id: P42898
gene_symbol: MTHFR
product_type: PROTEIN
status: INITIALIZED
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: >-
  MTHFR is the cytosolic, FAD-dependent, NAD(P)H-linked methylenetetrahydrofolate
  reductase (EC 1.5.1.53) that catalyses the committed, physiologically
  irreversible reduction of 5,10-methylenetetrahydrofolate (CH2-THF) to
  5-methyltetrahydrofolate (CH3-THF). Because CH3-THF is used exclusively by
  cobalamin-dependent methionine synthase (MTR) to remethylate homocysteine to
  methionine, MTHFR commits folate one-carbon units to the methionine cycle and
  represents the key regulatory node linking folate and methionine metabolism and,
  through methionine, biosynthesis of the universal methyl donor
  S-adenosylmethionine (SAM). The enzyme is a homodimer built from a conserved
  catalytic TIM-barrel domain that binds FAD and NADPH and a eukaryote-specific
  C-terminal SAM-binding regulatory domain; it is allosterically inhibited by its
  downstream product SAM (reversed by S-adenosylhomocysteine), and phosphorylation
  of an N-terminal serine-rich region increases sensitivity to SAM inhibition,
  allowing methylation status to feed back on flux through the enzyme. Loss of
  function causes severe MTHFR deficiency, an autosomal recessive homocystinuria
  with hyperhomocysteinemia and neurological disease, while the common
  thermolabile c.677C>T (p.Ala222Val) polymorphism reduces activity and is a
  folate-sensitive risk factor for hyperhomocysteinemia, vascular disease and
  neural tube defects.
alternative_products:
- name: '1'
  id: P42898-1
- name: '2'
  id: P42898-2
  sequence_note: VSP_053744
existing_annotations:
- term:
    id: GO:0004489
    label: methylenetetrahydrofolate reductase [NAD(P)H] activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: enables
  review:
    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.
    action: ACCEPT
    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.
    supported_by:
    - reference_id: PMID:29891918
      supporting_text: >-
        a reaction requiring FAD as a cofactor and NADPH as an electron donor
- term:
    id: GO:0071265
    label: L-methionine biosynthetic process
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: involved_in
  review:
    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).
    action: ACCEPT
    reason: >-
      Well-supported involvement in methionine biosynthesis/remethylation; the IBA
      call matches human and cross-species experimental evidence.
    supported_by:
    - reference_id: PMID:10551815
      supporting_text: complement its methionine auxotrophic phenotype in vivo
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: is_active_in
  review:
    summary: >-
      MTHFR is a soluble cytosolic enzyme of the folate/methionine cycles; the
      cytosolic localization is consistent across IBA, IEA and Reactome (TAS)
      sources.
    action: ACCEPT
    reason: >-
      Correct subcellular localization for this cytosolic one-carbon-metabolism
      enzyme.
- term:
    id: GO:0035999
    label: tetrahydrofolate interconversion
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: involved_in
  review:
    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".
    action: ACCEPT
    reason: >-
      Accurately describes the folate-cycle process in which the catalytic
      activity participates.
    supported_by:
    - reference_id: PMID:29891918
      supporting_text: >-
        a reaction requiring FAD as a cofactor and NADPH as an electron donor
- term:
    id: GO:0071949
    label: FAD binding
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: >-
      FAD binding is a genuine, experimentally supported molecular function of
      MTHFR (see also the GO:0050660 IDA below).
    supported_by:
    - reference_id: PMID:29891918
      supporting_text: >-
        a reaction requiring FAD as a cofactor and NADPH as an electron donor
- term:
    id: GO:0004489
    label: methylenetetrahydrofolate reductase [NAD(P)H] activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: >-
      Electronic (multi-method) assignment of the core catalytic activity,
      duplicating the IBA/IDA calls for the same MF term.
    action: ACCEPT
    reason: >-
      Correct catalytic activity; the IEA is redundant with experimentally
      supported calls but not wrong.
