id: Q9Y4U1
gene_symbol: MMACHC
product_type: PROTEIN
status: INITIALIZED
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: >-
  MMACHC (CblC; "cyanocobalamin reductase / alkylcobalamin dealkylase") is the cytosolic
  vitamin B12 (cobalamin) processing chaperone-enzyme that performs the first intracellular
  tailoring step on incoming dietary/circulating cobalamin. It removes the upper axial (beta)
  ligand of cob(III)alamin to generate a common cob(II)alamin/cob(I)alamin intermediate that
  is then partitioned to the two downstream B12-dependent enzymes: methylcobalamin for cytosolic
  methionine synthase (MTR) and 5'-deoxyadenosylcobalamin for mitochondrial methylmalonyl-CoA
  mutase (MMUT). Using a divergent NADPH-dependent flavin reductase (nitroreductase-like)
  scaffold with FAD or FMN as prosthetic group, it catalyzes reductive decyanation of
  cyanocobalamin (the inactive dietary form) to cob(II)alamin plus cyanide (EC 1.16.1.6), and,
  via an arginine-rich glutathione-binding pocket, glutathione-dependent dealkylation of
  alkylcobalamins (methyl- and adenosylcobalamin) by nucleophilic displacement of the alkyl
  group to yield cob(I)alamin and an S-alkylglutathione (EC 2.5.1.151); cysteine and
  homocysteine cannot substitute for glutathione. Cobalamin is bound in the base-off
  configuration. MMACHC binds cobalamin and interacts with the CblD protein MMADHC (which
  partitions cofactor between the methyl- and adenosylcobalamin routes) and with the lysosomal
  B12 exporters LMBD1/LMBRD1 and ABCD4 that deliver cobalamin to it, and it participates in a
  cytosolic multiprotein processing complex with MMADHC, MTR and MTRR. Loss of MMACHC function
  causes cblC disease (combined methylmalonic aciduria and homocystinuria), the most common
  inborn error of intracellular cobalamin metabolism, because both the adenosylcobalamin and
  methylcobalamin arms are simultaneously deprived of their cofactor.
existing_annotations:
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: is_active_in
  review:
    summary: >-
      Phylogenetic (PAN-GO) inference that MMACHC is active in the cytoplasm. MMACHC is a
      well-established cytosolic protein, so the localization is correct, but "cytoplasm" is a
      general parent of the more precise "cytosol" term that is directly supported experimentally.
    action: MODIFY
    reason: >-
      The cytoplasmic localization is correct but the more specific cytosol term (GO:0005829)
      is directly supported by immunofluorescence/fractionation and better represents where the
      enzyme acts.
    proposed_replacement_terms:
    - id: GO:0005829
      label: cytosol
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0009235
    label: cobalamin metabolic process
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: involved_in
  review:
    summary: >-
      Phylogenetic inference that MMACHC is involved in cobalamin metabolism. This is the central,
      well-supported biological process for MMACHC: it processes incoming cobalamin into the common
      intermediate feeding both active cofactor pools.
    action: ACCEPT
    reason: >-
      Correct and core. MMACHC catalyzes the first intracellular tailoring step of cobalamin, and
      IBA is consistent with the direct experimental evidence.
    supported_by:
    - reference_id: PMID:25809485
      supporting_text: assimilation of dietary cobalamin into the active cofactor forms
- term:
    id: GO:0032451
    label: demethylase activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: enables
  review:
    summary: >-
      Phylogenetic inference of demethylase activity, reflecting MMACHC's dealkylation of
      methylcobalamin. Mechanistically MMACHC removes the methyl group from cobalamin by
      glutathione-dependent nucleophilic S-alkyltransfer, not classic substrate demethylation;
      the essence (methyl removal) is right but the term flavor is imprecise.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      MMACHC's methylcobalamin activity is a glutathione S-alkyltransferase (EC 2.5.1.151) that
      transfers the methyl group to glutathione, better captured by the alkyl transferase term
      (GO:0016765) that GOA already applies. "Demethylase activity" (typically implying oxidative
      or hydrolytic demethylation of a substrate) mischaracterizes the chemistry.
    proposed_replacement_terms:
    - id: GO:0016765
      label: transferase activity, transferring alkyl or aryl (other than methyl) groups
    supported_by:
    - reference_id: PMID:19801555
      supporting_text: using the thiolate of glutathione for nucleophilic
    - reference_id: PMID:21697092
      supporting_text: the first example of an enzyme with glutathione transferase
- term:
    id: GO:0033787
    label: cyanocobalamin reductase (cyanide-eliminating) (NADP+) activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: enables
  review:
    summary: >-
      Phylogenetic inference of cyanocobalamin reductase (decyanase) activity. This is a core,
      directly demonstrated MMACHC molecular function: FAD/FMN + NADPH-dependent reductive
      decyanation of cyanocobalamin to cob(II)alamin.
    action: ACCEPT
    reason: >-
      Correct and core; IBA agrees with strong experimental evidence (EXP/IDA) for the decyanation
      reaction.
