MTHFD1 is the cytoplasmic C-1-tetrahydrofolate synthase (C1-THF synthase), a 935-residue trifunctional enzyme that interconverts the one-carbon oxidation states of tetrahydrofolate (THF). A single polypeptide, functioning as a homodimer, carries three catalytic activities in two structural modules: an N-terminal dehydrogenase/cyclohydrolase (D/C) domain providing NADP-dependent 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) and 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) activities, and a larger C-terminal domain providing 10-formyltetrahydrofolate synthetase / formate--tetrahydrofolate ligase (EC 6.3.4.3) activity. Together these reactions interconvert 5,10-methylene-THF, 5,10-methenyl-THF and 10-formyl-THF, generating the one-carbon-loaded folate cofactors required for de novo purine synthesis (10-formyl-THF), thymidylate synthesis and serine/glycine and methionine metabolism (5,10-methylene-THF, and downstream 5-methyl-THF for homocysteine remethylation). MTHFD1 is a soluble cytosolic enzyme, ubiquitously expressed and enriched in liver. Human variants cause or predispose to disorders of folate one-carbon metabolism, including folate-sensitive neural tube defect susceptibility (notably the common R653Q allele, also linked to congenital heart defects) and an autosomal-recessive combined immunodeficiency with megaloblastic anemia (MTHFD1 deficiency, CIMAH).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: Cytosol is the correct and core subcellular location for cytoplasmic C1-THF synthase. Supported by phylogenetic inference and by direct HPA immunofluorescence, and consistent with UniProt subcellular location. Supporting Evidence: file:human/MTHFD1/MTHFD1-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0004477 methenyltetrahydrofolate cyclohydrolase activity | IBA GO_REF:0000033 | ACCEPT | Summary: One of the three core catalytic activities (EC 3.5.4.9), carried by the N-terminal D/C domain. Phylogenetic inference is corroborated by direct experimental IDA annotations. Supporting Evidence: PMID:10828945 we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase. |
| GO:0035999 tetrahydrofolate interconversion | IBA GO_REF:0000033 | ACCEPT | Summary: Accurately captures the core biological process: interconversion of one-carbon-substituted THF species. This is the UniProt-assigned pathway and is supported experimentally. Supporting Evidence: file:human/MTHFD1/MTHFD1-uniprot.txt PATHWAY: One-carbon metabolism; tetrahydrofolate interconversion. |
| GO:0004488 methylenetetrahydrofolate dehydrogenase (NADP+) activity | IBA GO_REF:0000033 | ACCEPT | Summary: One of the three core catalytic activities (EC 1.5.1.5), carried by the N-terminal D/C domain. Phylogenetic inference is corroborated by direct experimental IDA annotations. Supporting Evidence: PMID:10828945 we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase. |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Root-level catalytic activity term. Not incorrect, but far too general given the three specific, experimentally verified catalytic MF annotations (EC 1.5.1.5, 3.5.4.9, 6.3.4.3). Reason: Uninformative parent term subsumed by the specific catalytic activities; adds no biological information. |
| GO:0004329 formate-tetrahydrofolate ligase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Correct MF (EC 6.3.4.3), the synthetase activity of the C-terminal domain. Electronic annotation redundant with the experimental IDA/IMP annotations for the same term. |
| GO:0004477 methenyltetrahydrofolate cyclohydrolase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Correct MF (EC 3.5.4.9). Electronic annotation redundant with the experimental IDA annotations for the same term. |
| GO:0004488 methylenetetrahydrofolate dehydrogenase (NADP+) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Correct MF (EC 1.5.1.5). Electronic annotation redundant with the experimental IDA annotations for the same term. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: The C-terminal formyltetrahydrofolate synthetase domain binds and hydrolyzes ATP; ATP binding is consistent with the ligase reaction (formate + THF + ATP -> 10-formyl-THF + ADP + Pi). Supporting but non-core (a component of the ligase MF), so kept as non-core. Supporting Evidence: file:human/MTHFD1/MTHFD1-uniprot.txt Reaction=(6S)-5,6,7,8-tetrahydrofolate + formate + ATP |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Cytoplasm is correct but less precise than the experimentally supported cytosol annotation. Retained as a correct, more-general localization. |
