Robert E. MacKenzie

Mitochondrial NAD-Dependent Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase Is Essential for Embryonic Development

ABSTRACT 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-tRNAfmet. One role postulated for NMDMC is to provide 10-formyltetrahydrofolate as a formyl group donor for the synthesis of this formylmethionyl-tRNAfmet. 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 MTHFD1 p.Arg653Gln variant alters enzyme function and increases risk for congenital heart defects.

Methylenetetrahydrofolate dehydrogenase)methenyltetrahydrofolate cyclohydrolase)formyltetrahydrofolate synthetase (MTHFD1) is a trifunctional enzyme that interconverts tetrahydrofolate (THF) derivatives for nucleotide synthesis. A common variant in MTHFD1, p.Arg653Gln (c.1958G>A), may increase the risk for neural tube defects (NTD). To examine the biological impact of this variant on MTHFD1 function, we measured enzyme activity and stability in vitro and assessed substrate flux in transfected mammalian cells. The purified Arg653Gln enzyme has normal substrate affinity but a 36% reduction in half)life at 42°C. Thermolability is reduced by magnesium adenosine triphosphate and eliminated by the substrate analog folate pentaglutamate, suggesting that folate status may modulate impact of the variant. The mutation reduces the metabolic activity of MTHFD1 within cells: formate incorporation into DNA in murine Mthfd1 knockout cells transfected with Arg653Gln is reduced by 26%±7.7% (P<0.05), compared to cells transfected with wild)type protein, indicating a disruption of de novo purine synthesis. We assessed the impact of the variant on risk for congenital heart defects (CHD) in a cohort of Quebec children (158 cases, 110 controls) and mothers of children with heart defects (199 cases, 105 controls). The 653QQ genotype in children is associated with increased risk for heart defects (odds ratio [OR], 2.11; 95% confidence interval [CI], 1.01–4.42), particularly Tetralogy of Fallot (OR, 3.60; 95% CI, 1.38–9.42) and aortic stenosis (OR, 3.13; 95% CI, 1.13–8.66). There was no effect of maternal genotype. Our results indicate that the Arg653Gln polymorphism decreases enzyme stability and increases risk for CHD. Further evaluation of this polymorphism in folate)related disorders and its potential interaction with folate status is warranted. Hum Mutat 0,1–9, 2008.

Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. A multifunctional protein from porcine liver.

Abstract Methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase from porcine liver have been co-purified more than 500-fold to apparent homogeneity. The inability of three sequential chromatographic procedures followed by affinity chromatography using NADP+- or ATP-substituted Sepharose to resolve the three activities demonstrates that they are physically associated. Molecular weight estimates of the native protein by gel filtration (Mr = 150,000) and by dodecyl sulfate gel electrophoresis (Mr = 100,000) indicate that the native structure is probably a single subunit. Since only one protein band is seen on dodecyl sulfate gels, it is concluded that the three activities are properties of a single polypeptide chain. The kinetic properties of the three activities are described, the most unusual feature being the susceptibility of the cyclohydrolase to competitive inhibition by NADP+, NAD+, ATP, and folate.

Tetrahydropteroylpolyglutamate derivatives as substrates of two multifunctional proteins with folate-dependent enzyme activities

Abstract Several of the folate-mediated reactions in eucaryotic cells are carried out by multifunctional proteins using the naturally occurring pteroylpolyglutamate derivatives. The compounds tetrahydropteroyl(glutamate) n where n = 1, 3, 5, or 7 were used to determine whether the additional glutamyl residues on the substrates provide kinetic advantages with two folate-dependent multifunctional protein. Methylenetetrahydrofolate dehydrogenase(EC 1.5.1.5)-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9)-formyltetrahydrofolate synthetase (EC 6.3.4.3) activities comprise a trifunctional protein, and formiminoglutamate:tetrahydrofolate formiminotransferase(EC 2.1.2.5)-formiminotetrahydrofolate cyclodeaminase (EC 4.3.1.40 for a bifunctional one. The dehydrogenase, transferase and synthetase were found to have 10–40-fold lower K m values for the tetrahydropteroylpolyglutamate derivatives with essentially unchanged values of V . Specificities with cyclodeaminase and cyclo-activities; hydrolase were determined by using pteroylglutamates as inhibitors of the activities; pteroylpentaglutamate is a 70-fold better inhibitor than folate with cyclodeaminase, but is only 10-fold better with cyclohydrolase. Because of the sequential nature of the enzymic activities in these multifunctional proteins, the tetrahydropteroylpolyglutamate substrates were examined to see if they provide a kinetic advantage by promoting transfer of folate intermediates between active sites on a single enzyme molecule. With the sequential transferase-deaminase activities, it was observed that the product of the transferase accumulates in the medium with tetrahydropteroylmonoglutamate as the substrate, but does not when the pentaglutamate is used. Chemical modification to selectively inactive the transferase and deaminase, followed by recombination, demonstrated that this kinetic property is observed because the intermediate formiminotetrahydropteroylpentaglutamate is transferred preferentially to the daminase site rather than equilibrating with the medium.

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.

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in transformed cells is a mitochondrial enzyme

Transformed mammalian cells express a unique bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in addition to the usual NADP-dependent dehydrogenase-cyclohydrolase-synthetase. Subcellular fractionation of murine cell lines revealed that the NAD-dependent dehydrogenase activity is located predominantly in mitochondria, while the NADP-dependent trifunctional dehydrogenase appears to exist only in the cytosol of these cells. Western analysis using monospecific polyclonal antisera confirms the subcellular distribution of these two proteins.

Formiminotransferase-cyclodeaminase from porcine liver: Purification and physical properties of the enzyme complex☆

Abstract A simple procedure for the purification of the formiminotransferase-cyclodeaminase enzyme complex is described. The crystalline preparation is homogeneous by ultracentrifugation and electrophoresis and appears to be composed of eight apparently identical subunits of about 6.4 × 10 4 daltons. Both enzyme activities migrate with the single protein band on electrophoresis and it is proposed that the activities are probably associated with different sites on one type of polypeptide chain.

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.

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.

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.