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.

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.

Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase.

Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceutical interest, as their function is crucial for amino acid and DNA biosynthesis. The crystal structure of the human cytosolic methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme has been determined previously with a bound NADP cofactor. While the substrate binding site was identified to be localized in a deep and rather hydrophobic cleft at the interface between two protein domains, the unambiguous assignment of catalytic residues was not possible. We succeeded in determining the crystal structures of three ternary DC301/NADP/inhibitor complexes. Investigation of these structures followed by site-directed mutagenesis studies allowed identification of the amino acids involved in catalysis by both enzyme activities. The inhibitors bind close to Lys56 and Tyr52, residues of a strictly conserved motif for active sites in dehydrogenases. While Lys56 is in a good position for chemical interaction with the substrate analogue, Tyr52 was found stacking against the inhibitors' aromatic rings and hence seems to be more important for proper positioning of the ligand than for catalysis. Also, Ser49 and/or Cys147 were found to possibly act as an activator for water in the cyclohydrolase step. These and the other residues (Gln100 and Asp125), with which contacts are made, are strictly conserved in THF dehydrogenases. On the basis of structural and mutagenesis data, we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase.

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.

Crystallization and preliminary X-ray analysis of the formiminotransferase domain from the bifunctional enzyme formiminotransferase-cyclodeaminase.

Formiminotransferase-cyclodeaminase (E.C. 2.1.2.5-E.C. 4.3.1.4) is a bifunctional enzyme involved in the histidine-degradation pathway which exhibits specificity for polyglutamylated folate substrates. The first function of the enzyme transfers the formimino group of formiminoglutamate to the N5 position of tetrahydrofolate, while the second function catalyses the cyclodeamination of the formimino group, yielding N5,10-methenyl-tetrahydrofolate, with efficient channeling of the intermediate between these activities. Initial studies have shown that the enzyme consists of eight identical subunits of 62 kDa each, arranged as a circular tetramer of dimers. It is this formation which results in two different dimeric interfaces, which are necessary for the two different activities. The identical subunits have been shown to consist of two domains, each of which can be obtained as dimers. The formiminotransferase domain has been crystallized in the presence of the substrate analogue folinic acid. The crystals belong to space group P212121, with unit-cell dimensions a = 64.4, b = 103.7, c = 122.3 A. Both a native data set and a mercurial derivative data set have been collected to 2.8 A resolution.

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.

Methenyltetrahydrofolate cyclohydrolase is rate limiting for the enzymatic conversion of 10-formyltetrahydrofolate to 5,10-methylenetetrahydrofolate in bifunctional dehydrogenase-cyclohydrolase enzymes.

The kinetic properties of three methylenetetrahydrofolate dehydrogenase-cyclohydrolase (D/C) enzymes (the NADP-dependent bifunctional domain of the human cytoplasmic trifunctional enzyme, the human mitochondrial NAD-dependent bifunctional enzyme, and the NAD(P)-dependent bifunctional enzyme from Photobacterium phosphoreum) were determined in both forward and reverse directions. In the forward direction, the enzymes possess widely different ratios of kcat C/Kcat D, but all channel methenylH4folate produced by the D activity to the C activity with approximately the same efficiency. A deuterium isotope effect is observed with the human NADP-dependent enzyme in both forward and reverse dehydrogenase assays, consistent with hydride transfer being rate limiting for the interconversion of methenyl- and methyleneH4folate. However, no kinetic isotope effect is observed for the overall reverse reaction (formylH4folate to methyleneH4folate). We devised an assay to measure the reverse cyclohydrolase activity independent of the dehydrogenase, and determined that the Kcat (overall reverse) for each enzyme is approximately equal to the Kcat for its reverse cyclohydrolase activity. Therefore, the rate-limiting step in the overall reverse reaction is not hydride transfer by the dehydrogenase, but the production of methenylH4folate catalyzed by the cyclohydrolase. The reverse cyclohydrolase activities of the NADP-dependent D/C and the P. phosphoreum enzymes, but not the mitochondrial NAD-dependent enzyme, can be stimulated 2-fold by the addition of 2',5'-ADP. The results suggest that the cyclohydrolases of the human NADP dependent and P. phosphoreum enzymes are optimized to catalyze the reverse reaction in the presence of bound coenzyme. These results imply that essentially all of the methenylH4folate produced by the cyclohydrolase in the reverse reaction is channeled to the dehydrogenase.

