Binding Modes of Xanthine-Derived Selective Allosteric Site Inhibitors of MTHFD2.

Methylenetetrahydrofolate dehydrogenase (MTHFD2) is a mitochondrial enzyme involved in 1 C metabolism that is upregulated in various cancer cells, but absent in normal proliferating cells. Xanthine derivatives are the first selective inhibitors of MTHFD2 which bind to its allosteric site. Xanthine derivatives (including the co-crystallized inhibitors) were herein interrogated by molecular/induced-fit docking, MM-GBSA binding free energy calculations and molecular dynamics simulations in both MTHFD2 and MTHFD1 (a close homolog expressed in healthy cells). The gained insights from our in silico protocol allowed us to study binding mode, key protein-ligand interactions and dynamic movement of the allosteric inhibitors, correlating with their experimental binding affinities, biological activities and selectivity for MTHFD2. The reported conformational changes with MTHFD2 upon binding of xanthine derivatives were furthermore evaluated and confirmed by RMSF analyses of the MD simulation trajectories. The results reported herein are expected to benefit in the rational design of selective MTHFD2 allosteric inhibitors.

The First Structure of Human MTHFD2L and Its Implications for the Development of Isoform-Selective Inhibitors.

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a mitochondrial 1-carbon metabolism enzyme, which is an attractive anticancer drug target as it is highly upregulated in cancer but is not expressed in healthy adult cells. Selective MTHFD2 inhibitors could therefore offer reduced side-effects during treatment, which are common with antifolate drugs that target other 1C-metabolism enzymes. This task is challenging however, as MTHFD2 shares high sequence identity with the constitutively expressed isozymes cytosolic MTHFD1 and mitochondrial MTHFD2L. In fact, one of the most potent MTHFD2 inhibitors reported to date, TH7299, is actually more active against MTHFD1 and MTHFD2L. While structures of MTHFD2 and MTHFD1 exist, no MTHFD2L structures are available. We determined the first structure of MTHFD2L and its complex with TH7299, which reveals the structural basis for its highly potent MTHFD2L inhibition. Detailed analysis of the MTHFD2L structure presented here clearly highlights the challenges associated with developing truly isoform-selective MTHFD2 inhibitors.

The catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2).

Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a new drug target that is expressed in cancer cells but not in normal adult cells, which provides an Achilles heel to selectively kill cancer cells. Despite the availability of crystal structures of MTHFD2 in the inhibitor- and cofactor-bound forms, key information is missing due to technical limitations, including (a) the location of absolutely required Mg2+ ion, and (b) the substrate-bound form of MTHFD2. Using computational modeling and simulations, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate [Formula: see text], while [Formula: see text] together with Arg233 stabilize the inorganic phosphate (Pi); (ii) Asp168 and three water molecules coordinate [Formula: see text], and [Formula: see text] further stabilizes Pi by forming a hydrogen bond with two oxygens of Pi; (iii) Arg201 directly coordinates the Pi; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of [Formula: see text], [Formula: see text] and Pi. Our computational study at the empirical valence bond level allowed us also to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution connected to the reorganization of the surrounding water molecules which further facilitates the subsequent cyclohydrolase activity. The cyclohydrolase activity then drives the hydration of the imidazoline ring and the ring opening in a concerted way. Furthermore, we have uncovered that two key residues, Ser197/Arg233, are important factors in determining the cofactor (NADP+/NAD+) preference of the dehydrogenase activity. Our work sheds new light on the structural and kinetic framework of MTHFD2, which will be helpful to design small molecule inhibitors that can be used for cancer treatment.

Crystal structure of Thermus thermophilus methylenetetrahydrofolate dehydrogenase and determinants of thermostability.