    supported_by:
    - reference_id: PMID:18779575
      supporting_text: the trafficking chaperone, MMACHC, catalyzes a
    - reference_id: PMID:19700356
      supporting_text: wild-type MMACHC is able to reductively decyanate
- term:
    id: GO:0071949
    label: FAD binding
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: enables
  review:
    summary: >-
      Phylogenetic inference of FAD binding. MMACHC is a divergent flavin reductase that uses FAD
      (or FMN) as a prosthetic group to catalyze reductive decyanation; FAD binding is directly
      demonstrated.
    action: ACCEPT
    reason: >-
      Correct and core cofactor-binding function, supported experimentally and by structure.
    supported_by:
    - reference_id: PMID:21697092
      supporting_text: can use FMN or FAD as a prosthetic group to catalyze reductive decyanation
- term:
    id: GO:0031419
    label: cobalamin binding
  evidence_type: IDA
  original_reference_id: PMID:19700356
  qualifier: enables
  review:
    summary: >-
      Cobalamin binding is a core molecular function directly demonstrated for MMACHC: it binds
      cyanocobalamin (and hydroxocobalamin) with tight affinity in the base-off configuration, and
      pathogenic mutations (e.g. G147D) abolish cobalamin binding. Present in UniProt (GO:0031419,
      IEA:UniProtKB-KW) but not yet in the curated GOA set, so added here as NEW.
    action: NEW
    reason: >-
      MMACHC's substrate-binding function (cobalamin binding) underlies both its decyanase and
      dealkylase activities and is directly evidenced structurally and biochemically; it should be
      an explicit annotation.
    supported_by:
    - reference_id: PMID:19700356
      supporting_text: MMACHC binds CNCbl in the base-off form
    - reference_id: PMID:22642810
      supporting_text: The encoded MMACHC protein binds intracellular Cbl derivatives with
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    summary: >-
      Electronic annotation to cytosol from the UniProt subcellular-location mapping. Consistent
      with the experimentally determined cytosolic localization of MMACHC.
    action: ACCEPT
    reason: >-
      Correct; matches direct experimental evidence for cytosolic localization.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0033787
    label: cyanocobalamin reductase (cyanide-eliminating) (NADP+) activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: >-
      Electronic annotation of cyanocobalamin reductase activity via RHEA:16113/EC:1.16.1.6
      mapping. This exactly matches the experimentally established decyanation reaction of MMACHC.
    action: ACCEPT
    reason: >-
      Correct EC/RHEA-to-GO mapping for a directly demonstrated catalytic activity; core function.
    supported_by:
    - reference_id: PMID:25809485
      supporting_text: catalyzes the elimination of the upper axial ligand in cobalamin
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:23825108
  qualifier: enables
  review:
    summary: >-
      IntAct IPI capturing the interaction of MMACHC with methionine synthase (MTR, Q99707), part
      of the cytosolic cobalamin-processing complex. The specific partner is biologically relevant
      but the bare "protein binding" term is uninformative.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Per curation guidelines, "protein binding" (GO:0005515) is uninformative; the informative
      content (MMACHC-MTR interaction) is captured by the interaction data and the process/complex
      annotations. Retain the underlying IPI evidence but do not treat as a core molecular function.
    supported_by:
    - reference_id: PMID:23825108
      supporting_text: interaction of MS with MMACHC may
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:33961781
  qualifier: enables
  review:
    summary: >-
      IntAct IPI from a proteome-scale interactome study capturing an interaction with CCT6B
      (Q92526, a CCT/TRiC chaperonin subunit). High-throughput, uninformative bare protein-binding
      annotation.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      "Protein binding" is uninformative and this is a high-throughput proteome-scale contact with
      a chaperonin subunit, not an established functional partnership. Keep the evidence but not as
      a core function.
    supported_by:
    - reference_id: PMID:33961781
      supporting_text: interactome
- term:
    id: GO:0009235
    label: cobalamin metabolic process
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9759218
  qualifier: involved_in
  review:
    summary: >-
      Reactome traceable-author annotation placing MMACHC in cobalamin metabolism. Concordant with
      the core biological role and with the IDA/IBA cobalamin-metabolic-process annotations.
    action: ACCEPT
    reason: >-
      Correct and core; Reactome pathway curation agrees with experimental evidence.
    supported_by:
    - reference_id: PMID:25809485
      supporting_text: assimilation of dietary cobalamin into the active cofactor forms
- term:
    id: GO:0033787
    label: cyanocobalamin reductase (cyanide-eliminating) (NADP+) activity
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-3149519
  qualifier: enables
  review:
    summary: >-
      Reactome TAS for the "MMACHC decyanates CNCbl" reaction, annotated to cyanocobalamin reductase
      activity. Matches the directly demonstrated decyanation function.
    action: ACCEPT
    reason: >-
      Correct and core; consistent with EXP/IDA evidence for decyanation.
    supported_by:
    - reference_id: PMID:18779575
      supporting_text: the trafficking chaperone, MMACHC, catalyzes a
- term:
    id: GO:0005654
    label: nucleoplasm
  evidence_type: IDA
  original_reference_id: GO_REF:0000052
  qualifier: located_in
  review:
    summary: >-
      HPA immunofluorescence IDA reporting a nucleoplasmic signal. MMACHC is a soluble cytosolic
      enzyme; dedicated localization studies place it in the cytoplasm/cytosol, not the nucleus.
