id: A7UXI1
gene_symbol: cao-2
product_type: PROTEIN
status: IN_PROGRESS
taxon:
  id: NCBITaxon:367110
  label: Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257
    / FGSC 987)
description: >-
  cao-2 encodes CAO-2 (NCU11424), the second carotenoid cleavage oxygenase (CCO) of Neurospora
  crassa and the paralog of cao-1. In contrast to cao-1 (a stilbenoid/resveratrol cleaver), CAO-2
  is a genuine carotenoid-cleaving enzyme: a torulene dioxygenase (EC 1.13.11.59) that catalyzes the
  committed cleavage step of the neurosporaxanthin biosynthetic pathway. It cleaves the C40 carotene
  torulene with molecular oxygen to yield the C35 apocarotenal 4'-apo-beta-carotenal plus
  3-methyl-2-butenal; the apocarotenal is subsequently oxidized to the carboxylic xanthophyll
  neurosporaxanthin by the aldehyde dehydrogenase YLO-1. CAO-2 is cytosolic, is specific for torulene
  (it does not cleave gamma-carotene in vitro), and its expression is induced by light in a WC-1/WC-2
  (White Collar) dependent manner, as expected for a structural gene of the carotenoid pathway.
  Disruption of cao-2 abolishes neurosporaxanthin production and causes torulene to accumulate.
existing_annotations:
- term:
    id: GO:0010436
    label: carotenoid dioxygenase activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: enables
  review:
    summary: >-
      Phylogenetic (IBA) annotation of carotenoid dioxygenase activity. For CAO-2 this is correct:
      the enzyme is a torulene dioxygenase (EC 1.13.11.59) that oxidatively cleaves the C40 carotene
      torulene, demonstrated directly with purified enzyme (PMID:17610084). No specific GO term for
      torulene dioxygenase activity exists, so this carotenoid-dioxygenase parent is the best available.
    action: ACCEPT
    reason: >-
      Core molecular function, correctly propagated by IBA and independently supported by direct
      experimental evidence. This is the positive-control counterpart to cao-1, where the identical
      family IBA term is refuted; here it is right. (Currently only IBA in GOA despite experimental
      characterization - an IDA upgrade from PMID:17610084 is warranted.)
    supported_by:
      - reference_id: PMID:17610084
        supporting_text: >-
          cleaved torulene to produce beta-apo-4'-carotenal, the corresponding aldehyde of
          neurosporaxanthin
      - reference_id: PMID:17610084
        supporting_text: >-
          lack of gamma-carotene-cleaving activity in vitro
- term:
    id: GO:0016121
    label: carotene catabolic process
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: involved_in
  review:
    summary: >-
      Phylogenetic (IBA) annotation of carotene catabolic process. Accurate in that the carotene
      torulene is consumed/cleaved by CAO-2, but partial: the informative biological process is the
      biosynthesis of the apocarotenoid neurosporaxanthin, of which this torulene cleavage is the
      committed step (disruption abolishes neurosporaxanthin and accumulates torulene, PMID:17610084).
    action: ACCEPT
    reason: >-
      Not wrong - torulene catabolism does occur - but it under-describes the role. The more
      informative process term is apocarotenoid biosynthetic process (GO:0043289), captured in
      core_functions; adding it (with IDA from PMID:17610084) is recommended.
    supported_by:
      - reference_id: PMID:17610084
        supporting_text: >-
          CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the
          neurosporaxanthin biosynthetic pathway
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    summary: >-
      Automated (IEA) cytosol annotation from UniProt subcellular-location mapping, consistent with
      the curated cytoplasmic/cytosolic localization of CAO-2.
    action: ACCEPT
    reason: >-
      Consistent core localization for this soluble cytosolic carotenoid oxygenase.
- term:
    id: GO:0016702
    label: oxidoreductase activity, acting on single donors with incorporation of
      molecular oxygen, incorporation of two atoms of oxygen
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: >-
      InterPro-based (IEA) dioxygenase MF term (incorporation of two oxygen atoms). Correct and
      consistent with the demonstrated torulene dioxygenase activity; a general parent of the
      carotenoid dioxygenase term.
    action: ACCEPT
    reason: >-
      Accurate general dioxygenase MF, corroborating GO:0010436. The specific activity is captured
      by the carotenoid dioxygenase term.