- term:
    id: GO:0006555
    label: L-methionine metabolic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: involved_in
  review:
    summary: >-
      InterPro2GO mapping placing MTHFR in methionine metabolism, consistent with
      its role generating the methyl donor for homocysteine remethylation.
    action: ACCEPT
    reason: >-
      Correct but broader parent of the more specific L-methionine biosynthetic
      process; acceptable as an IEA at this generality.
- term:
    id: GO:0106313
    label: methylenetetrahydrofolate reductase (NADPH) activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    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.
    action: ACCEPT
    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.
    supported_by:
    - reference_id: PMID:29891918
      supporting_text: >-
        a reaction requiring FAD as a cofactor and NADPH as an electron donor
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:28514442
  qualifier: enables
  review:
    summary: >-
      High-throughput AP-MS interactome (BioPlex 2.0) reporting a single MTHFR
      interaction partner (SMPD2). "protein binding" conveys no specific molecular
      function.
    action: MARK_AS_OVER_ANNOTATED
    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.
    supported_by:
    - reference_id: PMID:28514442
      supporting_text: >-
        the largest such network so far. With more than 56,000 candidate
        interactions
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:33961781
  qualifier: enables
  review:
    summary: >-
      The same SMPD2 interaction detected in the successor BioPlex 3.0 AP-MS
      network; again a generic "protein binding" call.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Uninformative bare "protein binding" from a high-throughput screen (and a
      duplicate of the BioPlex 2.0 interaction); over-annotated.
    supported_by:
    - reference_id: PMID:33961781
      supporting_text: >-
        BioPlex 3.0, results from affinity purification
- term:
    id: GO:0001666
    label: response to hypoxia
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    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.
    action: REMOVE
    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.
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: is_active_in
  review:
    summary: >-
      Electronic ortholog-based confirmation of cytosolic localization, consistent
      with the IBA and Reactome calls.
    action: ACCEPT
    reason: Correct cytosolic localization.
- term:
    id: GO:0009410
    label: response to xenobiotic stimulus
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    summary: >-
      Generic ortholog-projected stimulus-response term (Ensembl, from rat) with no
      specific mechanistic basis for human MTHFR.
    action: REMOVE
    reason: >-
      Over-propagated IEA "response to X" with no experimental support in human and
      no informative link to MTHFR function.
- term:
    id: GO:0033274
    label: response to vitamin B2
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.
- term:
    id: GO:0035999
    label: tetrahydrofolate interconversion
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: involved_in
  review:
    summary: >-
      Electronic (UniPathway UPA00193) assignment of the folate-interconversion
      process, duplicating the IBA/IDA calls for this term.
    action: ACCEPT
    reason: Correct folate-cycle process; redundant with experimental evidence.
- term:
    id: GO:0043200
    label: response to amino acid
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    summary: >-
      Generic ortholog-projected stimulus-response term (Ensembl, from rat) without
      a specific mechanistic basis for human MTHFR.
    action: REMOVE
    reason: >-
      Over-propagated IEA "response to X" lacking human experimental support and
      informative value.
- term:
    id: GO:0046500
    label: S-adenosylmethionine metabolic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.
    supported_by:
    - reference_id: PMID:29891918
      supporting_text: allosteric inhibition by its end product SAM
- term:
    id: GO:0051593
    label: response to folic acid
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    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.
    action: KEEP_AS_NON_CORE
    reason: >-
      Peripheral, plausible given the folate-cycle role and folate-sensitivity of
      the C677T variant, but not a core function.
- term:
    id: GO:0070555
    label: response to interleukin-1
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: involved_in
  review:
    summary: >-
      Generic ortholog-projected cytokine-response term (Ensembl, from rat) with no
      mechanistic connection to MTHFR catalysis.
    action: REMOVE
    reason: >-
      Over-propagated IEA "response to X" with no human experimental support and no
      informative value for MTHFR function.
