MTHFD2 (NMDMC) is a bifunctional mitochondrial enzyme of mitochondrial one-carbon (folate) metabolism. It is synthesized as a 350-residue precursor with an N-terminal mitochondrial transit peptide and localizes to the mitochondrial matrix. A single polypeptide carries two catalytic activities: an NAD-dependent methylenetetrahydrofolate dehydrogenase (EC 1.5.1.15) that oxidizes (6R)-5,10-methylene-tetrahydrofolate to 5,10-methenyl-tetrahydrofolate, and a methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) that hydrolyzes 5,10-methenyl-tetrahydrofolate to 10-formyl-tetrahydrofolate. Together these interconvert one-carbon-loaded folates, channeling one-carbon units derived from mitochondrial serine and glycine catabolism toward formate export and downstream biosynthesis (purine synthesis and mitochondrial protein synthesis). Unlike the cytosolic NADP-dependent trifunctional enzyme MTHFD1, the dehydrogenase of MTHFD2 is NAD-specific and uniquely requires an enzyme-magnesium complex together with inorganic phosphate to bind its NAD cofactor; it can use NADP only at markedly reduced efficiency. The enzyme is a homodimer. MTHFD2 is expressed mainly in developing and proliferating tissues and is strongly re-expressed in many cancers, where it is an actively pursued metabolic drug target.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004477 methenyltetrahydrofolate cyclohydrolase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Core molecular function. MTHFD2 is a bifunctional enzyme whose cyclohydrolase activity (EC 3.5.4.9) hydrolyzes 5,10-methenyl-THF to 10-formyl-THF. The phylogenetic (IBA) call is consistent with direct experimental evidence in human (PMID:16100107, PMID:8218174) and with the UniProt catalytic-activity annotation. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt Reaction=(6R)-5,10-methenyltetrahydrofolate + H2O = (6R)-10- file:human/MTHFD2/MTHFD2-uniprot.txt Belongs to the tetrahydrofolate dehydrogenase/cyclohydrolase family. |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Correct subcellular localization. MTHFD2 is a mitochondrial matrix enzyme, supported by UniProt (SUBCELLULAR LOCATION: Mitochondrion), HPA immunofluorescence (IDA), and the mitochondrial-proteome dataset. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion. |
| GO:0035999 tetrahydrofolate interconversion | IBA GO_REF:0000033 | ACCEPT | Summary: Core biological process. The two catalytic activities of MTHFD2 interconvert one-carbon-substituted tetrahydrofolates (5,10-methylene-THF to 5,10-methenyl-THF to 10-formyl-THF), which is precisely tetrahydrofolate interconversion. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt Reaction=(6R)-5,10-methylene-5,6,7,8-tetrahydrofolate + NAD(+) = (6R)- file:human/MTHFD2/MTHFD2-uniprot.txt Reaction=(6R)-5,10-methenyltetrahydrofolate + H2O = (6R)-10- |
| GO:0004487 methylenetetrahydrofolate dehydrogenase (NAD+) activity | IBA GO_REF:0000033 | ACCEPT | Summary: Core molecular function and the defining activity of MTHFD2. The dehydrogenase is NAD-specific (EC 1.5.1.15), distinguishing it from the NADP-dependent cytosolic enzyme. Well supported by direct experimental evidence (PMID:8218174 IDA, PMID:16100107 IMP). Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt Reaction=(6R)-5,10-methylene-5,6,7,8-tetrahydrofolate + NAD(+) = (6R)- PMID:16100107 requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. |
| GO:0004488 methylenetetrahydrofolate dehydrogenase (NADP+) activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Real but non-core activity. MTHFD2's dehydrogenase is NAD-specific; it can use NADP only at reduced efficiency (higher KM, ~8-fold lower Vmax). The NADP+ activity is a genuine measured in-vitro capability rather than the physiological function, so it is retained but marked non-core. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt Although its dehydrogenase activity is NAD-specific, it can |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Uninformative root-level catalytic term, fully subsumed by the specific dehydrogenase and cyclohydrolase molecular-function annotations. Marked as over-annotated rather than removed. |
| GO:0004477 methenyltetrahydrofolate cyclohydrolase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Correct cyclohydrolase activity, electronically inferred (RHEA:23700 / EC:3.5.4.9). Redundant with the experimental IDA annotation of the same term but consistent with it; accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt Reaction=(6R)-5,10-methenyltetrahydrofolate + H2O = (6R)-10- file:human/MTHFD2/MTHFD2-uniprot.txt Belongs to the tetrahydrofolate dehydrogenase/cyclohydrolase family. |
| GO:0004487 methylenetetrahydrofolate dehydrogenase (NAD+) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Correct NAD-dependent dehydrogenase activity, electronically inferred (RHEA:22892 / EC:1.5.1.15). Consistent with the experimental annotations; accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt EC=1.5.1.15 |