- term:
    id: GO:0043289
    label: apocarotenoid biosynthetic process
  evidence_type: IDA
  qualifier: involved_in
  review:
    summary: >-
      Proposed NEW annotation (not currently in GOA). CAO-2's torulene cleavage is the committed step
      of neurosporaxanthin (a C35 carboxylic apocarotenoid) biosynthesis; disruption abolishes
      neurosporaxanthin and accumulates torulene (PMID:17610084). This apocarotenoid-biosynthesis
      process role is the informative complement to the existing (accurate but partial) carotene
      catabolic process IBA.
    action: NEW
    reason: >-
      Captures the experimentally-established biosynthetic pathway role directly, addressing the
      under-curation of this experimentally-characterized gene (currently only IBA/IEA in GOA).
    supported_by:
      - reference_id: PMID:17610084
        supporting_text: >-
          CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the
          neurosporaxanthin biosynthetic pathway
core_functions:
- description: >-
    Torulene dioxygenase (EC 1.13.11.59): a non-heme iron carotenoid cleavage oxygenase that cleaves
    the C40 carotene torulene with O2 to give the C35 apocarotenal 4'-apo-beta-carotenal plus
    3-methyl-2-butenal - the committed step of neurosporaxanthin (apocarotenoid) biosynthesis. GO:0010436
    is the most specific available MF term (no torulene-dioxygenase term exists).
  molecular_function:
    id: GO:0010436
    label: carotenoid dioxygenase activity
  directly_involved_in:
    - id: GO:0043289
      label: apocarotenoid biosynthetic process
  locations:
    - id: GO:0005829
      label: cytosol
  supported_by:
    - reference_id: PMID:17610084
      supporting_text: >-
        CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the
        neurosporaxanthin biosynthetic pathway
proposed_new_terms:
- proposed_name: torulene dioxygenase activity
  proposed_definition: >-
    Catalysis of the reaction: torulene + O2 = 4'-apo-beta-carotenal + 3-methyl-2-butenal. Oxidative
    cleavage of the C40 carotene torulene at the 4',5' double bond by a non-heme iron carotenoid
    cleavage oxygenase, producing a C35 apocarotenal.
  justification: >-
    CAO-2 (and its ortholog CarT in Fusarium fujikuroi) has a specific, experimentally-defined
    activity (EC 1.13.11.59) for which no dedicated GO MF term exists; only the parent GO:0010436
    (carotenoid dioxygenase activity) is available. A specific term would let the neurosporaxanthin
    pathway step be annotated precisely.
  proposed_parent:
    id: GO:0010436
    label: carotenoid dioxygenase activity
  supported_by:
    - reference_id: PMID:17610084
      supporting_text: >-
        cleaved torulene to produce beta-apo-4'-carotenal, the corresponding aldehyde of
        neurosporaxanthin
suggested_questions:
- question: >-
    CAO-2 is experimentally characterized (disruption phenotype and purified-enzyme assay,
    PMID:17610084) yet carries only IBA/IEA GO annotations - should GOA add IDA annotations for
    torulene dioxygenase activity and apocarotenoid/neurosporaxanthin biosynthetic process?
- question: >-
    Since Neurospora lacks an identified retinal-forming enzyme and CAO-2's product beta-apo-4'-carotenal
    has been proposed as a candidate physiological chromophore of the NOP-1 rhodopsin, does CAO-2 (via
    this apocarotenal) contribute to NOP-1 photobiology in addition to its neurosporaxanthin role?
suggested_experiments:
- description: >-
    Structural determination of CAO-2 (no experimental structure exists, unlike cao-1) to define the
    carotenoid-binding cleft and the basis of torulene versus gamma-carotene selectivity, enabling a
    direct structural comparison with the stilbenoid-adapted cleft of CAO-1.
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: []
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Unlike in cao-1, the IBA carotenoid-dioxygenase and carotene-catabolic terms are CORRECT for
      CAO-2 (a genuine torulene dioxygenase). This paralog is the positive control showing the same
      family IBA is right for one paralog and wrong for the other.
- 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: PMID:17610084
  title: Identification of the gene responsible for torulene cleavage in the Neurospora
    carotenoid pathway.
  findings:
  - statement: >-
      CAO-2 is the torulene dioxygenase of the neurosporaxanthin biosynthetic pathway; purified enzyme
      cleaves torulene to beta-apo-4'-carotenal, and cao-2 disruption abolishes neurosporaxanthin and
      accumulates torulene.
    supporting_text: >-
      CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin
      biosynthetic pathway
  - statement: >-
      cao-2 expression is light-induced in a White Collar (WC-1/WC-2) dependent manner, the opposite of
      the light-independent cao-1.
    supporting_text: >-
      cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Primary genetic + biochemical characterization of CAO-2 (torulene-accumulating mutants, targeted
      disruption, purified-enzyme assay). Abstract-only cache, but the abstract is detailed and the
      claims are anchored to it. Basis for the recommended IDA upgrades.