MTHFR

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0004489 methylenetetrahydrofolate reductase [NAD(P)H] activity	IBA GO_REF:0000033	ACCEPT	Summary: Core catalytic molecular function. MTHFR reduces 5,10-methylene-THF to 5-methyl-THF using NAD(P)H as electron donor and FAD as cofactor; the phylogenetic (IBA) inference is fully consistent with direct human experimental evidence and is the primary function of the gene. Reason: This is the well-established, experimentally verified catalytic activity of MTHFR and is correctly captured at the right level of specificity by the pan-family IBA call. Supporting Evidence: PMID:29891918 a reaction requiring FAD as a cofactor and NADPH as an electron donor
GO:0071265 L-methionine biosynthetic process	IBA GO_REF:0000033	ACCEPT	Summary: MTHFR generates the CH3-THF methyl donor that methionine synthase uses to remethylate homocysteine to methionine, so the enzyme is upstream of methionine biosynthesis. Supported experimentally by yeast complementation (human MTHFR rescues the methionine auxotrophy of a met11 deletion). Reason: Well-supported involvement in methionine biosynthesis/remethylation; the IBA call matches human and cross-species experimental evidence. Supporting Evidence: PMID:10551815 complement its methionine auxotrophic phenotype in vivo
GO:0005829 cytosol	IBA GO_REF:0000033	ACCEPT	Summary: MTHFR is a soluble cytosolic enzyme of the folate/methionine cycles; the cytosolic localization is consistent across IBA, IEA and Reactome (TAS) sources. Reason: Correct subcellular localization for this cytosolic one-carbon-metabolism enzyme.
GO:0035999 tetrahydrofolate interconversion	IBA GO_REF:0000033	ACCEPT	Summary: MTHFR interconverts folate one-carbon forms (5,10-methylene-THF to 5-methyl-THF) within the folate cycle; UniProt assigns the pathway "One-carbon metabolism; tetrahydrofolate interconversion". Reason: Accurately describes the folate-cycle process in which the catalytic activity participates. Supporting Evidence: PMID:29891918 a reaction requiring FAD as a cofactor and NADPH as an electron donor
GO:0071949 FAD binding	IBA GO_REF:0000033	ACCEPT	Summary: MTHFR is a flavoprotein that binds FAD as an essential redox cofactor; confirmed by the human crystal structure and biochemical FAD-responsiveness of disease variants. Reason: FAD binding is a genuine, experimentally supported molecular function of MTHFR (see also the GO:0050660 IDA below). Supporting Evidence: PMID:29891918 a reaction requiring FAD as a cofactor and NADPH as an electron donor
GO:0004489 methylenetetrahydrofolate reductase [NAD(P)H] activity	IEA GO_REF:0000120	ACCEPT	Summary: Electronic (multi-method) assignment of the core catalytic activity, duplicating the IBA/IDA calls for the same MF term. Reason: Correct catalytic activity; the IEA is redundant with experimentally supported calls but not wrong.
GO:0006555 L-methionine metabolic process	IEA GO_REF:0000002	ACCEPT	Summary: InterPro2GO mapping placing MTHFR in methionine metabolism, consistent with its role generating the methyl donor for homocysteine remethylation. Reason: Correct but broader parent of the more specific L-methionine biosynthetic process; acceptable as an IEA at this generality.
GO:0106313 methylenetetrahydrofolate reductase (NADPH) activity	IEA GO_REF:0000120	ACCEPT	Summary: NADPH-specific catalytic activity term (RHEA:19817, EC:1.5.1.53). Human MTHFR strongly prefers NADPH over NADH (KM 35.5 uM vs 3760 uM), so this specific term is well justified. Reason: Correctly captures the NADPH-preferring catalytic activity confirmed for the human enzyme; more specific and biochemically accurate than the generic NAD(P)H term. Supporting Evidence: PMID:29891918 a reaction requiring FAD as a cofactor and NADPH as an electron donor
GO:0005515 protein binding	IPI PMID:28514442 Architecture of the human interactome defines protein commun...	MARK AS OVER ANNOTATED	Summary: High-throughput AP-MS interactome (BioPlex 2.0) reporting a single MTHFR interaction partner (SMPD2). "protein binding" conveys no specific molecular function. Reason: Bare "protein binding" from a proteome-scale AP-MS screen is uninformative about MTHFR function; retained as a recorded interaction but marked over-annotated rather than removed. Supporting Evidence: PMID:28514442 the largest such network so far. With more than 56,000 candidate interactions