| GO:0004488 methylenetetrahydrofolate dehydrogenase (NADP+) activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronically inferred NADP+ dehydrogenase activity. As above, this is a genuine but minor, reduced-efficiency activity of an NAD-specific enzyme; retained as non-core. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt Although its dehydrogenase activity is NAD-specific, it can |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Correct localization from the UniProt subcellular-location vocabulary mapping; consistent with experimental evidence. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion. |
| GO:0005759 mitochondrial matrix | IEA GO_REF:0000117 | ACCEPT | Summary: More specific and correct localization: MTHFD2 acts in the mitochondrial matrix (soluble folate-metabolic enzyme with a cleaved transit peptide). Consistent with Reactome TAS annotations to the same term. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion. |
| GO:0046655 folic acid metabolic process | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Broadly correct but less informative than tetrahydrofolate interconversion / tetrahydrofolate metabolic process, which capture the enzyme's specific one-carbon folate role. Retained as non-core. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a high-throughput binary interactome (Y2H) screen; the recorded partner is NAGK (Q9UJ70-2). This is uninformative as a molecular function and does not describe MTHFD2's catalytic role. Marked over-annotated rather than removed (experimental IPI). |
| GO:0046655 folic acid metabolic process | TAS Reactome:R-HSA-196757 | KEEP AS NON CORE | Summary: Reactome pathway-level assignment (Metabolism of folate and pterines). Correct but general; the specific tetrahydrofolate interconversion / metabolic-process terms are preferred as core. Retained as non-core. |
| GO:0004477 methenyltetrahydrofolate cyclohydrolase activity | TAS Reactome:R-HSA-6801328 | ACCEPT | Summary: Core cyclohydrolase activity, curated by Reactome as the reaction converting 5,10-methenyl-THF to 10-formyl-THF. Accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt Reaction=(6R)-5,10-methenyltetrahydrofolate + H2O = (6R)-10- file:human/MTHFD2/MTHFD2-uniprot.txt Belongs to the tetrahydrofolate dehydrogenase/cyclohydrolase family. |
| GO:0004487 methylenetetrahydrofolate dehydrogenase (NAD+) activity | TAS Reactome:R-HSA-6801462 | ACCEPT | Summary: Core NAD-dependent dehydrogenase activity, curated by Reactome as the oxidation of 5,10-methylene-THF to 5,10-methenyl-THF. Accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt EC=1.5.1.15 |
| GO:0005739 mitochondrion | IDA GO_REF:0000052 | ACCEPT | Summary: Direct immunofluorescence evidence (HPA) for mitochondrial localization; consistent with all other evidence. Accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: MTHFD2 was detected in a high-confidence quantitative human mitochondrial proteome, corroborating mitochondrial localization. High-throughput evidence that agrees with the curated location; accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion. |
| GO:0004477 methenyltetrahydrofolate cyclohydrolase activity | IDA PMID:16100107 Magnesium and phosphate ions enable NAD binding to methylene... | ACCEPT | Summary: Direct experimental evidence for the cyclohydrolase activity of human MTHFD2 (structural/mutagenesis study of the bifunctional enzyme). Core molecular function; accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt Reaction=(6R)-5,10-methenyltetrahydrofolate + H2O = (6R)-10- file:human/MTHFD2/MTHFD2-uniprot.txt Belongs to the tetrahydrofolate dehydrogenase/cyclohydrolase family. |
| GO:0000287 magnesium ion binding | IMP PMID:16100107 Magnesium and phosphate ions enable NAD binding to methylene... | ACCEPT | Summary: Well supported. Mutagenesis and modeling show MTHFD2 uniquely requires a magnesium ion (with inorganic phosphate) to bind NAD and support dehydrogenase activity; mutants affecting the Mg-coordinating residues reduce activity and Mg affinity. Core supporting function. Supporting Evidence: PMID:16100107 the magnesium ion interacts with both Pi and the aspartate |
| GO:0004487 methylenetetrahydrofolate dehydrogenase (NAD+) activity | IMP PMID:16100107 Magnesium and phosphate ions enable NAD binding to methylene... | ACCEPT | Summary: Direct mutagenesis evidence for NAD-dependent dehydrogenase activity; point mutants (e.g. D168, R201, D225, R233) cause loss of NAD-dependent dehydrogenase activity. Core molecular function; accept. Supporting Evidence: PMID:16100107 It is unique in its absolute requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. |