- term:
    id: GO:0035999
    label: tetrahydrofolate interconversion
  evidence_type: IGI
  original_reference_id: PMID:10551815
  qualifier: involved_in
  review:
    summary: >-
      Genetic-interaction evidence from complementation of the yeast met11 mutant by
      human MTHFR, demonstrating its role in folate one-carbon interconversion.
    action: ACCEPT
    reason: >-
      Supported by functional complementation restoring MTHFR activity in a
      met11-deleted yeast strain.
    supported_by:
    - reference_id: PMID:10551815
      supporting_text: complement its methionine auxotrophic phenotype in vivo
- term:
    id: GO:0071265
    label: L-methionine biosynthetic process
  evidence_type: IGI
  original_reference_id: PMID:10551815
  qualifier: acts_upstream_of
  review:
    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).
    action: ACCEPT
    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.
    supported_by:
    - reference_id: PMID:10551815
      supporting_text: complement its methionine auxotrophic phenotype in vivo
- term:
    id: GO:0106313
    label: methylenetetrahydrofolate reductase (NADPH) activity
  evidence_type: IMP
  original_reference_id: PMID:25736335
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: >-
      Mutational impact on measured NADPH-dependent reductase activity directly
      supports this catalytic MF term.
    supported_by:
    - reference_id: PMID:25736335
      supporting_text: found residual activity
    - reference_id: PMID:25736335
      supporting_text: reduced affinity for
- term:
    id: GO:0106313
    label: methylenetetrahydrofolate reductase (NADPH) activity
  evidence_type: IDA
  original_reference_id: PMID:29891918
  qualifier: enables
  review:
    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).
    action: ACCEPT
    reason: >-
      Direct experimental measurement of the core catalytic activity; the most
      authoritative support for the NADPH-specific MF term.
    supported_by:
    - reference_id: PMID:29891918
      supporting_text: >-
        a reaction requiring FAD as a cofactor and NADPH as an electron donor
- term:
    id: GO:0035999
    label: tetrahydrofolate interconversion
  evidence_type: IDA
  original_reference_id: PMID:29891918
  qualifier: involved_in
  review:
    summary: >-
      Direct assay of the reduction of 5,10-methylene-THF to 5-methyl-THF, the
      folate-interconversion reaction catalysed by MTHFR.
    action: ACCEPT
    reason: >-
      Directly demonstrated folate one-carbon interconversion activity.
    supported_by:
    - reference_id: PMID:29891918
      supporting_text: >-
        a reaction requiring FAD as a cofactor and NADPH as an electron donor
- term:
    id: GO:0070828
    label: heterochromatin organization
  evidence_type: IDA
  original_reference_id: PMID:24769206
  qualifier: acts_upstream_of
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.
    supported_by:
    - reference_id: PMID:24769206
      supporting_text: >-
        plays a role in the heterochromatin maintenance at the centromeric region
    - reference_id: PMID:24769206
      supporting_text: MTHFR expression resulted in a decrease of H3K9me3 levels
- term:
    id: GO:0001843
    label: neural tube closure
  evidence_type: IMP
  original_reference_id: PMID:25855017
  qualifier: acts_upstream_of
  review:
    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.
    action: KEEP_AS_NON_CORE
    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.
- term:
    id: GO:0001843
    label: neural tube closure
  evidence_type: IMP
  original_reference_id: PMID:29222906
  qualifier: acts_upstream_of
  review:
    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.
    action: KEEP_AS_NON_CORE
    reason: >-
      Downstream folate-sensitive developmental susceptibility, not a core
      function; retained as non-core.
- term:
    id: GO:0001843
    label: neural tube closure
  evidence_type: NAS
  original_reference_id: PMID:9349452
  qualifier: acts_upstream_of
  review:
    summary: >-
      Author-statement (NAS) evidence associating elevated homocysteine and the
      MTHFR C677T mutation with spina bifida, supporting an upstream role in neural
      tube closure.
    action: KEEP_AS_NON_CORE
    reason: >-
      Same downstream developmental association as the IMP calls; NAS evidence,
      retained as non-core.