GO:0005515 protein binding	IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling...	MARK AS OVER ANNOTATED	Summary: The same SMPD2 interaction detected in the successor BioPlex 3.0 AP-MS network; again a generic "protein binding" call. Reason: Uninformative bare "protein binding" from a high-throughput screen (and a duplicate of the BioPlex 2.0 interaction); over-annotated. Supporting Evidence: PMID:33961781 BioPlex 3.0, results from affinity purification
GO:0001666 response to hypoxia	IEA GO_REF:0000107	REMOVE	Summary: Automatic ortholog transfer (Ensembl Compara, from rat) of a generic stimulus response. Not a described function of human MTHFR and unsupported by the primary literature reviewed here. Reason: Over-propagated electronic ortholog-projected "response to X" annotation with no human experimental support and no clear mechanistic link to MTHFR catalysis; appropriate to remove as an incorrect IEA inference.
GO:0005829 cytosol	IEA GO_REF:0000107	ACCEPT	Summary: Electronic ortholog-based confirmation of cytosolic localization, consistent with the IBA and Reactome calls. Reason: Correct cytosolic localization.
GO:0009410 response to xenobiotic stimulus	IEA GO_REF:0000107	REMOVE	Summary: Generic ortholog-projected stimulus-response term (Ensembl, from rat) with no specific mechanistic basis for human MTHFR. Reason: Over-propagated IEA "response to X" with no experimental support in human and no informative link to MTHFR function.
GO:0033274 response to vitamin B2	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Ortholog-projected term. There is a real biochemical connection (riboflavin is the precursor of the FAD cofactor, and riboflavin status modulates the thermolabile variant), but this is a physiological modulation of an FAD-dependent enzyme rather than a discrete MTHFR-driven biological process. Reason: Peripheral and cofactor-related rather than a core function; retained as non-core given the plausible FAD/riboflavin link but not central to gene function.
GO:0035999 tetrahydrofolate interconversion	IEA GO_REF:0000120	ACCEPT	Summary: Electronic (UniPathway UPA00193) assignment of the folate-interconversion process, duplicating the IBA/IDA calls for this term. Reason: Correct folate-cycle process; redundant with experimental evidence.
GO:0043200 response to amino acid	IEA GO_REF:0000107	REMOVE	Summary: Generic ortholog-projected stimulus-response term (Ensembl, from rat) without a specific mechanistic basis for human MTHFR. Reason: Over-propagated IEA "response to X" lacking human experimental support and informative value.
GO:0046500 S-adenosylmethionine metabolic process	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: MTHFR is allosterically inhibited by SAM and, by producing CH3-THF, feeds the methionine cycle that generates SAM; it thus participates indirectly in SAM homeostasis. The ortholog-projected term captures a real regulatory connection but is downstream/indirect. Reason: Genuine but indirect/regulatory relationship to SAM metabolism (SAM is the allosteric inhibitor and a downstream product), not the enzyme's core catalytic function. Supporting Evidence: PMID:29891918 allosteric inhibition by its end product SAM
GO:0051593 response to folic acid	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Ortholog-projected term. MTHFR acts on folate-derived substrates and its thermolabile variant is folate-sensitive, so a response-to-folate connection is biologically plausible but peripheral and not directly demonstrated for the human protein here. Reason: Peripheral, plausible given the folate-cycle role and folate-sensitivity of the C677T variant, but not a core function.
GO:0070555 response to interleukin-1	IEA GO_REF:0000107	REMOVE	Summary: Generic ortholog-projected cytokine-response term (Ensembl, from rat) with no mechanistic connection to MTHFR catalysis. Reason: Over-propagated IEA "response to X" with no human experimental support and no informative value for MTHFR function.