      A nuclear pool is not part of its established biology.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Not a core or well-supported localization. The high-throughput antibody signal conflicts with
      dedicated subcellular-localization work showing MMACHC is cytoplasmic; treat as an
      over-annotation rather than removing the underlying HPA observation.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0033787
    label: cyanocobalamin reductase (cyanide-eliminating) (NADP+) activity
  evidence_type: EXP
  original_reference_id: PMID:18779575
  qualifier: enables
  review:
    summary: >-
      Direct experimental demonstration that MMACHC catalyzes reductive decyanation of
      cyanocobalamin, cleaving the Co-CN bond using NADPH-derived electrons to yield cob(II)alamin.
      This is the founding evidence for the decyanase activity.
    action: ACCEPT
    reason: >-
      Core molecular function, directly demonstrated in the paper that first defined the reaction.
    supported_by:
    - reference_id: PMID:18779575
      supporting_text: the trafficking chaperone, MMACHC, catalyzes a
    - reference_id: PMID:18779575
      supporting_text: The product, cob(II)alamin, is a known substrate for assimilation into
- term:
    id: GO:0033787
    label: cyanocobalamin reductase (cyanide-eliminating) (NADP+) activity
  evidence_type: EXP
  original_reference_id: PMID:21697092
  qualifier: enables
  review:
    summary: >-
      Structural and biochemical characterization confirming MMACHC's reductive decyanation of
      cyanocobalamin, using FMN or FAD as prosthetic group on a divergent flavin-reductase scaffold.
    action: ACCEPT
    reason: >-
      Core molecular function, directly demonstrated and structurally rationalized.
    supported_by:
    - reference_id: PMID:21697092
      supporting_text: the reductive decyanation of
    - reference_id: PMID:21697092
      supporting_text: can use FMN or FAD as a prosthetic group to catalyze reductive decyanation
- term:
    id: GO:0033787
    label: cyanocobalamin reductase (cyanide-eliminating) (NADP+) activity
  evidence_type: EXP
  original_reference_id: PMID:25809485
  qualifier: enables
  review:
    summary: >-
      Mutational/kinetic study confirming decyanation activity of MMACHC (CblC) and showing it is
      largely preserved in R161Q/R161G mutants (which instead lose dealkylation), reinforcing the
      decyanase assignment.
    action: ACCEPT
    reason: >-
      Core molecular function, directly demonstrated; decyanation is separable from dealkylation.
    supported_by:
    - reference_id: PMID:25809485
      supporting_text: decreased dealkylation but not decyanation
- term:
    id: GO:0016765
    label: transferase activity, transferring alkyl or aryl (other than methyl) groups
  evidence_type: EXP
  original_reference_id: PMID:19801555
  qualifier: enables
  review:
    summary: >-
      Direct demonstration that MMACHC dealkylates alkylcobalamins (methyl- and
      adenosylcobalamin) by using the thiolate of glutathione for nucleophilic displacement,
      generating cob(I)alamin and an S-alkylglutathione (EC 2.5.1.151). GOA maps this
      glutathione S-alkyltransferase chemistry to GO:0016765.
    action: ACCEPT
    reason: >-
      Core molecular function (the alkylcobalamin dealkylase / GSH S-alkyltransferase activity),
      directly demonstrated. No dedicated GO term for EC 2.5.1.151 exists, so this alkyltransferase
      term is the best available representation.
    supported_by:
    - reference_id: PMID:19801555
      supporting_text: using the thiolate of glutathione for nucleophilic
    - reference_id: PMID:19801555
      supporting_text: cysteine and homocysteine, cannot
- term:
    id: GO:0016765
    label: transferase activity, transferring alkyl or aryl (other than methyl) groups
  evidence_type: EXP
  original_reference_id: PMID:21697092
  qualifier: enables
  review:
    summary: >-
      Structural study confirming MMACHC's glutathione transferase (dealkylation) activity toward
      alkylcobalamins on a scaffold unrelated to the classical GST superfamily.
    action: ACCEPT
    reason: >-
      Core molecular function, directly demonstrated and structurally characterized.
    supported_by:
    - reference_id: PMID:21697092
      supporting_text: the dealkylation of alkylcobalamins
    - reference_id: PMID:21697092
      supporting_text: the first example of an enzyme with glutathione transferase
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IDA
  original_reference_id: PMID:23270877
  qualifier: located_in
  review:
    summary: >-
      Direct experimental determination (immunofluorescence and subcellular fractionation) that
      MMACHC is cytosolic. This is the definitive localization annotation for the protein.
    action: ACCEPT
    reason: >-
      Core cellular component; directly demonstrated cytosolic localization.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IDA
  original_reference_id: PMID:23270877
  qualifier: is_active_in
  review:
    summary: >-
      Direct evidence that MMACHC is active in the cytosol, where it performs cobalamin processing.