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.

Crystallization of the bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase domain of the human trifunctional enzyme.

Methylenetetrahydrofolate([H4] folate) dehydrogenase (D) and methenyl[H4] folate cyclohydrolase (C) coexist as a bifunctional enzyme (DC) or as the amino-terminal domain of a trifunctional enzyme (DCS) where the third activity is 10-formyl[H4]folate synthetase (S). Two crystal forms of the DC domain of the human cytosolic DCS enzyme have been grown from polyethyleneglycol solution. The monoclinic P2(1) crystals diffract to 2.8 A with a = 72.5 A, b = 68.5 A, c = 125.2 A, and beta = 91.8 degrees but were found to be twinned. The orthorhombic P2(1)2(1)2(1) crystals diffract to 2.5 A with a = 67.7 A, b = 135.9 A, c = 61.6 A, and contain two molecules per asymmetric unit.

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.

Interhospital transfer of the critically ill trauma patient: the potential role of a specialist transport team in a trauma system.

A specialist transfer team based in the regional intensive therapy unit (ITU) at the Western Infirmary, Glasgow, acts as a central interhospital retrieval team for Glasgow and the west of Scotland. The establishment of trauma systems has been proposed. This paper describes the activities of the specialist transfer team to illustrate the potential role of a central retrieval team within such a system.

Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase.

The bifunctional dehydrogenase/cyclohydrolase domain of the human NADP-dependent trifunctional methyleneH4folate dehydrogenase/methenylH4folate cyclohydrolase/formylH4folate synthetase (H4folate = tetrahydrofolate) catalyzes two sequential reactions involved in the interconversion of H4folate derivatives. We have established by equilibrium dialysis that a single H4folate-binding site exists per monomer of the dimeric domain and that the presence of nucleotides has two unexpected effects on H4folate substrate binding. Nucleotides containing a 5'-phosphate cause positive cooperativity in the binding of methyleneH4folate but not of 10-formylH4folate, and NADP increases the affinity for 10-formylH4folate by a factor of 25. The results indicate that dinucleotide preferentially binds before 10-formylH4folate in the reverse cyclohydrolase reaction, and this mechanism increases the efficiency of conversion of 10-formylH4folate to methyleneH4folate. 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 Keq = 16 for ([10- formylH4folate][NADPH])/([methyleneH4folate][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-formylH4folate synthesis. The results of the binding and kinetic studies indicate that the bifunctional nature of the methyleneH4folate dehydrogenase/methenylH4folate cyclohdrolase domain is designed to optimize the overall reverse reactions in vivo.

The two monofunctional domains of octameric formiminotransferase-cyclodeaminase exist as dimers.

Formiminotransferase-cyclodeaminase is a bifunctional enzyme arranged as a circular tetramer of dimers that exhibits the ability to efficiently channel polyglutamylated folate between catalytic sites. Through deletion mutagenesis we demonstrate that each subunit consists of an N-terminal transferase active domain and a C-terminal deaminase active domain separated by a linker sequence of minimally eight residues. The full-length enzyme and both isolated domains have been expressed as C-terminally histidine-tagged proteins. Both domains self-dimerize, providing direct evidence for the existence of two types of subunit interfaces. The results suggest that both the transferase and the deaminase activities are dependent on the formation of specific subunit interfaces. Because channeling is not observed between isolated domains, only the octamer appears able to directly transfer pentaglutamylated intermediate between active sites.

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.

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.