| GO:0005829 cytosol | IEA GO_REF:0000117 | ACCEPT | Summary: Correct core location; electronic (ARBA) annotation redundant with the HPA IDA cytosol annotation. |
| GO:0046655 folic acid metabolic process | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Folate (folic acid) metabolism is the pathway MTHFD1 operates in. Correct but broad; redundant with the experimental IMP annotation. Downstream/pathway-level rather than the precise molecular function, so non-core. |
| GO:0005515 protein binding | IPI PMID:24169621 Elucidating novel hepatitis C virus-host interactions using ... | MARK AS OVER ANNOTATED | Summary: Bare 'protein binding' from a high-throughput HCV host-interactome MS/RNAi screen. This is uninformative about molecular function and the partner interaction has no established functional relevance to MTHFD1's enzymatic role. Reason: Non-specific protein-binding term with no functional interpretation; per policy, bare protein-binding IPI is flagged as over-annotation rather than removed. |
| GO:0001780 neutrophil homeostasis | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Ortholog-transferred phenotypic process from mouse Mthfd1. Plausible as a downstream consequence of impaired folate one-carbon metabolism (hematologic phenotypes in deficiency), but not a molecular function of the enzyme. Kept as non-core. |
| GO:0001843 neural tube closure | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Neural tube closure is a well-established folate-sensitive developmental process to which MTHFD1 variants contribute (NTD susceptibility). This is a downstream, whole-organism developmental role, not the core molecular function; retained as non-core. |
| GO:0006164 purine nucleotide biosynthetic process | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: MTHFD1 supplies 10-formyl-THF for de novo purine biosynthesis; this is a genuine downstream metabolic role supported experimentally (formate incorporation into DNA reduced by the R653Q variant). Pathway-level, so non-core. Supporting Evidence: PMID:18767138 indicating a disruption of de novo purine synthesis. |
| GO:0007507 heart development | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Ortholog-transferred developmental process; consistent with the R653Q association with congenital heart defects. Whole-organism developmental role, not a molecular function. |
| GO:0009257 10-formyltetrahydrofolate biosynthetic process | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: 10-formyl-THF biosynthesis is a direct product-forming role of both the synthetase and cyclohydrolase activities. Correct and supported experimentally; a specific metabolic output of the enzyme's core chemistry. Kept as non-core (downstream of the MF). |
| GO:0019346 transsulfuration | IEA GO_REF:0000107 | MARK AS OVER ANNOTATED | Summary: Transsulfuration is the homocysteine -> cystathionine -> cysteine pathway (CBS/CTH). MTHFD1 functions in folate one-carbon metabolism and homocysteine REMETHYLATION, not in transsulfuration. This ortholog-transferred annotation is mis-scoped to the wrong pathway. Reason: MTHFD1 does not act in the transsulfuration pathway; the term likely conflates folate/homocysteine metabolism with the distinct transsulfuration branch. IEA ortholog transfer, flagged as over-annotation. |
| GO:0035999 tetrahydrofolate interconversion | IEA GO_REF:0000120 | ACCEPT | Summary: Core process; electronic annotation redundant with the experimental IDA/IBA annotations. |
| GO:0061053 somite development | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Ortholog-transferred developmental process from mouse Mthfd1. Downstream, whole-organism developmental phenotype of impaired folate metabolism, not a molecular function. Non-core. |