      Same study as the located_in cytosol annotation, with the active-in qualifier reflecting the
      site of catalysis.
    action: ACCEPT
    reason: >-
      Core cellular component with appropriate is_active_in qualifier; cytosol is where MMACHC
      carries out cobalamin processing.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0016491
    label: oxidoreductase activity
  evidence_type: IDA
  original_reference_id: PMID:21697092
  qualifier: enables
  review:
    summary: >-
      Oxidoreductase activity reflecting the reductive decyanation reaction of MMACHC. Correct but
      a general parent of the specific cyanocobalamin reductase term.
    action: MODIFY
    reason: >-
      The essence is correct (MMACHC is a reductase) but the term is too general; the specific
      cyanocobalamin reductase term (GO:0033787) is the informative representation of this activity.
    proposed_replacement_terms:
    - id: GO:0033787
      label: cyanocobalamin reductase (cyanide-eliminating) (NADP+) activity
    supported_by:
    - reference_id: PMID:21697092
      supporting_text: the reductive decyanation of
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:27771510
  qualifier: enables
  review:
    summary: >-
      IPI capturing MMACHC interactions within the cytosolic cobalamin-processing complex
      (with MTR/Q99707, MMADHC/Q9H3L0, MTRR/Q9UBK8). Biologically important partners, but bare
      "protein binding" is uninformative.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      "Protein binding" (GO:0005515) conveys no functional specificity; the meaningful content (the
      MMACHC-MMADHC-MTR-MTRR processing complex) is better captured elsewhere. Retain the IPI
      evidence but not as a core molecular function.
    supported_by:
    - reference_id: PMID:27771510
      supporting_text: at least MS, MSR, MMACHC and MMADHC
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:25535791
  qualifier: enables
  review:
    summary: >-
      IPI capturing MMACHC interactions with the lysosomal cobalamin exporters LMBD1/LMBRD1
      (Q9NUN5) and ABCD4 (O14678), which deliver cobalamin to cytosolic MMACHC. Meaningful partners,
      but the bare protein-binding term is uninformative.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      "Protein binding" is uninformative; the biologically relevant LMBD1/ABCD4 interactions are
      captured by the interaction data. Keep the evidence, not as a core molecular function.
    supported_by:
    - reference_id: PMID:25535791
      supporting_text: interacts with LMBD1 and ABCD4 with low nanomolar affinity
- term:
    id: GO:0006749
    label: glutathione metabolic process
  evidence_type: IDA
  original_reference_id: PMID:19801555
  qualifier: involved_in
  review:
    summary: >-
      IDA linking MMACHC to glutathione metabolism because its alkylcobalamin dealkylase reaction
      consumes glutathione (as nucleophile) to form S-alkylglutathione. This is a genuine but
      ancillary consequence of the core cobalamin-processing chemistry rather than a primary role
      in glutathione homeostasis.
    action: KEEP_AS_NON_CORE
    reason: >-
      MMACHC uses glutathione as a co-substrate in dealkylation, so involvement in glutathione
      metabolism is defensible, but MMACHC's core role is cobalamin processing, not glutathione
      metabolism per se.
    supported_by:
    - reference_id: PMID:19801555
      supporting_text: using the thiolate of glutathione for nucleophilic
- term:
    id: GO:0006749
    label: glutathione metabolic process
  evidence_type: IDA
  original_reference_id: PMID:22642810
  qualifier: involved_in
  review:
    summary: >-
      Structural IDA corroborating glutathione involvement: MMACHC has an arginine-rich pocket that
      binds GSH for dealkylation. As above, glutathione is a co-substrate of the core dealkylation
      reaction rather than the protein's primary metabolic role.
    action: KEEP_AS_NON_CORE
    reason: >-
      Glutathione is consumed in the dealkylation reaction; annotation is defensible but ancillary
      to the core cobalamin-processing function.
    supported_by:
    - reference_id: PMID:22642810
      supporting_text: arginine-rich pocket close to the Cbl
- term:
    id: GO:0009235
    label: cobalamin metabolic process
  evidence_type: IDA
  original_reference_id: PMID:19700356
  qualifier: involved_in
  review:
    summary: >-
      Direct experimental evidence placing MMACHC in cobalamin metabolism: wild-type MMACHC binds
      cyanocobalamin base-off and reductively decyanates it to cob(II)alamin, the precursor for the
      active cofactor forms.
    action: ACCEPT
    reason: >-
      Core biological process, directly demonstrated.
    supported_by:
    - reference_id: PMID:19700356
      supporting_text: wild-type MMACHC is able to reductively decyanate
- term:
    id: GO:0009235
    label: cobalamin metabolic process
  evidence_type: IDA
  original_reference_id: PMID:22642810
  qualifier: involved_in
  review:
    summary: >-
      Direct evidence (structure + biochemistry) that MMACHC processes cobalamin derivatives,
      exhibiting FMN-dependent decyanase and GSH-dependent dealkylase activities central to
      cobalamin metabolism.
    action: ACCEPT
    reason: >-
      Core biological process, directly demonstrated.
    supported_by:
    - reference_id: PMID:22642810
      supporting_text: dealkylase activity toward alkyl-Cbls
- term:
    id: GO:0016491
    label: oxidoreductase activity
  evidence_type: IDA
  original_reference_id: PMID:19801555
  qualifier: enables
  review:
    summary: >-
      Oxidoreductase activity annotation reflecting MMACHC's reductive processing of cobalamin.