GO:0035999 tetrahydrofolate interconversion	IGI PMID:10551815 Functional characterization of human methylenetetrahydrofola...	ACCEPT	Summary: Genetic-interaction evidence from complementation of the yeast met11 mutant by human MTHFR, demonstrating its role in folate one-carbon interconversion. Reason: Supported by functional complementation restoring MTHFR activity in a met11-deleted yeast strain. Supporting Evidence: PMID:10551815 complement its methionine auxotrophic phenotype in vivo
GO:0071265 L-methionine biosynthetic process	IGI PMID:10551815 Functional characterization of human methylenetetrahydrofola...	ACCEPT	Summary: Human MTHFR complements the methionine auxotrophy of a yeast MET11 deletion, placing it genetically upstream of methionine biosynthesis (it supplies the CH3-THF methyl donor rather than synthesizing methionine directly). Reason: The acts_upstream_of qualifier correctly reflects that MTHFR provides the methyl donor for the methionine-synthase step rather than catalysing methionine formation itself; supported by yeast complementation. Supporting Evidence: PMID:10551815 complement its methionine auxotrophic phenotype in vivo
GO:0106313 methylenetetrahydrofolate reductase (NADPH) activity	IMP PMID:25736335 Insights into severe 5,10-methylenetetrahydrofolate reductas...	ACCEPT	Summary: Enzymatic characterization of 72 MTHFR-deficient patient fibroblast lines using an assay in the physiological direction; disease mutations reduce residual MTHFR (NADPH-dependent) activity, with many showing reduced NADPH affinity. Reason: Mutational impact on measured NADPH-dependent reductase activity directly supports this catalytic MF term. Supporting Evidence: PMID:25736335 found residual activity PMID:25736335 reduced affinity for
GO:0106313 methylenetetrahydrofolate reductase (NADPH) activity	IDA PMID:29891918 Structural basis for the regulation of human 5,10-methylenet...	ACCEPT	Summary: Direct biochemical assay of recombinant human MTHFR established the NADPH-dependent reductase activity and its kinetic parameters (KM for CH2-THF and NADPH; strong NADPH over NADH preference). Reason: Direct experimental measurement of the core catalytic activity; the most authoritative support for the NADPH-specific MF term. Supporting Evidence: PMID:29891918 a reaction requiring FAD as a cofactor and NADPH as an electron donor
GO:0035999 tetrahydrofolate interconversion	IDA PMID:29891918 Structural basis for the regulation of human 5,10-methylenet...	ACCEPT	Summary: Direct assay of the reduction of 5,10-methylene-THF to 5-methyl-THF, the folate-interconversion reaction catalysed by MTHFR. Reason: Directly demonstrated folate one-carbon interconversion activity. Supporting Evidence: PMID:29891918 a reaction requiring FAD as a cofactor and NADPH as an electron donor
GO:0070828 heterochromatin organization	IDA PMID:24769206 MTHFR promotes heterochromatin maintenance.	KEEP AS NON CORE	Summary: MTHFR knockdown decreased H3K9me3 and derepressed centromeric heterochromatin markers, and CDK1/Cyclin B1 phosphorylates MTHFR at T34 during mitosis; the authors conclude MTHFR contributes to centromeric heterochromatin maintenance. This is an indirect, downstream consequence of MTHFR's role in supplying methyl groups (via CH3-THF -> methionine -> SAM) for histone methylation. Reason: A genuine but indirect (acts_upstream_of) systemic effect mediated through the enzyme's methyl-donor output, not a core molecular function; retained as non-core. Supporting Evidence: PMID:24769206 plays a role in the heterochromatin maintenance at the centromeric region PMID:24769206 MTHFR expression resulted in a decrease of H3K9me3 levels
GO:0001843 neural tube closure	IMP PMID:25855017 Variants in MTHFR gene and neural tube defects susceptibilit...	KEEP AS NON CORE	Summary: Genetic-association evidence linking MTHFR variants to neural tube defect susceptibility. Reduced MTHFR activity elevates homocysteine and perturbs folate/methylation status, an established folate-sensitive risk pathway for failure of neural tube closure. Reason: Downstream developmental/disease-susceptibility phenotype (acts_upstream_of) arising from reduced one-carbon metabolism, not a core molecular function of the enzyme; retained as non-core.