| GO:0046655 folic acid metabolic process | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Folate metabolic process is the correct pathway. The acts_upstream_of qualifier (ISS from mouse) frames the enzyme as upstream of folate metabolism outcomes; correct but broad and redundant with the experimental IMP annotation. Non-core. |
| GO:0005829 cytosol | IDA GO_REF:0000052 | ACCEPT | Summary: Direct HPA immunofluorescence localizing MTHFD1 to the cytosol. This is the core, experimentally verified subcellular location. |
| GO:0004488 methylenetetrahydrofolate dehydrogenase (NADP+) activity | IDA PMID:1881876 Expression of active domains of a human folate-dependent tri... | ACCEPT | Summary: Direct experimental demonstration of the NADP-dependent methylenetetrahydrofolate dehydrogenase activity (EC 1.5.1.5) via independent expression of the active D/C domain. Core catalytic function. Supporting Evidence: PMID:1881876 Both domains formed active enzymes thereby demonstrating their ability to fold independently. |
| GO:0004329 formate-tetrahydrofolate ligase activity | IMP PMID:25633902 Characterization and review of MTHFD1 deficiency: four new p... | ACCEPT | Summary: Formate--tetrahydrofolate ligase / 10-formyl-THF synthetase activity (EC 6.3.4.3), inferred from the impaired one-carbon flux (reduced methionine formation from formate) in MTHFD1-deficiency patient cells. Core catalytic function. Supporting Evidence: PMID:25633902 Patient fibroblast studies revealed severely reduced methionine formation from [(14)C]-formate |
| GO:0046655 folic acid metabolic process | IMP PMID:25633902 Characterization and review of MTHFD1 deficiency: four new p... | KEEP AS NON CORE | Summary: MTHFD1 deficiency perturbs folate one-carbon flux (methionine formation from formate, responsive to folic/folinic acid), supporting involvement in folate metabolism. Broad, pathway-level process, so non-core. Supporting Evidence: PMID:25633902 was responsive to folic and folinic acid. |
| GO:0071265 L-methionine biosynthetic process | IMP PMID:25633902 Characterization and review of MTHFD1 deficiency: four new p... | KEEP AS NON CORE | Summary: MTHFD1 acts upstream of methionine biosynthesis: patient fibroblasts show severely reduced methionine formation from [14C]-formate. This is a genuine downstream metabolic consequence (via one-carbon supply for homocysteine remethylation), not the core molecular function. Supporting Evidence: PMID:25633902 Patient fibroblast studies revealed severely reduced methionine formation from [(14)C]-formate |
| GO:0004329 formate-tetrahydrofolate ligase activity | IDA PMID:18767138 The MTHFD1 p.Arg653Gln variant alters enzyme function and in... | ACCEPT | Summary: Direct biochemical measurement of the formyltetrahydrofolate synthetase / formate--THF ligase activity (EC 6.3.4.3) of the purified enzyme. Core catalytic function. Supporting Evidence: file:human/MTHFD1/MTHFD1-uniprot.txt Reaction=(6S)-5,6,7,8-tetrahydrofolate + formate + ATP |
| GO:0004477 methenyltetrahydrofolate cyclohydrolase activity | IDA PMID:10828945 Structures of three inhibitor complexes provide insight into... | ACCEPT | Summary: Direct structural/mechanistic evidence for the methenyltetrahydrofolate cyclohydrolase activity (EC 3.5.4.9) of the human D/C domain. Core catalytic function. Supporting Evidence: PMID:10828945 we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase. |
| GO:0004488 methylenetetrahydrofolate dehydrogenase (NADP+) activity | IDA PMID:10828945 Structures of three inhibitor complexes provide insight into... | ACCEPT | Summary: Direct structural/mechanistic evidence for the NADP-dependent dehydrogenase activity (EC 1.5.1.5) of the human D/C domain. Core catalytic function. Supporting Evidence: PMID:10828945 we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase. |
| GO:0004488 methylenetetrahydrofolate dehydrogenase (NADP+) activity | IDA PMID:18767138 The MTHFD1 p.Arg653Gln variant alters enzyme function and in... | ACCEPT | Summary: Direct measurement of the methylenetetrahydrofolate dehydrogenase (NADP+) activity of the purified enzyme (EC 1.5.1.5). Core catalytic function; redundant with the other IDA annotations to the same term. |