      Correct but a general parent of the specific cyanocobalamin reductase activity.
    action: MODIFY
    reason: >-
      Too general; the specific cyanocobalamin reductase term (GO:0033787) informatively captures
      MMACHC's reductase activity.
    proposed_replacement_terms:
    - id: GO:0033787
      label: cyanocobalamin reductase (cyanide-eliminating) (NADP+) activity
    supported_by:
    - reference_id: PMID:19801555
      supporting_text: transferase activity of MMACHC
- term:
    id: GO:0032451
    label: demethylase activity
  evidence_type: IDA
  original_reference_id: PMID:19801555
  qualifier: enables
  review:
    summary: >-
      IDA annotating demethylase activity based on MMACHC dealkylating methylcobalamin. The reaction
      is a glutathione-dependent S-alkyl (methyl) transfer to glutathione, not oxidative/hydrolytic
      substrate demethylation; the essence (methyl removal from cobalamin) is right but the term is
      the wrong chemistry.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      The chemistry is a glutathione S-alkyltransferase (EC 2.5.1.151), better represented by the
      alkyl transferase term (GO:0016765) that GOA already applies from the same paper. Treat
      demethylase activity as an imprecise over-annotation of the methylcobalamin dealkylation.
    proposed_replacement_terms:
    - id: GO:0016765
      label: transferase activity, transferring alkyl or aryl (other than methyl) groups
    supported_by:
    - reference_id: PMID:19801555
      supporting_text: using the thiolate of glutathione for nucleophilic
- term:
    id: GO:0033787
    label: cyanocobalamin reductase (cyanide-eliminating) (NADP+) activity
  evidence_type: IDA
  original_reference_id: PMID:19700356
  qualifier: enables
  review:
    summary: >-
      Direct experimental evidence for cyanocobalamin reductase (decyanase) activity: wild-type
      MMACHC reductively decyanates CNCbl to cob(II)alamin given NADPH and FAD.
    action: ACCEPT
    reason: >-
      Core molecular function, directly demonstrated.
    supported_by:
    - reference_id: PMID:19700356
      supporting_text: wild-type MMACHC is able to reductively decyanate
- term:
    id: GO:0042803
    label: protein homodimerization activity
  evidence_type: IPI
  original_reference_id: PMID:22642810
  qualifier: enables
  review:
    summary: >-
      MMACHC forms a domain-swapped homodimer (reciprocal exchange of a conserved PNRRP loop);
      dimerization is triggered by binding FMN or adenosylcobalamin and is required for proper
      dealkylation activity. Directly demonstrated by structure and solution studies.
    action: ACCEPT
    reason: >-
      Substrate/cofactor-triggered homodimerization is a genuine, functionally relevant molecular
      property directly demonstrated for MMACHC.
    supported_by:
    - reference_id: PMID:22642810
      supporting_text: dimerize to mediate the reciprocal
- term:
    id: GO:0043295
    label: glutathione binding
  evidence_type: IDA
  original_reference_id: PMID:22642810
  qualifier: enables
  review:
    summary: >-
      MMACHC has an arginine-rich pocket adjacent to the cobalamin site that binds glutathione,
      required for the dealkylation reaction; mutation of the conserved arginines disrupts GSH
      binding and dealkylation. Directly demonstrated structurally.
    action: ACCEPT
    reason: >-
      Glutathione binding is a genuine, mechanistically essential molecular function underpinning
      the alkylcobalamin dealkylase activity.
    supported_by:
    - reference_id: PMID:22642810
      supporting_text: arginine-rich pocket close to the Cbl
- term:
    id: GO:0070988
    label: demethylation
  evidence_type: IDA
  original_reference_id: PMID:19801555
  qualifier: involved_in
  review:
    summary: >-
      Biological-process annotation of demethylation, again reflecting methylcobalamin dealkylation.
      As with GO:0032451, the underlying reaction is glutathione-dependent S-alkyl transfer, so the
      generic "demethylation" process term imprecisely characterizes the cobalamin-processing role.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      The process is more accurately the cobalamin metabolic/dealkylation process; generic
      demethylation over-annotates the methylcobalamin dealkylation step and is redundant with the
      cobalamin metabolic process annotations.
    supported_by:
    - reference_id: PMID:19801555
      supporting_text: using the thiolate of glutathione for nucleophilic
- term:
    id: GO:0071949
    label: FAD binding
  evidence_type: IDA
  original_reference_id: PMID:19700356
  qualifier: enables
  review:
    summary: >-
      Direct evidence that MMACHC uses FAD (with NADPH) for reductive decyanation of cyanocobalamin.