The elucidation of mechanisms behind the thermostability of proteins is extremely important both from the theoretical and applied perspective. Here we report the crystal structure of methylenetetrahydrofolate dehydrogenase (MTHFD) from Thermus thermophilus HB8, a thermophilic model organism. Molecular dynamics trajectory analysis of this protein at different temperatures (303 K, 333 K and 363 K) was compared with homologous proteins from the less temperature resistant organism Thermoplasma acidophilum and the mesophilic organism Acinetobacter baumannii using several data reduction techniques like principal component analysis (PCA), residue interaction network (RIN) analysis and rotamer analysis. These methods enabled the determination of important residues for the thermostability of this enzyme. The description of rotamer distributions by Gini coefficients and Kullback-Leibler (KL) divergence both revealed significant correlations with temperature. The emerging view seems to indicate that a static salt bridge/charged residue network plays a fundamental role in the temperature resistance of Thermus thermophilus MTHFD by enhancing both electrostatic interactions and entropic energy dispersion. Furthermore, this analysis uncovered a relationship between residue mutations and evolutionary pressure acting on thermophilic organisms and thus could be of use for the design of future thermostable enzymes.

Molecular structure of a 5,10-methylenetetrahydrofolate dehydrogenase from the silkworm Bombyx mori.

The enzyme 5,10-methylenetetrahydrofolate dehydrogenase (MTHFD) is essential for the production of certain amino acids (glycine, serine, and methionine) and nucleic acids (thymidylate and purine). Here, we identified a cDNA encoding this enzyme from the silkworm Bombyx mori. The recombinant B. mori MTHFD (bmMTHFD) expressed in Escherichia coli recognized 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate as substrate in the presence of NADP + as well as NAD +. The bmMTHFD structure was determined at a resolution of 1.75 Å by X-ray crystallography. Site-directed mutagenesis indicated that the amino acid residue Tyr49 contributed to its catalytic activity. Our findings provide insight into the mechanism underlying the activity of MTHFD from B. mori and potentially other insects and may therefore facilitate the development of inhibitors specific to MTHFD as insecticides.

An assessment of three human methylenetetrahydrofolate dehydrogenase/cyclohydrolase-ligand complexes following further refinement.

The enzymes involved in folate metabolism are key drug targets for cell-growth modulation, and accurate crystallographic structures provide templates to be exploited for structure-based ligand design. In this context, three ternary complex structures of human methylenetetrahydrofolate dehydrogenase/cyclohydrolase have been published [Schmidt et al. (2000), Biochemistry, 39, 6325-6335] and potentially represent starting points for the development of new antifolate inhibitors. However, an inspection of the models and the deposited data revealed deficiencies and raised questions about the validity of the structures. A number of inconsistencies relating to the publication were also identified. Additional refinement was carried out with the deposited data, seeking to improve the models and to then validate the complex structures or correct the record. In one case, the inclusion of the inhibitor in the structure was supported and alterations to the model allowed details of enzyme-ligand interactions to be described that had not previously been discussed. For one weak inhibitor, the data suggested that the ligand may adopt two poses in the binding site, both with few interactions with the enzyme. In the third case, that of a potent inhibitor, inconsistencies were noted in the assignment of the chemical structure and there was no evidence to support the inclusion of the ligand in the active site.

Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor.

To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD+ and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment. Cancer Res; 77(4); 937-48. ©2017 AACR.

Physiological role of FolD (methylenetetrahydrofolate dehydrogenase), FchA (methenyltetrahydrofolate cyclohydrolase) and Fhs (formyltetrahydrofolate synthetase) from Clostridium perfringens in a heterologous model of Escherichia coli.

Most organisms possess bifunctional FolD [5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrahdrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (EcoFolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (CpeFolD) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of CpeFolD is about five times more efficient than that of EcoFolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is ∼ 10 times more efficient than that of EcoFolD. Kinetic parameters for CpeFhs were also determined for utilization of all of its substrates. Both CpeFolD and CpeFchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of CpeFolD and CpeFchA is also necessary to rescue an E. coli folD deletion strain (harbouring CpeFhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.

Characterization of 2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl Ureido Based Inhibitors of Trypanosoma brucei FolD and Testing for Antiparasitic Activity.

The bifunctional enzyme N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclo hydrolase (FolD) is essential for growth in Trypanosomatidae. We sought to develop inhibitors of Trypanosoma brucei FolD (TbFolD) as potential antiparasitic agents. Compound 2 was synthesized, and the molecular structure was unequivocally assigned through X-ray crystallography of the intermediate compound 3. Compound 2 showed an IC50 of 2.2 µM, against TbFolD and displayed antiparasitic activity against T. brucei (IC50 49 µM). Using compound 2, we were able to obtain the first X-ray structure of TbFolD in the presence of NADP(+) and the inhibitor, which then guided the rational design of a new series of potent TbFolD inhibitors.

