dehydrogenase activities from both enzymes can be activated by inorganic phosphate. Furthermore, an analysis of multiply aligned dehydrogenase-cyclohydrolase sequences from 11 species revealed that bacterial and mitochondrial enzymes are more closely related to each other than to the dehydrogenase-cyclohydrolase domains from eukaryotic trifunctional enzymes, and that the bacterial and mitochondrial enzymes share a common point of divergence. Since the NADP+ cofactor is kinetically favoured by a factor of 18 over NAD+, and is therefore likely to be the preferred in vivo cofactor, we propose that the P. phosphoreum enzyme and the human mitochondrial enzyme evolved from a common ancestral dehydrogenase-cyclohydrolase with dual cofactor specificity, but that cofactor preference in these two enzymes diverged in response to different metabolic requirements.

Methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase from porcine liver. Isolation of a dehydrogenase-cyclohydrolase fragment from the multifunctional enzyme.

Tryptic digestion of a multifunctional enzyme from porcine liver containing methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.5), methenyltetrahydrofolate cyclohydrolase (5,10-methenyltetrahydrofolate 5-hydrolase, EC 3.5.4.9) and formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase, EC 6.3.4.3) activities destroys the synthetase. A fragment containing both dehydrogenase and cyclohydrolase activities has been isolated by affinity chromatography on an NADP+-Sepharose affinity column. The purified fragment is homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis where its molecular weight was determined as 33 000 +/- 1200 compared with 100 000 for the undigested protein. The cyclohydrolase activity retains sensitivity to inhibition by NADP+, MgATP and ATP.

Formiminotransferase cyclodeaminase from porcine liver. An octomeric enzyme containing bifunctional polypeptides.

The forminiminotransferase-cyclodeaminase complex contains 2.8% carbohydrate and is composed of identical subunits based on molecular weight, isoelectric focusing and the number of CNBr-generated peptides. Electron microscopy demonstrated that the complex is a ring-shaped molecule when negatively stained both in solution and in crystalline structures. Rotational reinforcement of electron micrographs reveals that the molecule is composed of eight subunits arranged in a planar ring. These results indicate that each polypeptide of the complex must contain both enzymic activities.

Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase from porcine liver. Interaction between the dehydrogenase and cyclohydrolase activities of the multifunctional enzyme.

Methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.5) one of the activities of a trifunctional folate-dependent enzyme from porcine liver, uses an ordered kinetic mechannism as determined from initial velocity, product inhibition and dead-end inhibition studies. The final product released from the dehydrogenase is methenyltetrahydrofolate. However, from the time course of appearance of products it is observed that the methenyltetrahydrofolate, rather than equilibrating with the solution, is converted preferentially to formyltetrahydrofolate by the cyclohydrolase, (5,10-methenyletrahydrofolate 5-hydrolase (decyclizing), EC 3.5.4.9) demonstrating a functional interaction between these two enzymic activites.

NAD(+)-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase: detection of the mRNA in normal murine tissues and transcriptional regulation of the gene in cell lines.

NAD(+)-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase, a nuclear-encoded mitochondrial bifunctional enzyme, is detectable in extracts of immortalized and transformed cells but not in most adult tissues (Mejia, N.R. and MacKenzie, R.E. (1985) J. Biol. Chem. 260, 14616-14620). Normal tissues contain low levels of the mRNA, with the exception of thymus and especially testis which contain much higher amounts. The protein could not be detected in any of these normal tissues either by Western analysis or by enzyme activity assay. The elevated level of the mRNA in testis is not dependent on active spermatogenesis. Oncogenic transformation of NIH 3T3 fibroblasts by the Ha-ras oncogene did not significantly affect the steady state level of the dehydrogenase-cyclohydrolase mRNA. The gene in quiescent Balb/c 3T3 fibroblasts is induced by mitogens such as serum and phorbol esters and requires de novo transcription. Post-transcriptionally, the mRNA is stabilized by factors in serum such as insulin-like growth factor-1. The intracellular location of the enzyme and its regulation of expression are consistent with its proposed role in mitochondrial biogenesis.

Human liver methenyltetrahydrofolate synthetase: improved purification and increased affinity for folate polyglutamate substrates.

