NAD-dependent Methylenetetrahydrofolate Dehydrogenase-methenyltetrahydrofolate Cyclohydrolase Research Articles

Mthfd2 Modulates Mitochondrial Function and DNA Repair to Maintain the Pluripotency of Mouse Stem Cells.

SummaryThe pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells.

Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. We found that ubiquitous Nmdmc overexpression enhanced Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body was sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincided with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan.

Magnesium and Phosphate Ions Enable NAD Binding to Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase

The mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) is believed to have evolved from a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase. It is unique in its absolute requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. To enable us to investigate the roles of these ions, a homology model of human NMDMC was constructed based on the structures of three homologous proteins. The model supports the hypothesis that the absolutely required Pi can bind in close proximity to the 2'-hydroxyl of NAD through interactions with Arg166 and Arg198. The characterization of mutants of Arg166, Asp190, and Arg198 show that Arg166 is primarily responsible for Pi binding, while Arg198 plays a secondary role, assisting in binding and properly orienting the ion in the cofactor binding site. Asp190 helps to properly position Arg166. Mutants of Asp133 suggest that the magnesium ion interacts with both Pi and the aspartate side chain and plays a role in positioning Pi and NAD. NMDMC uses Pi and magnesium to adapt an NADP binding site for NAD binding. This adaptation represents a novel variation of the classic Rossmann fold.

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.

Mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase

This chapter discusses mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase. The assays are based on the measurement of methenyltetrahydrofolate, which absorbs maximally at 350–355 nm. Eukaryotes contain methylenetetrahydrofolate dehydrogenase enzymes in both cytoplasmic and mitochondrial compartments. Saccharomyces cerevisiae has two distinct genes that encode different trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase (D) (EC1.5.1.5)-methenyltetrahydrofolate cyclohydrolase (C) (EC3.5.4.9)-10-formyltetrahydrofolate synthetase (S) [formate-tetrahydrofolate ligase (EC6.3.4.3)] enzymes. In contrast, higher eukaryotes express a cytoplasmic NADP-dependent D/C/S enzyme and a nuclear-encoded mitochondrial bifunctional NAD-dependent D/C that is unusual in which it requires both Mg 2+ and inorganic phosphate for activity. The NAD-dependent dehydrogenase activity, originally detected in Ehrlich ascites tumor cells, was later found in all immortalized mammalian cell lines tested but not in differentiated adult tissues. Both NADP- and NAD-dependent activities are present in the tissues of various insects.

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.

The isolation and characterization of a Drosophila gene encoding a putative NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase

Mammalian NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase is a bifunctional mitochondrial enzyme expressed in most established cell lines but only in developing normal tissues. We report the cloning and molecular characterization of a Drosophila gene ( DNMDMC) that encodes a protein with 56% identity to the mammalian bifunctional protein. Like the mammalian bifunctional proteins, the Drosophila protein contains a putative mitochondrial targeting sequence and its transcripts are expressed in developing tissues. Unlike its mammalian homologs, DNMDMC is expressed at high levels in adult tissues. DNMDMC maps to polytene chromosome band 85C, is encoded in three exons, and is closely flanked by two additional genes.

Expression of human NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in Escherichia coli: Purification and partial characterization

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is a bifunctional enzyme synthesized as a 37-kDa precursor that is imported into the mitochondria of embryonic and transformed mammalian cells. The cDNA encoding the human bifunctional enzyme was modified to remove nucleotides corresponding to the mitochondrial targeting sequence and was subcloned into a procaryotic expression vector under the control of the T7 RNA polymerase promoter. The soluble dehydrogenase-cyclohydrolase was expressed in Escherichia coli at levels up to 150-fold higher than those found in transformed mammalian cells. Forms of the recombinant enzyme with one, three, or seven additional amino-terminal residues were purified to homogeneity and shown to have similar kinetic properties. Investigation of the absolute requirement of the enzyme for Mg 2+ using fluorescence quenching indicates that this ion binds in the absence of substrates.