| GO:0006164 purine nucleotide biosynthetic process | IMP PMID:18767138 The MTHFD1 p.Arg653Gln variant alters enzyme function and in... | KEEP AS NON CORE | Summary: The R653Q variant reduces formate incorporation into DNA in Mthfd1-knockout cells, demonstrating that MTHFD1 acts upstream of de novo purine synthesis by supplying 10-formyl-THF. Downstream metabolic role, non-core. Supporting Evidence: PMID:18767138 indicating a disruption of de novo purine synthesis. |
| GO:0035999 tetrahydrofolate interconversion | IDA PMID:10828945 Structures of three inhibitor complexes provide insight into... | ACCEPT | Summary: Core biological process: interconversion of one-carbon-substituted THF forms, directly demonstrated by the characterized dehydrogenase/cyclohydrolase chemistry. Supporting Evidence: file:human/MTHFD1/MTHFD1-uniprot.txt PATHWAY: One-carbon metabolism; tetrahydrofolate interconversion. |
| GO:0004329 formate-tetrahydrofolate ligase activity | IDA PMID:1881876 Expression of active domains of a human folate-dependent tri... | ACCEPT | Summary: Direct evidence for the formyltetrahydrofolate synthetase / formate--THF ligase activity (EC 6.3.4.3) from independent expression of the active synthetase domain. Core catalytic function. Supporting Evidence: PMID:1881876 Both domains formed active enzymes thereby demonstrating their ability to fold independently. |
| GO:0004477 methenyltetrahydrofolate cyclohydrolase activity | IDA PMID:1881876 Expression of active domains of a human folate-dependent tri... | ACCEPT | Summary: Direct evidence for the methenyltetrahydrofolate cyclohydrolase activity (EC 3.5.4.9) from the active D/C domain. Core catalytic function. Supporting Evidence: PMID:1881876 Both domains formed active enzymes thereby demonstrating their ability to fold independently. |
| GO:0009257 10-formyltetrahydrofolate biosynthetic process | IDA PMID:1881876 Expression of active domains of a human folate-dependent tri... | KEEP AS NON CORE | Summary: 10-formyl-THF is the product of both the synthetase (formate + THF) and cyclohydrolase (5,10-methenyl-THF) reactions carried by MTHFD1. Correct downstream metabolic output; non-core. |
| GO:0035999 tetrahydrofolate interconversion | IDA PMID:1881876 Expression of active domains of a human folate-dependent tri... | ACCEPT | Summary: Core process, directly supported by demonstration of the three interconverting activities. |
| GO:0001843 neural tube closure | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: ISS ortholog transfer for the folate-sensitive developmental role in neural tube closure. Downstream developmental process (NTD susceptibility), non-core. |
| GO:0048702 embryonic neurocranium morphogenesis | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Ortholog-transferred embryonic craniofacial morphogenesis role. A whole-organism developmental phenotype of folate one-carbon insufficiency, not a molecular function. Non-core. |
| GO:0048703 embryonic viscerocranium morphogenesis | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Ortholog-transferred embryonic craniofacial morphogenesis role. Whole-organism developmental phenotype, not a molecular function. Non-core. |
| GO:0004329 formate-tetrahydrofolate ligase activity | ISS GO_REF:0000024 | ACCEPT | Summary: Correct core MF (EC 6.3.4.3) transferred by sequence similarity from mouse Mthfd1; redundant with the experimental IDA/IMP annotations. |
| GO:0006164 purine nucleotide biosynthetic process | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Purine biosynthesis role transferred from mouse Mthfd1; supported experimentally in human cells. Downstream metabolic process, non-core. |
| GO:0007507 heart development | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Ortholog-transferred heart-development role; consistent with the R653Q congenital heart defect association. Whole-organism developmental process, non-core. |
| GO:0009257 10-formyltetrahydrofolate biosynthetic process | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Correct downstream metabolic output (10-formyl-THF), transferred by similarity; redundant with the IDA annotation. Non-core. |
| GO:0035999 tetrahydrofolate interconversion | ISS GO_REF:0000024 | ACCEPT | Summary: Core process transferred by similarity; redundant with the experimental IDA/IBA annotations. |