- term:
    id: GO:0004489
    label: methylenetetrahydrofolate reductase [NAD(P)H] activity
  evidence_type: IGI
  original_reference_id: PMID:10551815
  qualifier: enables
  review:
    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.
    action: ACCEPT
    reason: >-
      Functional complementation directly demonstrates the reductase activity.
    supported_by:
    - reference_id: PMID:10551815
      supporting_text: reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate
- term:
    id: GO:0004489
    label: methylenetetrahydrofolate reductase [NAD(P)H] activity
  evidence_type: IDA
  original_reference_id: PMID:12673793
  qualifier: enables
  review:
    summary: >-
      Direct enzymatic characterization of expressed human MTHFR wild-type and
      mutant alleles measuring reductase activity; one variant (N324S) showed FAD
      responsiveness.
    action: ACCEPT
    reason: >-
      Direct measurement of the core catalytic reductase activity.
    supported_by:
    - reference_id: PMID:12673793
      supporting_text: >-
        5-methyltetrahydrofolate, a major methyl donor for homocysteine
        remethylation to
- term:
    id: GO:0004489
    label: methylenetetrahydrofolate reductase [NAD(P)H] activity
  evidence_type: IMP
  original_reference_id: PMID:12673793
  qualifier: enables
  review:
    summary: >-
      Missense mutations in expressed human MTHFR decreased measured reductase
      activity (to 10-36% of control), supporting the catalytic MF via mutational
      impact.
    action: ACCEPT
    reason: >-
      Mutational reduction of enzyme activity supports the catalytic function.
    supported_by:
    - reference_id: PMID:12673793
      supporting_text: >-
        flavin adenine dinucleotide (FAD) responsiveness in vitro
- term:
    id: GO:0004489
    label: methylenetetrahydrofolate reductase [NAD(P)H] activity
  evidence_type: IDA
  original_reference_id: PMID:24769206
  qualifier: enables
  review:
    summary: >-
      MTHFR immunocomplex purified from cells exhibited reductase activity (reduced
      in mitotic/phosphorylated state), supporting the catalytic MF.
    action: ACCEPT
    reason: >-
      Direct assay of MTHFR enzymatic activity from purified immunocomplex supports
      the reductase function.
    supported_by:
    - reference_id: PMID:24769206
      supporting_text: >-
        catalyzes the reduction of 5,10-methylenetetrahydrofolate to
- term:
    id: GO:0035999
    label: tetrahydrofolate interconversion
  evidence_type: IDA
  original_reference_id: PMID:12673793
  qualifier: involved_in
  review:
    summary: >-
      Direct assay of the folate-interconversion reaction (reduction of
      methylene-THF to methyl-THF) for wild-type and mutant human MTHFR.
    action: ACCEPT
    reason: Directly demonstrated folate-cycle interconversion activity.
    supported_by:
    - reference_id: PMID:12673793
      supporting_text: >-
        5-methyltetrahydrofolate, a major methyl donor for homocysteine
        remethylation to
- term:
    id: GO:0035999
    label: tetrahydrofolate interconversion
  evidence_type: IMP
  original_reference_id: PMID:12673793
  qualifier: involved_in
  review:
    summary: >-
      Mutational impact on the folate-interconversion reaction (reduced enzyme
      activity in severe-deficiency alleles).
    action: ACCEPT
    reason: Mutational impact supports participation in folate interconversion.
    supported_by:
    - reference_id: PMID:12673793
      supporting_text: >-
        flavin adenine dinucleotide (FAD) responsiveness in vitro
- term:
    id: GO:0044877
    label: protein-containing complex binding
  evidence_type: IPI
  original_reference_id: PMID:24769206
  qualifier: enables
  review:
    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.
    action: KEEP_AS_NON_CORE
    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".
    supported_by:
    - reference_id: PMID:29891918
      supporting_text: >-
        provides the predominant interface for MTHFR
- term:
    id: GO:0050660
    label: flavin adenine dinucleotide binding
  evidence_type: IDA
  original_reference_id: PMID:12673793
  qualifier: enables
  review:
    summary: >-
      MTHFR binds FAD as its essential redox cofactor; a severe-deficiency variant
      (N324S) showed FAD responsiveness in vitro, directly implicating FAD binding.