GO:0001843 neural tube closure	IMP PMID:29222906 Low maternal folate concentrations and maternal MTHFR C677T ...	KEEP AS NON CORE	Summary: Case-control study associating the maternal MTHFR C677T polymorphism and low folate with increased neural tube defect risk in offspring, consistent with MTHFR acting upstream of neural tube closure via folate/homocysteine status. Reason: Downstream folate-sensitive developmental susceptibility, not a core function; retained as non-core.
GO:0001843 neural tube closure	NAS PMID:9349452 Elevated plasma total homocysteine and C677T mutation of the...	KEEP AS NON CORE	Summary: Author-statement (NAS) evidence associating elevated homocysteine and the MTHFR C677T mutation with spina bifida, supporting an upstream role in neural tube closure. Reason: Same downstream developmental association as the IMP calls; NAS evidence, retained as non-core.
GO:0004489 methylenetetrahydrofolate reductase [NAD(P)H] activity	IGI PMID:10551815 Functional characterization of human methylenetetrahydrofola...	ACCEPT	Summary: Genetic-interaction (yeast met11 complementation) support for the core MTHFR catalytic activity; human MTHFR restores MTHFR activity in vitro in the met11-deleted strain. Reason: Functional complementation directly demonstrates the reductase activity. Supporting Evidence: PMID:10551815 reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate
GO:0004489 methylenetetrahydrofolate reductase [NAD(P)H] activity	IDA PMID:12673793 Characterization of mutations in severe methylenetetrahydrof...	ACCEPT	Summary: Direct enzymatic characterization of expressed human MTHFR wild-type and mutant alleles measuring reductase activity; one variant (N324S) showed FAD responsiveness. Reason: Direct measurement of the core catalytic reductase activity. Supporting Evidence: PMID:12673793 5-methyltetrahydrofolate, a major methyl donor for homocysteine remethylation to
GO:0004489 methylenetetrahydrofolate reductase [NAD(P)H] activity	IMP PMID:12673793 Characterization of mutations in severe methylenetetrahydrof...	ACCEPT	Summary: Missense mutations in expressed human MTHFR decreased measured reductase activity (to 10-36% of control), supporting the catalytic MF via mutational impact. Reason: Mutational reduction of enzyme activity supports the catalytic function. Supporting Evidence: PMID:12673793 flavin adenine dinucleotide (FAD) responsiveness in vitro
GO:0004489 methylenetetrahydrofolate reductase [NAD(P)H] activity	IDA PMID:24769206 MTHFR promotes heterochromatin maintenance.	ACCEPT	Summary: MTHFR immunocomplex purified from cells exhibited reductase activity (reduced in mitotic/phosphorylated state), supporting the catalytic MF. Reason: Direct assay of MTHFR enzymatic activity from purified immunocomplex supports the reductase function. Supporting Evidence: PMID:24769206 catalyzes the reduction of 5,10-methylenetetrahydrofolate to
GO:0035999 tetrahydrofolate interconversion	IDA PMID:12673793 Characterization of mutations in severe methylenetetrahydrof...	ACCEPT	Summary: Direct assay of the folate-interconversion reaction (reduction of methylene-THF to methyl-THF) for wild-type and mutant human MTHFR. Reason: Directly demonstrated folate-cycle interconversion activity. Supporting Evidence: PMID:12673793 5-methyltetrahydrofolate, a major methyl donor for homocysteine remethylation to
GO:0035999 tetrahydrofolate interconversion	IMP PMID:12673793 Characterization of mutations in severe methylenetetrahydrof...	ACCEPT	Summary: Mutational impact on the folate-interconversion reaction (reduced enzyme activity in severe-deficiency alleles). Reason: Mutational impact supports participation in folate interconversion. Supporting Evidence: PMID:12673793 flavin adenine dinucleotide (FAD) responsiveness in vitro