| GO:0004488 methylenetetrahydrofolate dehydrogenase (NADP+) activity | IMP PMID:16100107 Magnesium and phosphate ions enable NAD binding to methylene... | KEEP AS NON CORE | Summary: Mutagenesis also affects NADP-dependent dehydrogenase activity, confirming the enzyme can use NADP. As this is the minor, reduced-efficiency activity of an NAD-specific enzyme, it is retained as non-core. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt Although its dehydrogenase activity is NAD-specific, it can |
| GO:0042301 phosphate ion binding | IMP PMID:16100107 Magnesium and phosphate ions enable NAD binding to methylene... | ACCEPT | Summary: Well supported. Inorganic phosphate is absolutely required to support dehydrogenase activity; mutagenesis identifies the Pi-binding residues (Arg166/Arg198 in model numbering) that adapt an NADP site for NAD binding. Core supporting function. Supporting Evidence: PMID:16100107 It is unique in its absolute requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-6801328 | ACCEPT | Summary: Correct specific matrix localization, curated by Reactome. Accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion. |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-6801462 | ACCEPT | Summary: Correct specific matrix localization, curated by Reactome (duplicate of the R-HSA-6801328 assignment). Accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion. |
| GO:0005576 extracellular region | HDA PMID:22664934 Comparison of tear protein levels in breast cancer patients ... | MARK AS OVER ANNOTATED | Summary: From a mass-spectrometry survey of tear fluid in breast cancer patients. MTHFD2 is a soluble mitochondrial matrix enzyme; its detection in a biofluid proteome reflects release from cells, not a functional extracellular localization. Retained (experimental HDA) but flagged as over-annotation of a bulk-detection secretome hit. |
| GO:0000287 magnesium ion binding | IDA PMID:8218174 NAD-dependent methylenetetrahydrofolate dehydrogenase-methen... | ACCEPT | Summary: Direct biochemical evidence that the enzyme has an absolute requirement for Mg2+, which affects NAD affinity. Core supporting function; accept. Supporting Evidence: PMID:8218174 unique in its absolute requirement for Mg2+ and inorganic phosphate. |
| GO:0004477 methenyltetrahydrofolate cyclohydrolase activity | ISS PMID:8218174 NAD-dependent methylenetetrahydrofolate dehydrogenase-methen... | ACCEPT | Summary: Cyclohydrolase activity inferred by sequence similarity (to MTHFD1, P09440). Consistent with, and superseded by, the direct experimental annotation of the same term. Accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt Reaction=(6R)-5,10-methenyltetrahydrofolate + H2O = (6R)-10- file:human/MTHFD2/MTHFD2-uniprot.txt Belongs to the tetrahydrofolate dehydrogenase/cyclohydrolase family. |
| GO:0004487 methylenetetrahydrofolate dehydrogenase (NAD+) activity | IDA PMID:8218174 NAD-dependent methylenetetrahydrofolate dehydrogenase-methen... | ACCEPT | Summary: Direct biochemical characterization of the recombinant human enzyme established the NAD-dependent methylenetetrahydrofolate dehydrogenase activity. Core molecular function; accept. Supporting Evidence: PMID:8218174 The recombinant human bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase |
| GO:0004488 methylenetetrahydrofolate dehydrogenase (NADP+) activity | IDA PMID:8218174 NAD-dependent methylenetetrahydrofolate dehydrogenase-methen... | KEEP AS NON CORE | Summary: The enzyme can use NADP in place of NAD but with much higher KM and lower Vmax; this is a real but minor activity of an NAD-specific enzyme. Retained as non-core. Supporting Evidence: PMID:8218174 The NAD cofactor can be replaced by NADP with a much higher KM and lower VMAX. |
| GO:0005739 mitochondrion | ISS PMID:8218174 NAD-dependent methylenetetrahydrofolate dehydrogenase-methen... | ACCEPT | Summary: Mitochondrial localization inferred by similarity (mammalian homolog of the yeast mitochondrial MIS1 enzyme). Consistent with the direct and high-throughput evidence for mitochondrial localization. Accept. Supporting Evidence: file:human/MTHFD2/MTHFD2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion. |
| GO:0042301 phosphate ion binding | IDA PMID:8218174 NAD-dependent methylenetetrahydrofolate dehydrogenase-methen... | ACCEPT | Summary: Direct evidence for an absolute inorganic-phosphate requirement affecting NAD binding. Core supporting function; accept. Supporting Evidence: PMID:8218174 unique in its absolute requirement for Mg2+ and inorganic phosphate. |
| GO:0046653 tetrahydrofolate metabolic process | IDA PMID:8218174 NAD-dependent methylenetetrahydrofolate dehydrogenase-methen... | ACCEPT | Summary: Core biological process. The enzyme's characterized activities place it in tetrahydrofolate (one-carbon folate) metabolism, providing 10-formyl-THF for downstream synthesis. Accept. Supporting Evidence: PMID:8218174 Its role is proposed to be in providing formyltetrahydrofolate for the synthesis of formylmethionyl transfer RNA |
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)