Binding of the 2',5'-ADP subsite stimulates cyclohydrolase activity of human NADP(+)-dependent methylenetetrahydrofolate dehydrogenase/cyclohydrolase.

The bifunctional dehydrogenase/cyclohydrolase domain of the human trifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase catalyzes two sequential reactions with significant channeling of the intermediate, methenyltetrahydrofolate. Equilibrium dialysis established that a single, high-affinity NADP+ binding site exists per monomer of the dimeric enzyme. Kinetic characterization of NADP+ binding to the dehydrogenase using analogs as inhibitors demonstrated that affinity for this substrate is due almost exclusively to binding at the 2',5'-ADP subsite. The same structural specificities for binding are exhibited by these analogs in their effects on the cyclohydrolase. Both NADP+ and its 3-aminopyridine analog AADP partially inhibit the activity of the cyclohydrolase when assayed with added methenyltetrahydrofolate as substrate. However, under the same conditions, the cyclohydrolase is actually activated by 2',5'-ADP; activation requires the presence of the 5'-phosphate since 2'-AMP binds but does not activate. Nicotinamide ribose monophosphate (NMN) has no detectable effect either alone or in combination with 2',5'-ADP. The results are consistent with the existence of a shared dehydrogenase/cyclohydrolase active site proximal to the 2',5'-ADP subsite. NADP+ reduces the rate of the fully activated cyclohydrolase by 2-fold. Inhibition appears to be due to the loosely bound nicotinamide ring interacting with the common folate subsite, resulting in only partial inhibition by NADP+. The interaction of 2',5'-ADP with the cyclohydrolase suggests a potential role for this portion of the molecule in promoting the efficiency of the channeling of endogenously generated methenyltetrahydrofolate.

The nucleotide sequence of porcine formiminotransferase cyclodeaminase. Expression and purification from Escherichia coli.

We have isolated and characterized cDNA clones encoding the porcine liver octameric enzyme, 5-formiminotetrahydrofolate:L-glutamate N-formiminotransferase (EC 2.1.2.5)-formiminotetrahydrofolate cyclodeaminase (EC 4.3.1.4). The cDNA encodes a novel amino acid sequence of 541 residues which contains exact matches to two sequences derived by automated sequence analysis of CNBr cleavage fragments isolated from the porcine enzyme. The recombinant enzyme has been expressed as a soluble protein in Escherichia coli at levels 4-fold higher than those observed in liver, and is bifunctional, displaying both transferase and deaminase activities. With a calculated subunit molecular mass of 58,926 Da, it is similar in size to the enzyme isolated from porcine liver. Purification of the enzyme from E. coli involves chromatography on a novel polyglutamate column which might interact with the folylpolyglutamate binding site of the protein. The purified recombinant enzyme has a transferase specific activity of 39-41 units/mg/min.

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene.

The recombinant human bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is unique in its absolute requirement for Mg2+ and inorganic phosphate. Both ions affect the affinity of the enzyme for NAD and have no effect on the binding of methylenetetrahydrofolate. The NAD cofactor can be replaced by NADP with a much higher KM and lower VMAX. Kinetic investigation using NADP supports the role of Mg2+ in dinucleotide binding and illustrates that the 2'-phosphate can substitute for phosphate in this process. The human NAD-dependent bifunctional enzyme has a 44% amino acid sequence identity with the dehydrogenase-cyclohydrolase domain of the yeast mitochondrial NADP-dependent trifunctional enzyme encoded by the MIS1 gene, compared to 37% identity with the corresponding domain of the cytosolic trifunctional enzyme. The sequence comparison and the kinetic properties suggest that the NAD bifunctional enzyme is the mammalian homolog of the yeast mitochondrial trifunctional enzyme, which has evolved a unique use of inorganic phosphate to change its dinucleotide specificity from NADP to NAD. Its role is proposed to be in providing formyltetrahydrofolate for the synthesis of formylmethionyl transfer RNA required for the initiation of protein synthesis in mitochondria.