Methenyltetrahydrofolate synthetase (5-formyltetrahydrofolate cyclodehydrase (cyclo-ligase) (ADP-forming) EC 6.3.3.2) catalyzes the ATP- and Mg2+-dependent transformation of 5-formyltetrahydrofolate (leucovorin) to 5,10-methenyltetrahydrofolate. The enzyme has been purified 49,000-fold from human liver by a two-column procedure with Blue Sepharose followed by folinate-Sepharose chromatography. It appears as a single band both on SDS-polyacrylamide gel electrophoresis (Mr 27,000) and on isoelectric focusing (pI = 7.0) and is monomeric, with a molecular weight of 27,000 on gel filtration. Initial-velocity studies suggest that the enzyme catalyzes a sequential mechanism and at 30 degrees C and pH 6.0 the turnover number is 1000 min-1. The enzyme has a higher affinity for its pentaglutamate substrate (Km = 0.6 microM) than for the monoglutamate (Km = 2 microM). The antifolate methotrexate has no inhibitory effect at concentrations up to 350 microM, while methotrexate pentaglutamate is a competitive inhibitor with a Ki = 15 microM. Similarly, dihydrofolate monoglutamate is a weak inhibitor with a Ki = 50 microM, while the pentaglutamate is a potent competitive inhibitor with a Ki of 3.8 microM. Thus, dihydrofolate and methotrexate pentaglutamates could regulate enzyme activity and help explain why leucovorin fails to rescue cells from high concentrations of methotrexate.

Formyltetrahydrofolate dehydrogenase-hydrolase from pig liver: simultaneous assay of the activities.

The bifunctional folate-dependent enzyme, 10-formyltetrahydrofolate dehydrogenase-hydrolase (10-formyltetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.6), has been purified to homogeneity from pig liver. Its amino acid composition was determined and gave a calculated v of 0.735 ml/g; a molecular weight of 92500 for the protein subunit was determined as well. Spectrophotometric, fluorescence emission and radiochemical methods were devised to assay the activities. Quantitative separation of carbon dioxide and formate produced by the dehydrogenase and the hydrolase reactions, respectively, demonstrated that both activities occur simultaneously. This fact, together with a 5-fold difference in the Km values for the folate substrate, strongly suggests that these two activities are functions of different sites. The possible role of polyglutamate specificity for the preferential selection of one of the activities under physiological conditions was ruled out when both proved to have similar specificities, as determined by sensitivity to inhibition by tetrahydropteroylpolyglutamates.

An improved procedure for the purification of formiminotransferase-cyclodeaminase from pig liver. Kinetics of the transferase activity with tetrahydropteroylpolyglutamates.

Formiminotransferase-cyclodeaminase is stabilized and activated approx. 40% in the presence of low concentrations (equal or less than 0.2%) of Triton X-100, possibly because the average hydrophobicity (1.10 kcal per residue) and the frequency of large non-polar side-chains (0.34) of this protein are both somewhat higher than average. This stabilization enabled us to develop a new purification procedure for the enzyme using chromatography on Matrex Gel Orange A and heparin-Sepharose columns in the presence of Triton X-100. This procedure is easier, much more reproducible, and gives slightly higher yield than the previous method described by Drury, et al. Further investigations of the role of tetrahydropteroylpolyglutamates with formiminotransferase-cyclodeaminase reveal that the use of polyglutamylated folate substrates does not change the mechanism of the transferase reaction, but decreases the K(m) for formininoglutamate, the second substrate, more than 10-fold, bringing it closer to the expected physiological concentration.

Channeling efficiency in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase domain: the effects of site-directed mutagenesis of NADP binding residues.

The three-dimensional structure of the dehydrogenase-cyclohydrolase bifunctional domain of the human trifunctional enzyme indicates that Arg-173 and Ser-197 are within 3 A of the 2'-phosphate of bound NADP. Site-directed mutagenesis confirms that Arg-173 is essential for efficient binding and cannot be substituted by lysine. R173A and R173K have detectable dehydrogenase activity, but the K(m) values for NADP are increased by at least 500-fold. The S197A mutant has a K(m) for NADP that is only 20-fold higher than wild-type, indicating that it plays a supporting role. Forward and reverse cyclohydrolase activities of all the mutants were unchanged, except that the reverse cyclohydrolase activity of mutants that bind NADP poorly, or lack Ser-197, cannot be stimulated by 2',5'-ADP. The 50% channeling efficiency in the forward direction is not improved by the addition of exogenous NADPH and cannot be explained by premature dissociation of the dinucleotide from the ternary complex. As well, channeling is unaffected in mutants that exhibit a wide range of dinucleotide binding. Given that dinucleotide binding is unrelated to substrate channeling efficiency in the D/C domain, we propose that the difference in forward and reverse channeling efficiencies can be explained solely by the movement of the methenylH(4)folate between two overlapping subsites to which it has different binding affinities.