Mitochondrial Methylenetetrahydrofolate Dehydrogenase (MTHFD2) Overexpression Is Associated with Tumor Cell Proliferation and Is a Novel Target for Drug Development.

Rapidly proliferating tumors attempt to meet the demands for nucleotide biosynthesis by upregulating folate pathways that provide the building blocks for pyrimidine and purine biosynthesis. In particular, the key role of mitochondrial folate enzymes in providing formate for de novo purine synthesis and for providing the one-carbon moiety for thymidylate synthesis has been recognized in recent studies. We have shown a significant correlation between the upregulation of the mitochondrial folate enzymes, high proliferation rates, and sensitivity to the folate antagonist methotrexate (MTX). Burkitt lymphoma and diffuse large-cell lymphoma tumor specimens have the highest levels of mitochondrial folate enzyme expression and are known to be sensitive to treatment with MTX. A key enzyme upregulated in rapidly proliferating tumors but not in normal adult cells is the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase (MTHFD2). This perspective outlines the rationale for specific targeting of MTHFD2 and compares known and generated crystal structures of MTHFD2 and closely related enzymes as a molecular basis for developing therapeutic agents against MTHFD2. Importantly, the development of selective inhibitors of mitochondrial methylenetetrahydrofolate dehydrogenase is expected to have substantial activity, and this perspective supports the investigation and development of MTHFD2 inhibitors for anticancer therapy.

Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis.

An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been identified. The proband presented with SCID, megaloblastic anemia, and neurologic abnormalities, but the causal metabolic impairment is unknown. SCID has been associated with impaired purine nucleotide metabolism, whereas megaloblastic anemia has been associated with impaired de novo thymidylate (dTMP) biosynthesis. MTHFD1 functions to condense formate with tetrahydrofolate and serves as the primary entry point of single carbons into folate-dependent one-carbon metabolism in the cytosol. In this study, we examined the impact of MTHFD1 loss of function on folate-dependent purine, dTMP, and methionine biosynthesis in fibroblasts from the proband with MTHFD1 deficiency. The flux of formate incorporation into methionine and dTMP was decreased by 90% and 50%, respectively, whereas formate flux through de novo purine biosynthesis was unaffected. Patient fibroblasts exhibited enriched MTHFD1 in the nucleus, elevated uracil in DNA, lower rates of de novo dTMP synthesis, and increased salvage pathway dTMP biosynthesis relative to control fibroblasts. These results provide evidence that impaired nuclear de novo dTMP biosynthesis can lead to both megaloblastic anemia and SCID in MTHFD1 deficiency.

An NTD-associated polymorphism in the 3' UTR of MTHFD1L can affect disease risk by altering miRNA binding.

Maternal folate levels and polymorphisms in folate-related genes are known risk factors for neural tube defects (NTDs). SNPs in the mitochondrial folate gene MTHFD1L are associated with the risk of NTDs. We investigated whether different alleles of SNP rs7646 in the 3' UTR of MTHFD1L can be differentially regulated by microRNAs affecting MTHFD1L expression. We previously reported that miR-9 targets MTHFD1L and now we identify miR-197 as an additional miRNA regulator. Both of these miRNAs have predicted binding sites in the MTHFD1L 3' UTR in the region containing SNP rs7646. We have determined whether the alleles of SNP rs7646 (A/G) and miRNA expression levels affect miRNA binding preferences for the MTHFD1L 3' UTR and consequently MTHFD1L expression. Our results indicate that miR-9 and miR-197 specifically downregulate MTHFD1L levels in HEK293 and MCF-7 cells and that SNPrs7646 significantly affects miR-197 binding affinity to the MTHFD1L 3' UTR, causing more efficient posttranscriptional gene repression in the presence of the allele that is associated with increased risk of NTDs. These results reveal that the association of SNP rs7646 and NTD risk involves differences in microRNA regulation and, highlights the importance of genotype-dependent differential microRNA regulation in relation to human disease risk.

Acinetobacter baumannii FolD ligand complexes --potent inhibitors of folate metabolism and a re-evaluation of the structure of LY374571.