| GO:0061053 somite development | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Ortholog-transferred somite-development role. Whole-organism developmental phenotype, not a molecular function. Non-core. |
| GO:0070062 extracellular exosome | HDA PMID:23533145 In-depth proteomic analyses of exosomes isolated from expres... | KEEP AS NON CORE | Summary: Detection in exosome proteomics. MTHFD1 is a soluble cytosolic enzyme; presence in exosome fractions is incidental/high-throughput and does not represent its functional location. Kept as non-core rather than removed (HDA). |
| GO:0016020 membrane | HDA PMID:19946888 Defining the membrane proteome of NK cells. | KEEP AS NON CORE | Summary: Detection in a membrane-proteome MS study. MTHFD1 is cytosolic and lacks membrane-spanning features; membrane co-fractionation is a common artifact. Non-core (HDA). |
| GO:0070062 extracellular exosome | HDA PMID:19056867 Large-scale proteomics and phosphoproteomics of urinary exos... | KEEP AS NON CORE | Summary: Incidental detection in urinary-exosome proteomics; not the functional location of this cytosolic enzyme. Non-core (HDA). |
| GO:0070062 extracellular exosome | HDA PMID:20458337 MHC class II-associated proteins in B-cell exosomes and pote... | KEEP AS NON CORE | Summary: Incidental detection in B-cell exosome proteomics; not the functional location. Non-core (HDA). |
| GO:0005829 cytosol | TAS Reactome:R-HSA-200644 | ACCEPT | Summary: Reactome-curated cytosolic localization for the dehydrogenase reaction; correct core location. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-200661 | ACCEPT | Summary: Reactome-curated cytosolic localization; correct core location. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-200711 | ACCEPT | Summary: Reactome-curated cytosolic localization for the synthetase reaction; correct core location. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-200718 | ACCEPT | Summary: Reactome-curated cytosolic localization for the dehydrogenase reaction; correct core location. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-200740 | ACCEPT | Summary: Reactome-curated cytosolic localization for the cyclohydrolase reaction; correct core location. |
| GO:0005739 mitochondrion | TAS PMID:3528153 Purification and characterization of a mitochondrial isozyme... | MARK AS OVER ANNOTATED | Summary: Mislocalization. The cited paper (PMID:3528153) characterizes the YEAST MITOCHONDRIAL C1-THF synthase isozyme (the paralog corresponding to human MTHFD2/MTHFD1L) and explicitly states that cytoplasmic C1-THF synthase is NOT mitochondrial. Human cytoplasmic MTHFD1 is cytosolic; the mitochondrial one-carbon enzymes are the separate MTHFD2/MTHFD2L/MTHFD1L gene products. Reason: Mitochondrial location is incorrect for the cytoplasmic MTHFD1 and derives from a paper on a distinct mitochondrial isozyme. Retained (not removed) as a curator TAS annotation, flagged as a mislocalization/over-annotation. Supporting Evidence: PMID:3528153 Cell fractionation experiments show that the isozyme, but not C1-tetrahydrofolate |
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Download this section (compressed HTML)Q: Are any of the three MTHFD1 catalytic activities differentially rate-limiting for specific one-carbon outputs (purine vs thymidylate vs methionine) under physiological or folate-depleted conditions in humans?
Q: Does MTHFD1 participate in substrate channeling or transient metabolon assembly with other cytosolic folate enzymes (e.g. SHMT1, TYMS, ATIC), and does the R653Q allele perturb this?
Experiment: Domain-selective activity assays and formate-flux (13C-formate) tracing in MTHFD1-null cells reconstituted with wild-type versus disease variants (S49F, R173C, R653Q) to quantify each activity's contribution to purine, thymidylate, and methionine synthesis.
Experiment: Structural determination of the full-length human MTHFD1 homodimer (currently only the D/C domain is crystallized) to define inter-domain organization and potential channeling of the C-terminal synthetase domain.
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