    action: ACCEPT
    reason: >-
      FAD binding is a genuine, experimentally supported molecular function
      confirmed both biochemically and by the human crystal structure.
    supported_by:
    - reference_id: PMID:12673793
      supporting_text: >-
        flavin adenine dinucleotide (FAD) responsiveness in vitro
- term:
    id: GO:0035999
    label: tetrahydrofolate interconversion
  evidence_type: IMP
  original_reference_id: PMID:25736335
  qualifier: involved_in
  review:
    summary: >-
      Enzymatic characterization of 72 patient fibroblast lines shows disease
      mutations reduce MTHFR activity in the physiological (folate-interconversion)
      direction.
    action: ACCEPT
    reason: >-
      Mutational impact on the physiological-direction assay supports participation
      in folate interconversion.
    supported_by:
    - reference_id: PMID:25736335
      supporting_text: found residual activity
- term:
    id: GO:0050667
    label: homocysteine metabolic process
  evidence_type: IDA
  original_reference_id: PMID:20031578
  qualifier: involved_in
  review:
    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.
    action: KEEP_AS_NON_CORE
    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).
    supported_by:
    - reference_id: PMID:20031578
      supporting_text: >-
        a cosubstrate for homocysteine remethylation to methionine by methionine
        synthase
- term:
    id: GO:0072341
    label: modified amino acid binding
  evidence_type: IDA
  original_reference_id: PMID:20031578
  qualifier: enables
  review:
    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.
    action: MARK_AS_OVER_ANNOTATED
    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.
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-200676
  qualifier: located_in
  review:
    summary: >-
      Reactome traceable-author-statement placing the MTHFR-catalysed reduction of
      5,10-methylene-THF-PG to 5-methyl-THF-PG in the cytosol.
    action: ACCEPT
    reason: >-
      Correct cytosolic localization from a curated pathway source, consistent with
      IBA/IEA evidence.
core_functions:
- description: >-
    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.
  molecular_function:
    id: GO:0004489
    label: methylenetetrahydrofolate reductase [NAD(P)H] activity
  directly_involved_in:
  - id: GO:0035999
    label: tetrahydrofolate interconversion
  locations:
  - id: GO:0005829
    label: cytosol
  supported_by:
  - reference_id: PMID:29891918
    supporting_text: >-
      a reaction requiring FAD as a cofactor and NADPH as an electron donor
- description: >-
    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.
  molecular_function:
    id: GO:0004489
    label: methylenetetrahydrofolate reductase [NAD(P)H] activity
  directly_involved_in:
  - id: GO:0071265
    label: L-methionine biosynthetic process
  locations:
  - id: GO:0005829
    label: cytosol
  supported_by:
  - reference_id: PMID:10551815
    supporting_text: complement its methionine auxotrophic phenotype in vivo
- description: >-
    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.
  molecular_function:
    id: GO:0050660
    label: flavin adenine dinucleotide binding
  locations:
  - id: GO:0005829
    label: cytosol
  supported_by:
  - reference_id: PMID:12673793
    supporting_text: >-
      flavin adenine dinucleotide (FAD) responsiveness in vitro
proposed_new_terms: []
suggested_questions:
- question: >-
    What are the physiological kinases and signalling contexts that phosphorylate
    the N-terminal serine-rich region of human MTHFR, and how does this tune SAM
    sensitivity in vivo?
- question: >-
    To what extent are the reported roles of MTHFR in centromeric heterochromatin
    maintenance mediated solely through SAM-dependent histone methylation versus a
    more direct nuclear function?
suggested_experiments:
- description: >-
    Quantitative in-cell measurement of MTHFR flux and the SAM/SAH ratio in
    phospho-site mutants to test how N-terminal phosphorylation modulates
    SAM-mediated allosteric inhibition under varying folate and methionine status.
- description: >-
    Structure-function analysis of the inter-domain linker (e.g. disease variants
    such as p.His354Tyr) using cryo-EM to capture SAM/SAH-dependent conformational
    states and their effect on FAD retention and catalysis.