The bifunctional N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DHCH or FolD), which is widely distributed in prokaryotes and eukaryotes, is involved in the biosynthesis of folate cofactors that are essential for growth and cellular development. The enzyme activities represent a potential antimicrobial drug target. We have characterized the kinetic properties of FolD from the Gram-negative pathogen Acinetobacter baumanni and determined high-resolution crystal structures of complexes with a cofactor and two potent inhibitors. The data reveal new details with respect to the molecular basis of catalysis and potent inhibition. A unexpected finding was that our crystallographic data revealed a different structure for LY374571 (an inhibitor studied as an antifolate) than that previously published. The implications of this observation are discussed.

The crystal structure of Leishmania major N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclohydrolase and assessment of a potential drug target.

Three enzyme activities in the protozoan Leishmania major, namely N(5),N(10)-methylenetetrahydrofolate dehydrogenase/N(5),N(10)-methenyltetrahydrofolate cyclohydrolase (DHCH) and N(10)-formyltetrahydrofolate ligase (FTL) produce the essential intermediate N(10)-formyltetrahydrofolate. Although trypanosomatids possess at least one functional DHCH, the same is not true for FTL, which is absent in Trypanosoma brucei. Here, we present the 2.7 Å resolution crystal structure of the bifunctional apo-DHCH from L. major, which is a potential drug target. Sequence alignments show that the cytosolic enzymes found in trypanosomatids share a high level of identity of approximately 60%. Additionally, residues that interact and participate in catalysis in the human homologue are conserved amongst trypanosomatid sequences and this may complicate attempts to derive potent, parasite specific DHCH inhibitors.

Crystallization and preliminary X-ray diffraction analysis of 5,10- methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum DSM 1728.

The methylenetetrahydrofolate dehydrogenase/ cyclohydrolase (MTHFDC) from the thermoacidophilic archaeon Thermoplasma acidophilum is a 30.6 kDa molecular-mass enzyme that sequentially catalyzes the conversion of formyltetrahydrofolate to methylenetetrahydrofolate, with a preference for NADP as a cofactor, rather than NAD. In order to elucidate the functional and structural features of MTHFDC from archaeons at a molecular level, it was overexpressed in Escherichia coli and crystallized in the presence of its cofactor, NADP, at 295 K using polyethylene glycol (PEG) 4000 as a precipitant. The crystal is a member of the monoclinic space group P21, with the following unit cell parameters: a=66.333 A, b=52.868 A, c=86.099 A, and beta= 97.570o, and diffracts to a resolution of at least 2.40 A at the synchrotron. Assuming a dimer in the crystallographic asymmetric unit, the calculated Matthews parameter (VM) was 2.44 A3/Da and the solvent content was 49.7%.

Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase.

A human mitochondrial isozyme of C1-tetrahydrofolate (THF) synthase was previously identified by its similarity to the human cytoplasmic C1-THF synthase. All C1-THF synthases characterized to date, from yeast to human, are trifunctional, containing the activities of 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. Here we report on the enzymatic characterization of the recombinant human mitochondrial isozyme. Enzyme assays of purified human mitochondrial C1-THF synthase protein revealed only the presence of 10-formyl-THF synthetase activity. Gel filtration and crosslinking studies indicated that human mitochondrial C1-THF synthase exists as a homodimer in solution. Steady-state kinetic characterization of the 10-formyl-THF synthetase activity was performed using (6R,S)-H4-PteGlu1, (6R,S)-H4-PteGlu3, and (6R,S)-H4-PteGlu5 substrates. The (6R,S)-H4-PteGlun Km dropped from greater than 500 microM for the monoglutamate to 15 microM and 3.6 microM for the tri- and pentaglutamates, respectively. The Km values for formate and ATP also are lowered when THF polyglutamates are used. The formate Km dropped 79-fold and the ATP Km dropped more than 5-fold when (6R,S)-H4-PteGlu5 was used as the substrate in place of (6R,S)-H4-PteGlu1.

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.

Disruption of the mthfd1 gene reveals a monofunctional 10-formyltetrahydrofolate synthetase in mammalian mitochondria.

