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.

Novel deazaxanthine-based DPP-4 inhibitors have been identified that are potent (IC(50) <10nM) and highly selective versus other dipeptidyl peptidases. Their synthesis and SAR are reported, along with initial efforts to improve the PK profile through decoration of the deazaxanthine core. Optimisation of compound 3a resulted in the identification of compound (S)-4i, which displayed an improved in vitro and ADME profile. Further enhancements to the PK profile were possible by changing from the deazahypoxanthine to the deazaxanthine template, culminating in compound 12g, which displayed good ex vivo DPP-4 inhibition and a superior PK profile in rat, suggestive of once daily dosing in man.

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. © 2008 Wiley)Liss, Inc.

Folate-mediated metabolism involves enzyme-catalyzed reactions that occur in the cytoplasmic, mitochondrial, and nuclear compartments in mammalian cells. Which of the folate-dependent enzymes are expressed in these compartments depends on the stage of development, cell type, cell cycle, and whether or not the cell is transformed. Mitochondria become formate-generating organelles in cells and tissues expressing the MTHFD2 and MTHFD1L genes. The products of these nuclear genes were derived from trifunctional precursor proteins, expressing methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase, and formyttetrahydrofolate synthetase activities. The MTHFD2 protein is a bifunctional protein with dehydrogenase and cyclohydrolase activities that arose from a trifunctional precursor through the loss of the synthetase domain and a novel adaptation to NAD rather than NADP specificity for the dehydrogenase. The MTHFD1L protein retains the size of its trifunctional precursor, but through the mutation of critical residues, both the dehydrogenase and cyclohydrolase activities have been silenced. MTHFD1L is thus a monofunctional formyltetrahydrofolate synthetase. This review discusses the properties and functions of these mitochondrial proteins and their rote in supporting cytosolic purine synthesis during embryonic development and in cells undergoing rapid growth. (C) 2008 Elsevier Inc.

In eukaryotes, folate metabolism is compartmentalized between the cytoplasm and organelles. The folate pathways of mitochondria are adapted to serve the metabolism of the organism. In yeast, mitochondria support cytoplasmic purine synthesis through the generation of formate. This pathway is important but not essential for survival, consistent with the flexibility of yeast metabolism. In plants, the mitochondrial pathways support photorespiration by generating serine from glycine. This pathway is essential under photosynthetic conditions and the enzyme expression varies with photosynthetic activity. In mammals, the expression of the mitochondrial enzymes varies in tissues and during development. In embryos, mitochondria supply formate and glycine for purine synthesis, a process essential for survival; in adult tissues, flux through mitochondria can favor serine production. The differences in the folate pathways of mitochondria depending on species, tissues and developmental stages, profoundly alter the nature of their metabolic contribution.

Appropriately substituted silicon diols have been shown to be inhibitors of both aspartyl and metallo proteases. Using the knowledge that the active site of serine proteases consists of two domains, an extended binding site where non-covalent binding interactions occur and a catalytic site where covalent bond-forming and bond-breaking reactions take place, silicon diol compounds were designed and synthesized as potent human leucocyte elastase inhibitors. The peptido-mimetic analog 9 is a potent human elastase inhibitor that provides key interactions at both the prime (C-terminal) and non-prime (N-terminal) sites within the enzyme and demonstrates that the silicon diol presents itself as an important privileged structure within the framework of protease inhibitors.

Whether the highly dynamic structure of the vimentin intermediate filament (IF) cytoskeleton responds to cues from cellular organelles, and what proteins might participate in such events is largely unknown. We have shown previously that the Golgi protein formiminotransferase cyclodeaminase (FTCD) binds to vimentin filaments in vivo and in vitro, and that overexpression of FTCD causes dramatic rearrangements of the vimentin IF cytoskeleton (Gao and Sztul, J. Cell Biol. 152, 877 - 894, 2001). Using real-time imaging, we now show that FTCD causes bundling of individual thinner vimentin filaments into fibers and that the bundling always originates at the Golgi. FTCD appears to be the molecular "glue" since FTCD cross-links vimentin filaments in vitro. To initiate the analysis of structural determinants required for FTCD function in vimentin dynamics, we used structure-based design to generate individual formiminotransferase (FT) and cyclodeaminase (CD) domains, and to produce an enzymatically inactive FTCD. We show that the intact octameric structure is required for FTCD binding to vimentin filaments and for promoting filament assembly, but that eliminating enzymatic activity does not affect FTCD effects on the vimentin cytoskeleton. Our findings indicate that the Golgi protein FTCD is a potent modulator of the vimentin IF cytoskeleton, and suggest that the Golgi might act as a reservoir for proteins that regulate cytoskeletal dynamics.