      FAD binding is a core cofactor function of this flavin-reductase-family enzyme.
    action: ACCEPT
    reason: >-
      Core cofactor-binding molecular function, directly demonstrated.
    supported_by:
    - reference_id: PMID:19700356
      supporting_text: wild-type MMACHC is able to reductively decyanate
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:23415655
  qualifier: enables
  review:
    summary: >-
      IPI capturing the MMACHC (CblC) - MMADHC (CblD, Q9H3L0) interaction, an adapter partnership
      that partitions cobalamin cofactor between the AdoCbl and MeCbl routes. Biologically important
      but the bare protein-binding term is uninformative.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      "Protein binding" is uninformative; the meaningful MMACHC-MMADHC adapter interaction is
      captured by the interaction/complex information. Keep the IPI evidence, not as a core MF.
    supported_by:
    - reference_id: PMID:23415655
      supporting_text: adapter function for CblD
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IDA
  original_reference_id: PMID:23270877
  qualifier: located_in
  review:
    summary: >-
      Direct immunofluorescence/fractionation evidence that MMACHC is cytoplasmic. Correct, though
      the more precise cytosol term (also annotated from this same study) better represents the
      soluble localization.
    action: MODIFY
    reason: >-
      Correct localization but "cytoplasm" is a general parent; the specific cytosol term
      (GO:0005829) is directly supported and more informative.
    proposed_replacement_terms:
    - id: GO:0005829
      label: cytosol
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0009235
    label: cobalamin metabolic process
  evidence_type: IMP
  original_reference_id: PMID:23825108
  qualifier: involved_in
  review:
    summary: >-
      IMP evidence: in cblC cells, MMACHC mutations disrupt formation of cob(II)alamin, and the
      MMACHC-methionine-synthase interaction is implicated in regulating cellular cobalamin
      processing, supporting MMACHC's role in cobalamin metabolism.
    action: ACCEPT
    reason: >-
      Core biological process; loss/mutation of MMACHC impairs cobalamin processing, consistent with
      the direct catalytic evidence.
    supported_by:
    - reference_id: PMID:23825108
      supporting_text: interaction of MS with MMACHC may
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-3318576
  qualifier: located_in
  review:
    summary: >-
      Reactome TAS localizing MMACHC to the cytosol (in the "Defective MMACHC does not reduce Cbl"
      reaction context). Consistent with the experimentally established cytosolic localization.
    action: ACCEPT
    reason: >-
      Correct cytosolic localization; consistent with the direct IDA cytosol evidence.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-3318590
  qualifier: located_in
  review:
    summary: >-
      Reactome TAS cytosol annotation (in the "Defective MMACHC does not decyanate CNCbl" reaction
      context). Consistent with the established cytosolic localization.
    action: ACCEPT
    reason: >-
      Correct cytosolic localization; consistent with the direct IDA cytosol evidence.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-3095889
  qualifier: located_in
  review:
    summary: >-
      Reactome TAS cytosol annotation (MMACHC dealkylates RCbl reaction context). Consistent with
      the established cytosolic localization.
    action: ACCEPT
    reason: >-
      Correct cytosolic localization; consistent with the direct IDA cytosol evidence.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-3149494
  qualifier: located_in
  review:
    summary: >-
      Reactome TAS cytosol annotation (MMACHC:cob(II)alamin binds MMADHC reaction context).
      Consistent with the established cytosolic localization.
    action: ACCEPT
    reason: >-
      Correct cytosolic localization; consistent with the direct IDA cytosol evidence.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-3149519
  qualifier: located_in
  review:
    summary: >-
      Reactome TAS cytosol annotation (MMACHC decyanates CNCbl reaction context). Consistent with
      the established cytosolic localization.
    action: ACCEPT
    reason: >-
      Correct cytosolic localization; consistent with the direct IDA cytosol evidence.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-3149563
  qualifier: located_in
  review:
    summary: >-
      Reactome TAS cytosol annotation (MMADHC targets transport of cytosolic cob(II)alamin to
      mitochondria reaction context). Consistent with the established cytosolic localization of
      MMACHC.
    action: ACCEPT
    reason: >-
      Correct cytosolic localization; consistent with the direct IDA cytosol evidence.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-3204318
  qualifier: located_in
  review:
    summary: >-
      Reactome TAS cytosol annotation (cob(II)alamin transferred from MMACHC:MMADHC:cob(II)alamin
      to MTRR:MTR reaction context). Consistent with the established cytosolic localization.
    action: ACCEPT
    reason: >-
      Correct cytosolic localization; consistent with the direct IDA cytosol evidence.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-3318571
  qualifier: located_in
  review:
    summary: >-
      Reactome TAS cytosol annotation (Defective MMADHC does not bind MMACHC:B12r reaction context).
      Consistent with the established cytosolic localization.
    action: ACCEPT
    reason: >-
      Correct cytosolic localization; consistent with the direct IDA cytosol evidence.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9759549
  qualifier: located_in
  review:
    summary: >-
      Reactome TAS cytosol annotation (Cob(I)alamin bound to MMACHC is oxidized to cob(II)alamin
      reaction context). Consistent with the established cytosolic localization.
    action: ACCEPT
    reason: >-
      Correct cytosolic localization; consistent with the direct IDA cytosol evidence.
    supported_by:
    - reference_id: PMID:23270877
      supporting_text: cytoplasmic while MMADHC is both mitochondrial and
core_functions:
- description: >-
    Reductive decyanation of cyanocobalamin: cleaves the Co-CN bond of dietary cyanocob(III)alamin
    using FAD/FMN and NADPH to produce cob(II)alamin plus cyanide, generating the common
    intermediate for downstream active-cofactor synthesis (EC 1.16.1.6).