The Mthfd1 gene encoding the cytoplasmic methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase enzyme (DCS) was inactivated in embryonic stem cells. The null embryonic stem cells were used to generate spontaneously immortalized fibroblast cell lines that exhibit the expected purine auxotrophy. Elimination of these cytoplasmic activities allowed for the accurate assessment of similar activities encoded by other genes in these cells. A low level of 10-formyltetrahydrofolate synthetase was detected and was shown to be localized to mitochondria. However, NADP-dependent methylenetetrahydrofolate dehydrogenase activity was not detected. Northern blot analysis suggests that a recently identified mitochondrial DCS (Prasannan, P., Pike, S., Peng, K., Shane, B., and Appling, D. R. (2003) J. Biol. Chem. 278, 43178-43187) is responsible for the synthetase activity. The lack of NADP-dependent dehydrogenase activity suggests that this RNA may encode a monofunctional synthetase. Moreover, examination of the primary structure of this novel protein revealed mutations in key residues required for dehydrogenase and cyclohydrolase activities. This monofunctional synthetase completes the pathway for the production of formate from formyltetrahydrofolate in the mitochondria in our model of mammalian one-carbon folate metabolism in embryonic and transformed cells.

A novel mitochondrial C1-tetrahydrofolate synthetase is upregulated in human colon adenocarcinoma.

To seek the genes involved in the development of colorectal cancer, we analyzed the microarray gene expression profiles of human normal and cancerous colon tissues using the BioExpress database platform. Through the analysis we found one gene named DKFZp586G1517 that was upregulated in colon adenocarcinomas. The full-length cDNA of the DKFZp586G1517 cloned by polymerase chain reaction (PCR) encodes a protein with 978 amino acids, which is homologous to the human cytosolic C(1)-tetrahydrofolate synthetase and contains a mitochondrial target signal at N-terminus. The gene product expressed in 293 cells was localized in mitochondria and processed at the predicted signal cleavage site, supporting the idea that DKFZp586G1517 is a novel mitochondrial C(1)-tetrahydrofolate synthetase (mtC(1)-THFS). The overexpression of mtC(1)-THFS in 293 cells stimulated the colony formation. These results suggest that mtC(1)-THFS may participate in the progression of colorectal cancer by conferring growth advantage and could be a new molecular target for cancer therapy.

Human mitochondrial C1-tetrahydrofolate synthase: gene structure, tissue distribution of the mRNA, and immunolocalization in Chinese hamster ovary calls.

C1-tetrahydrofolate (THF) synthase is a trifunctional enzyme found in eukaryotes that contains the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The cytoplasmic isozyme of C1-THF synthase is well characterized in a number of mammals, including humans; but a mitochondrial isozyme has been previously identified only in the yeast Saccharomyces. Here, we report the identification and characterization of the human gene encoding a functional mitochondrial C1-THF synthase. The gene spans 236 kilobase pairs on chromosome 6 and consists of 28 exons plus one alternative exon. The gene encodes a protein of 978 amino acids, including an N-terminal mitochondrial targeting sequence. The mitochondrial isozyme is 61% identical to the human cytoplasmic isozyme. Expression of the gene was detected in most human tissues, but transcripts were highest in placenta, thymus, and brain. Two mRNAs were detected, a 3.6-kb transcript and a 1.1-kb transcript, and both transcripts were observed in varying ratios in each tissue. The shorter transcript results from an alternative splicing event, where exon 7 is spliced to exon 8a instead of exon 8. Exon 8a is derived from an exonized Alu sequence, sharing no homology with exon 8 of the long transcript, and encodes just 15 amino acids followed by a stop codon and a polyadenylation signal. This short transcript potentially encodes a bifunctional enzyme lacking 10-formyl-THF synthetase activity. Both transcripts initiate at the same 5'-site, 107 nucleotides up-stream of the ATG start codon. The full-length (2934 bp) cDNA fused to a C-terminal V5 epitope tag was expressed in Chinese hamster ovary cells. Immunoblots of subfractionated cells revealed a 107-kDa protein only in the mitochondrial fractions of these cells, confirming the mitochondrial localization of the protein. Yeast cells expressing the full-length human cDNA exhibited elevated 10-formyl-THF synthetase activity, confirming its identification as the human mitochondrial C1-THF synthase.