GO:0044877 protein-containing complex binding	IPI PMID:24769206 MTHFR promotes heterochromatin maintenance.	KEEP AS NON CORE	Summary: IPI evidence with ComplexPortal CPX-2007 (the MTHFR homodimer). MTHFR is an obligate homodimer, so this reflects self-association within its own catalytic complex rather than binding to a distinct multiprotein complex. Reason: Captures the functionally relevant homodimerization but is a structural property, not the enzyme's core catalytic function; retained as non-core and more informative than bare "protein binding". Supporting Evidence: PMID:29891918 provides the predominant interface for MTHFR
GO:0050660 flavin adenine dinucleotide binding	IDA PMID:12673793 Characterization of mutations in severe methylenetetrahydrof...	ACCEPT	Summary: MTHFR binds FAD as its essential redox cofactor; a severe-deficiency variant (N324S) showed FAD responsiveness in vitro, directly implicating FAD binding. Reason: FAD binding is a genuine, experimentally supported molecular function confirmed both biochemically and by the human crystal structure. Supporting Evidence: PMID:12673793 flavin adenine dinucleotide (FAD) responsiveness in vitro
GO:0035999 tetrahydrofolate interconversion	IMP PMID:25736335 Insights into severe 5,10-methylenetetrahydrofolate reductas...	ACCEPT	Summary: Enzymatic characterization of 72 patient fibroblast lines shows disease mutations reduce MTHFR activity in the physiological (folate-interconversion) direction. Reason: Mutational impact on the physiological-direction assay supports participation in folate interconversion. Supporting Evidence: PMID:25736335 found residual activity
GO:0050667 homocysteine metabolic process	IDA PMID:20031578 Novel associations of CPS1, MUT, NOX4, and DPEP1 with plasma...	KEEP AS NON CORE	Summary: MTHFR indirectly controls homocysteine levels: by producing CH3-THF it supplies the methyl donor for homocysteine remethylation to methionine, and reduced MTHFR activity raises plasma homocysteine. The cited reference, however, is a genome-wide association study of plasma homocysteine and provides population-genetic (not direct in vitro binding/activity) evidence, so the IDA evidence code is a poor fit for this reference. Reason: The biological relationship (MTHFR upstream of homocysteine remethylation) is real and worth retaining as non-core, but it is an indirect metabolic role; the IDA/reference pairing is weak (the paper is a GWAS, not a direct assay). Supporting Evidence: PMID:20031578 a cosubstrate for homocysteine remethylation to methionine by methionine synthase
GO:0072341 modified amino acid binding	IDA PMID:20031578 Novel associations of CPS1, MUT, NOX4, and DPEP1 with plasma...	MARK AS OVER ANNOTATED	Summary: The cited reference is a genome-wide association study of plasma homocysteine; it contains no direct assay of MTHFR binding to a modified amino acid. There is no experimental basis in this reference for an IDA "modified amino acid binding" molecular function, and homocysteine is a substrate of methionine synthase, not of MTHFR. Reason: The reference does not support a direct modified-amino-acid binding function for MTHFR (it is a GWAS), and MTHFR does not bind homocysteine; the IDA MF call is unsupported and over-annotated. Not removed outright per policy on experimental-code annotations whose full evidence cannot be fully verified.
GO:0005829 cytosol	TAS Reactome:R-HSA-200676	ACCEPT	Summary: Reactome traceable-author-statement placing the MTHFR-catalysed reduction of 5,10-methylene-THF-PG to 5-methyl-THF-PG in the cytosol. Reason: Correct cytosolic localization from a curated pathway source, consistent with IBA/IEA evidence.

Gene Description

Existing Annotations Review

Core Functions

Cytosolic, FAD-dependent, NADPH-preferring methylenetetrahydrofolate reductase that catalyses the committed reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, channeling folate one-carbon units into methionine remethylation.

By generating 5-methyltetrahydrofolate, the sole methyl donor used by methionine synthase, MTHFR acts upstream of homocysteine remethylation and L-methionine biosynthesis, coupling the folate and methionine cycles.

Flavoprotein that binds FAD as an essential redox cofactor within its catalytic TIM-barrel domain, required for electron transfer from NADPH to the folate substrate.

References

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Notes

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