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO
    terms
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000107
  title: Automatic transfer of experimentally verified manual GO annotation data to
    orthologs using Ensembl Compara
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:10551815
  title: Functional characterization of human methylenetetrahydrofolate reductase
    in Saccharomyces cerevisiae.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Yeast met11 complementation by human MTHFR; supports the reductase activity
      and upstream role in methionine biosynthesis.
- id: PMID:12673793
  title: Characterization of mutations in severe methylenetetrahydrofolate reductase
    deficiency reveals an FAD-responsive mutation.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Direct enzymatic characterization of wild-type/mutant MTHFR; documents
      FAD responsiveness. Abstract-only in cache but abstract supports the calls.
- id: PMID:20031578
  title: 'Novel associations of CPS1, MUT, NOX4, and DPEP1 with plasma homocysteine
    in a healthy population: a genome-wide evaluation of 13 974 participants in the
    Women''s Genome Health Study.'
  findings: []
  reference_review:
    relevance: MEDIUM
    correctness: MISCITED
    review_notes: >-
      A GWAS of plasma homocysteine that confirms the MTHFR locus association. It
      does NOT provide direct (IDA) evidence for MTHFR "modified amino acid binding"
      or a direct homocysteine-metabolic assay; the IDA annotations sourced to it
      are over-attributed.
- id: PMID:24769206
  title: MTHFR promotes heterochromatin maintenance.
  findings: []
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Shows CDK1/Cyclin B1 phosphorylates MTHFR at T34 and that MTHFR knockdown
      lowers H3K9me3; supports an indirect (methyl-donor-mediated) heterochromatin
      role and reductase activity of the purified immunocomplex.
- id: PMID:25736335
  title: 'Insights into severe 5,10-methylenetetrahydrofolate reductase deficiency:
    molecular genetic and enzymatic characterization of 76 patients.'
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Enzymatic characterization of 72 patient fibroblast lines in the physiological
      direction; supports the NADPH-dependent catalytic activity and folate
      interconversion via mutational impact.
- id: PMID:25855017
  title: Variants in MTHFR gene and neural tube defects susceptibility in China.
  findings: []
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Genetic-association support for MTHFR acting upstream of neural tube closure
      (folate-sensitive susceptibility), a downstream/non-core role.
- id: PMID:28514442
  title: Architecture of the human interactome defines protein communities and disease
    networks.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: >-
      BioPlex 2.0 high-throughput AP-MS; source of a generic "protein binding"
      (SMPD2) call, uninformative for MTHFR molecular function.
- id: PMID:29222906
  title: 'Low maternal folate concentrations and maternal MTHFR C677T polymorphism
    are associated with an increased risk for neural tube defects in offspring: a
    case-control study among Pakistani case and control mothers.'
  findings: []
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Case-control association of maternal C677T with offspring NTD risk; supports
      the downstream/non-core neural tube closure annotation.
- id: PMID:29891918
  title: Structural basis for the regulation of human 5,10-methylenetetrahydrofolate
    reductase by phosphorylation and S-adenosylmethionine inhibition.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Definitive human structural + biochemical study: catalytic reductase activity,
      FAD cofactor, NADPH preference, homodimer, SAM allosteric inhibition, and
      phosphoregulation. Primary source for the core functions.
- id: PMID:33961781
  title: Dual proteome-scale networks reveal cell-specific remodeling of the human
    interactome.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: >-
      BioPlex 3.0 high-throughput AP-MS; duplicate source of the generic "protein
      binding" (SMPD2) interaction, uninformative for MTHFR molecular function.
- id: PMID:9349452
  title: Elevated plasma total homocysteine and C677T mutation of the methylenetetrahydrofolate
    reductase gene in patients with spina bifida.
  findings: []
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Associates C677T and elevated homocysteine with spina bifida; NAS support for
      the downstream/non-core neural tube closure annotation.
- id: Reactome:R-HSA-200676
  title: MTHFR dimer reduces 5,10-methylene-THFPG to 5-methyl-THFPG
  findings: []