Structural organization of the murine gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.

The structural organization of the entire nuclear gene (NMDMC) encoding the mitochondrial (mt) NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase enzyme (NMDMC) was determined by analysis of clones obtained from a lambda EMBL3 murine genomic DNA library. The gene is approx. 13 kb in length and contains eight exons and seven introns. All exon/intron splice junctions follow the GT/AG rule. The amino acid presequence, which is essential for transport of the NMDMC enzyme precursor into mt, is encoded almost entirely in the first exon. Two major transcriptional start points (tsp), located 33 and 75 nucleotides upstream from the AUG start codon, were revealed by S1 nuclease mapping and RNase protection analyses. The immediate 5'-flanking region of the first exon contains one CAAT box, a TATA-like box and three sites homologous to the consensus sequence for the binding of transcription factor Sp1.

Transcriptional regulation of murine NADP(+)-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase.

The cytosolic NADP(+)-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase is ubiquitously expressed in all mouse tissues and cell lines examined. Northern analyses of the RNA indicated that there is an extensive variation in the levels of mRNA in different tissues. However, the gene is refractory to induction by serum, phorbol esters or growth factors in cultured fibroblasts. The mRNA of the NADP(+)-dependent trifunctional enzyme is stabilized post-transcriptionally by insulin-like growth factor-1.

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is targeted to the cytoplasm in insect cell lines.

Cytosolic NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase synthetase and the mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) are differentially expressed during insect development although both enzymes are detectable at all stages. In contrast, cell lines derived from a variety of insect species express high levels of NMDMC but undetectable levels of the NADP-dependent enzyme. Northern analysis indicates the NMDMC message is expressed at levels 50-100 times higher in a Drosophila cell line compared to adult flies. RNase protection showed the predominance of shortened transcripts that require initiation at a downstream AUG producing a truncated protein that lacks a mitochondrial targeting sequence. These changes in expression effectively exchange the cytosolic NADP-dependent dehydrogenase for one with NAD specificity.

Methylenetetrahydrofolate dehydrogenases in normal and transformed mammalian cells.

Transformed mammalian cells express both the usual NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase as well as the bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase. Antisera to these proteins do not crossreact, and Western blots of cell extracts indicate that there is no inactive form of the NAD-dependent enzyme in normal tissues. Immunofluorescence studies suggest a cytosolic location for the NAD-dependent enzyme, although sequence homology seen in the N-terminal 10 residues indicates a closer relationship with the mitochondrial form of the yeast NADP dependent trifunctional enzyme.

Interaction of tetrahydropteroylpolyglutamates with two folate-dependent multifunctional enzymes.

The naturally occurring pteroylpolyglutamate derivatives are substrates for the folate-mediated reactions in cells, including the reactions catalyzed by two multifunctional folate dependent enzymes in eucaryotes. The appropriate derivatives of tetrahydropteroyl (glutamate)n where n = 1, 3, 5, or 7 were used to determine the specificity for, and kinetic advantages of the extra glutamyl residues with two multifunctional proteins from pig liver: methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase, and formiminotransferase-formininotetrahydrofolate cyclodeaminase. Specificity for the polyglutamate derivatives ranged from 10- to 70-fold as indicated from Km values or from the ability to inhibit the five different enzyme activities. With the sequential activities of the transferase-deaminase enzyme, it was demonstrated that when the tetrahydropteroyl pentaglutamate is used as a substrate, the intermediate formimino-compound does not accumulate in the medium. That this kinetic observation is due to preferential transfer of the pentaglutamate- but not monoglutamate intermediate from transferase to deaminase sites without its release from the enzyme molecule was supported by three types of experiments. Chemical modification to yield monofunctional derivatives of the transferase-deaminase affected the kinetics of the recombined activities only with the pentaglutamate substrate, causing a lag in the appearance of final product. Inhibition studies demonstrated that the deaminase activity could preferentially be inhibited only with the monoglutamate substrate. The deaminase activity with the monoglutamate substrate was increased by providing elevated formiminotetrahydrofolate in the assay mixture; no effect was observed when the reaction was carried out with pentaglutamate. Preliminary binding studies indicate a single folate site per subunit of the octameric enzyme, suggesting a type of combined transferase-deaminase site.