  molecular_function:
    id: GO:0033787
    label: cyanocobalamin reductase (cyanide-eliminating) (NADP+) activity
  directly_involved_in:
  - id: GO:0009235
    label: cobalamin metabolic process
  locations:
  - id: GO:0005829
    label: cytosol
  supported_by:
  - reference_id: PMID:18779575
    supporting_text: the trafficking chaperone, MMACHC, catalyzes a
  - reference_id: PMID:19700356
    supporting_text: wild-type MMACHC is able to reductively decyanate
- description: >-
    Glutathione-dependent dealkylation of alkylcobalamins: transfers the alkyl group (methyl from
    methylcobalamin, adenosyl from adenosylcobalamin) to the thiolate of glutathione by nucleophilic
    displacement, yielding cob(I)alamin and an S-alkylglutathione (EC 2.5.1.151), so incoming
    alkylcobalamins are converted to a common intermediate for both cofactor pools.
  molecular_function:
    id: GO:0016765
    label: transferase activity, transferring alkyl or aryl (other than methyl) groups
  directly_involved_in:
  - id: GO:0009235
    label: cobalamin metabolic process
  locations:
  - id: GO:0005829
    label: cytosol
  supported_by:
  - reference_id: PMID:19801555
    supporting_text: using the thiolate of glutathione for nucleophilic
  - reference_id: PMID:21697092
    supporting_text: the first example of an enzyme with glutathione transferase
- description: >-
    Cobalamin binding: binds incoming cob(III)alamin derivatives in the base-off configuration
    (dimethylbenzimidazole displaced from the cobalt), positioning the substrate for decyanation or
    dealkylation.
  molecular_function:
    id: GO:0031419
    label: cobalamin binding
  directly_involved_in:
  - id: GO:0009235
    label: cobalamin metabolic process
  locations:
  - id: GO:0005829
    label: cytosol
  supported_by:
  - reference_id: PMID:19700356
    supporting_text: MMACHC binds CNCbl in the base-off form
- description: >-
    Glutathione binding: an arginine-rich pocket adjacent to the cobalamin site binds glutathione,
    the co-substrate required for the alkylcobalamin dealkylase reaction.
  molecular_function:
    id: GO:0043295
    label: glutathione binding
  locations:
  - id: GO:0005829
    label: cytosol
  supported_by:
  - reference_id: PMID:22642810
    supporting_text: arginine-rich pocket close to the Cbl
- description: >-
    FAD binding: binds the flavin prosthetic group (FAD or FMN) required for NADPH-dependent
    reductive decyanation of cyanocobalamin.
  molecular_function:
    id: GO:0071949
    label: FAD binding
  locations:
  - id: GO:0005829
    label: cytosol
  supported_by:
  - reference_id: PMID:19700356
    supporting_text: wild-type MMACHC is able to reductively decyanate
  - reference_id: PMID:21697092
    supporting_text: can use FMN or FAD as a prosthetic group to catalyze reductive decyanation
proposed_new_terms:
- proposed_name: alkylcobalamin:glutathione S-alkyltransferase activity
  proposed_definition: >-
    Catalysis of the reaction: an R-cob(III)alamin + glutathione = cob(I)alamin + an S-substituted
    glutathione + H(+). This is the EC 2.5.1.151 activity of MMACHC/CblC that dealkylates
    alkylcobalamins (methylcobalamin, adenosylcobalamin) using glutathione as the nucleophile.
  justification: >-
    GO currently has no molecular-function term specific to EC 2.5.1.151; MMACHC's dealkylase
    activity is mapped to the general parent GO:0016765 (transferase, transferring alkyl or aryl
    other than methyl) and, imprecisely, to demethylase activity. A dedicated term would allow
    accurate annotation of this well-characterized cobalamin-processing chemistry.
  proposed_parent:
    id: GO:0016765
    label: transferase activity, transferring alkyl or aryl (other than methyl) groups
suggested_questions:
- question: >-
    What are the physiological electron donor(s) and cytosolic flavoprotein oxidoreductase
    partner(s) that supply reducing equivalents to MMACHC for reductive decyanation in vivo?
- question: >-
    How is cobalamin handed off from MMACHC to MMADHC and the downstream MTR (methyl branch) versus
    the mitochondrial MMUT (adenosyl branch), and what governs the partitioning between the two arms?
suggested_experiments:
- description: >-
    Structure/cryo-EM of the cytosolic MMACHC-MMADHC-MTR(-MTRR) processing complex with bound
    cobalamin to define the cofactor hand-off pathway and the structural basis of partitioning.
- description: >-
    Cell-based flux assays in MMACHC-null cells complemented with decyanation- versus
    dealkylation-selective mutants (e.g. R161Q) to dissect the relative in vivo contributions of the
    two catalytic activities to MeCbl and AdoCbl pools.
references:
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000044
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
    vocabulary mapping, accompanied by conservative changes to GO terms applied by
    UniProt
  findings: []
- id: GO_REF:0000052
  title: Gene Ontology annotation based on curation of immunofluorescence data
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:18779575
  title: Decyanation of vitamin B12 by a trafficking chaperone.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Founding paper establishing MMACHC reductive decyanation of cyanocobalamin to cob(II)alamin;
      directly supports the core cyanocobalamin reductase function and the "most common inborn error"
      framing. Abstract-only cache, PubMed-verified title.