Analysis of the promoter region of the gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase methenyltetrahydrofolate cyclohydrolase

Sequence analysis of the 5'-flanking region of the gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) revealed several putative cis-regulatory elements. To delineate the function of these regulatory elements, various deletion mutants of the 5'-flanking region were connected to the reporter gene chloramphenicol acetyltransferase (CAT) and promoter activity was measured in transient transfection assays. Transfection experiments performed with the sequence extending from -508 to +59 produced a high-level transient expression of the CAT gene in BALB/c 3T3-SV-T2 and NIH 3T3 cells. Removal of the sequence from +16 to +59 which includes the second transcription start point at +43, a TATA-like box and 5'-untranslated sequences abolished the promoter activity. Deletion analysis of 5'-upstream sequences revealed that the region from positions -55 to +59 is sufficient to mediate a high CAT activity comparable to the level obtained with the construct -508/+59. Within this region are found a CAAT box, a TATA-like box and two putative GC boxes. A functional analysis of the promoter showed that the sequence from -55 to +59 is sufficient to respond to stimulation by serum.

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 λMBL3 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 Sl 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 Spl.

Isolation and Characterization of cDNA Clones Encoding the Murine NAD-dependent Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase

Forty cDNA clones corresponding to the bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase enzyme were isolated from a mouse lambda gt11 library. Two classes of cDNA clones were shown by Northern analysis to correspond to the two mRNA species of 1.7 and 2.0 kilobases present in transformed cells but not in normal tissues and that apparently are derived from alternate polyadenylation signals. The 1050-base pair coding region encodes a protein of 350 amino acids which contains a putative mitochondrial-targeting signal peptide of 34 amino acids following the initiator methionine. The 20 amino acids immediately following the signal peptide correspond exactly to those determined by sequence analysis of the amino terminus of the purified protein. The derived amino acid sequence of the NAD-dependent dehydrogenase-cyclohydrolase shows extensive homology with the corresponding amino-terminal sequence of the trifunctional NADP-dependent dehydrogenase-cyclohydrolase-synthetase enzyme from human cells (approximately 40%), yeast cytosol (approximately 36%), and yeast mitochondria (approximately 45%).

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.

The activities of the NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells are kinetically independent.

The bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells has very different kinetic properties from the larger NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase present in all mammalian cells. The NAD-dependent dehydrogenase is unique in that it requires formation of a magnesium.enzyme complex to allow addition of the first substrate, NAD+. It catalyzes an equilibrium ordered kinetic mechanism that has methylenetetrahydrofolate as the last reactant to add and NADH as the last product released. The NADP-dependent dehydrogenase has the same order of addition of substrates, but NADPH is released prior to methenyltetrahydrofolate. The dehydrogenase-cyclohydrolase activities of both enzymes channel methenyltetrahydropteroylglutamate intermediates with the same efficiency which is unaffected by the number of glutamyl residues in the methylenetetrahydrofolate substrate. However, the cyclohydrolase activity of the bifunctional protein is kinetically independent of its dehydrogenase activity, as supported by its lack of inhibition by NAD+, whereas NADP+ strongly inhibits that of the NADP-dependent enzyme. This difference is further demonstrated by the observation that conversion of formyltetrahydrofolate to methylenetetrahydrofolate in the presence of reduced pyridine nucleotide is catalyzed readily only by the bifunctional enzyme.

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells. Purification and properties.

NAD-dependent methylenetetrahydrofolate dehydrogenase is expressed in transformed or established mammalian cell lines in vitro but only in the developmental tissues of normal adult animals (Mejia, N. R. and MacKenzie, R. E. (1985) J. Biol. Chem. 260, 14616-14620). The enzyme, which contains methenyltetrahydrofolate cyclohydrolase activity as well, has been purified 6000-fold from Ehrlich ascites tumor cells. The preparation is homogeneous by sodium dodecyl sulfate gel electrophoresis (Mr = 34,000), and results from cross-linking with bis(sulfosuccinimidyl)suberate are consistent with a dimeric structure (Mr = 68,000) for the native bifunctional enzyme. The dehydrogenase is specific for NAD and requires both a divalent cation, Mg2+ or Mn2+, for activity and as well is stimulated by inorganic phosphate. When compared to the usual NADP-dependent methylenetetrahydrofolate dehydrogenase from mouse liver, the NAD-dependent dehydrogenase activity of the murine tumor enzyme shows a greater affinity for the polyglutamate forms of folate.