The bifunctional dehydrogenase/cyclohydrolase domain of the human NADP-dependent trifunctional methyleneH(4)folate dehydrogenase/methenylH(4)folate cyclohydrolase/formylH(4)folate synthetase (H(4)folate tetrahydrofolate) catalyzes two sequential reactions involved in the interconversion of H(4)folate derivatives. We have established by equilibrium dialysis that a single H(4)folate-binding site exists per monomer of the dimeric domain and that the presence of nucleotides has two unexpected effects on H(4)folate substrate binding. Nucleotides containing a 5'-phosphate cause positive cooperativity in the binding of methyleneH(4)folate but not of 10-formylH(4)folate, and NADP increases the affinity for 10-formylH(4)folate by a factor of 25. The results indicate that dinucleotide preferentially binds before 10-formylH(4)folate in the reverse cyclohydrolase reaction, and this mechanism increases the efficiency of conversion of 10-formylH(4)folate to methyleneH(4)folate. We report new kinetic data that are also consistent with a steady-state random mechanism for this enzyme, To assess whether the enzyme functions at equilibrium in vivo, we determined the overall chemical equilibrium constant of K-eq = 16 for ([10-formylH(4)folate] [NADPH])/([methyleneH(4)folate][NADP]). Using this value and reported ratios of free dinucleotides and folate derivatives in vivo, we estimate that the cytosolic dehydrogenase/cyclohydrolase reactions exist near the equilibrium position. However, the NAD-dependent dehydrogenase/cyclohydrolase reactions in mitochondria are far from equilibrium and are poised toward 10-formylH(4)folate synthesis. The results of the binding and kinetic studies indicate that the bifunctional nature of the methyleneH(4)folate dehydrogenase/methenylH(4)folate cyclohydrolase domain is designed to optimize the overall reverse reactions in vivo.

Folic acid exists in mammalian cells with a poly-gamma-glutamate tail that may regulate the flux of folates through the various cellular pathways. The substrate polyglutamate specificity of methylenetetrahydrofolate dehydrogenase from pig liver has been examined by using a competitive method and measuring apparent tritium kinetic isotope effects on Vmax/Km for methylenetetrahydrofolate. This competitive method yields very accurate ratios of Km values for alternate substrates of an enzyme and may also be applied to reactions with no isotope effect. In combination with published data from our own and other laboratories, the kinetic parameters of methylenetetrahydrofolate dehydrogenase were used to calculate the initial velocities of pig liver methylenetetrahydrofolate dehydrogenase, thymidylate synthase, and methylenetetrahydrofolate reductase, at physiological concentrations of substrates and enzymes. These calculations suggest that the cellular concentration of methylenetetrahydrofolate may regulate the flux of this metabolite into the pathways leading to nucleotide biosynthesis and methionine regeneration. An increase in the cellular level of methylenetetrahydrofolate would permit more one-carbon units to be directed toward nucleotide biosynthesis.

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°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 μM) than for the monoglutamate (Km = 2 μM). The antifolate methotrexate has no inhibitory effect at concentrations up to 350 μM, while methotrexate pentaglutamate is a competitive inhibitor with a Km = 15 μM. Similarly, dihydrofolate monoglutamate is a weak inhibitor with a Kt = 50 μM, while the pentaglutamate is a potent competitive inhibitor with a Ki of 3.8 μM. Thus, dihydrofolate and methotrexate pentaglutamates could regulate enzyme activity and help explain why leucovorin fails to rescue cells from high concentrations of methotrexate.