- id: PMID:19700356
  title: Mechanism of vitamin B12-responsiveness in cblC methylmalonic aciduria with
    homocystinuria.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Shows base-off cobalamin binding, NADPH/FAD-dependent decyanation, and combined MMA +
      homocystinuria in cblC; supports decyanase activity, cobalamin binding, FAD binding, and the
      disease framing.
- id: PMID:19801555
  title: A human vitamin B12 trafficking protein uses glutathione transferase activity
    for processing alkylcobalamins.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Defines the glutathione-dependent alkylcobalamin dealkylase (S-alkyltransferase) activity
      (EC 2.5.1.151) and that cysteine/homocysteine cannot substitute; supports GO:0016765 and the
      demethylase/demethylation over-annotation reasoning.
- id: PMID:21697092
  title: Structural basis of multifunctionality in a vitamin B12-processing enzyme.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Crystal structures (with MeCbl) establishing the divergent flavin-reductase scaffold, FMN/FAD
      use, and dual decyanation/dealkylation chemistry; supports decyanase, alkyltransferase, and FAD
      binding functions.
- id: PMID:22642810
  title: Structure of MMACHC reveals an arginine-rich pocket and a domain-swapped
    dimer for its B12 processing function.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Defines the arginine-rich glutathione-binding pocket, GSH-dependent dealkylation, and the
      substrate/cofactor-triggered domain-swapped homodimer; supports glutathione binding, dealkylase
      activity, and homodimerization annotations.
- id: PMID:23270877
  title: Subcellular location of MMACHC and MMADHC, two human proteins central to
    intracellular vitamin B(12) metabolism.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Definitive subcellular-localization study showing MMACHC is cytoplasmic/cytosolic (vs the
      partly mitochondrial MMADHC); supports the cytosol core location and argues against a nuclear
      pool.
- id: PMID:23415655
  title: The C-terminal domain of CblD interacts with CblC and influences intracellular
    cobalamin partitioning.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Establishes the MMACHC(CblC)-MMADHC(CblD) interaction and CblD's adapter role in partitioning
      cobalamin between AdoCbl and MeCbl routes; supports the protein-binding IPI (as MMADHC partner).
- id: PMID:23825108
  title: 'Interaction between methionine synthase isoforms and MMACHC: characterization
    in cblG-variant, cblG and cblC inherited causes of megaloblastic anaemia.'
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Documents MMACHC-methionine synthase interaction and that cblC mutations disrupt cob(II)alamin
      formation; supports the cobalamin metabolic process IMP and the MTR protein-binding IPI.
- id: PMID:25535791
  title: 'Purification and interaction analyses of two human lysosomal vitamin B12
    transporters: LMBD1 and ABCD4.'
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Shows cytoplasmic MMACHC interacts with lysosomal exporters LMBD1 and ABCD4 with nanomolar
      affinity, delivering cobalamin to the cytosol; supports the corresponding protein-binding IPI.
- id: PMID:25809485
  title: Pathogenic mutations differentially affect the catalytic activities of the
    human B12-processing chaperone CblC and increase futile redox cycling.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Confirms decyanation vs dealkylation are separable (R161Q/G lose dealkylation but retain
      decyanation) and that MMACHC removes the upper axial ligand of incoming cobalamin; supports the
      decyanase function and disease mechanism.
- id: PMID:27771510
  title: Methionine synthase and methionine synthase reductase interact with MMACHC
    and with MMADHC.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Establishes the cytosolic multiprotein cobalamin-processing complex (MS/MTR, MSR/MTRR, MMACHC,
      MMADHC); supports the MTR/MMADHC/MTRR protein-binding IPI and the process context.
- 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: >-
      Proteome-scale interactome (HuRI/BioPlex-type) reporting a high-throughput MMACHC-CCT6B
      contact; correctly cited for the IPI but a non-functional/uninformative interaction, hence the
      protein-binding annotation is marked over-annotated.
- id: Reactome:R-HSA-3095889
  title: MMACHC dealkylates RCbl
  findings: []
- id: Reactome:R-HSA-3149494
  title: MMACHC:cob(II)alamin binds MMADHC
  findings: []
- id: Reactome:R-HSA-3149519
  title: MMACHC decyanates CNCbl
  findings: []
- id: Reactome:R-HSA-3149563
  title: MMADHC targets transport of cytosolic cob(II)alamin to mitochondria
  findings: []
- id: Reactome:R-HSA-3204318
  title: cob(II)alamin is transferred from MMACHC:MMADHC:cob(II)alamin to MTRR:MTR
  findings: []
- id: Reactome:R-HSA-3318571
  title: Defective MMADHC does not bind MMACHC:B12r
  findings: []
- id: Reactome:R-HSA-3318576
  title: Defective MMACHC does not reduce Cbl
  findings: []
- id: Reactome:R-HSA-3318590
  title: Defective MMACHC does not decyanate CNCbl
  findings: []
- id: Reactome:R-HSA-9759218
  title: Cobalamin (Cbl) metabolism
  findings: []
- id: Reactome:R-HSA-9759549
  title: Cob(I)alamin bound to MMACHC is oxidized to cob(II)alamin
  findings: []