id: Q7S860
gene_symbol: cao-1
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-1 encodes CAO-1 (NCU07008), one of two carotenoid cleavage oxygenase (CCO)
  family members in Neurospora crassa. Despite its family name and "carotenoid
  cleavage oxygenase 1" designation, CAO-1 does not act on carotenoids. It is a
  mononuclear non-heme Fe(II) dioxygenase that oxidatively cleaves the interphenyl
  Calpha-Cbeta double bond of the stilbenoid resveratrol, and of its dihydroxy
  derivative piceatannol, producing aromatic aldehydes (trans-resveratrol yields
  3,5-dihydroxybenzaldehyde plus 4-hydroxybenzaldehyde; piceatannol yields
  3,5-dihydroxybenzaldehyde plus 3,4-dihydroxybenzaldehyde). The enzyme uses a
  four-His-coordinated Fe(II) center shared with carotenoid-cleaving CCOs but has a
  distinct substrate-binding cleft that accommodates stilbenoids rather than
  carotenoids. Biologically CAO-1 acts in stilbene catabolism: expression is strongly
  and specifically induced by resveratrol (a plant phytoalexin) and, unlike genuine
  carotenoid-pathway genes, is not regulated by light. It was originally proposed to
  supply retinal by cleaving beta-carotene, but heterologously expressed CAO-1 does not
  convert beta-carotene or any apocarotenoid tested, so it is not part of carotenoid
  metabolism.
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) propagation of a carotenoid dioxygenase activity from the CCO
      family. This is directly contradicted by experimental characterization: purified
      CAO-1 does not cleave beta-carotene or any carotenoid/apocarotenoid tested. The
      true molecular function is stilbenoid (resveratrol/piceatannol) cleavage, captured
      by the IDA GO:0016702 annotations.
    action: REMOVE
    reason: >-
      An IBA inference refuted by direct experimental evidence (PMID:23893079,
      PMID:28493664). CAO-1 has no demonstrable carotenoid-cleaving activity; keeping this
      term would misrepresent the enzyme's molecular function. GOA already carries a
      corrective NOT carotenoid-metabolism annotation from the same work. Root cause is at
      the PANTHER level: cao-1 is placed in subfamily PTHR10543:SF89, labelled "carotenoid
      9,10(9',10')-cleavage dioxygenase 1", which is functionally heterogeneous - it lumps
      genuine carotenoid cleavers (Arabidopsis CCD1), stilbenoid/resveratrol cleavers
      (U. maydis RCO1, B. fuckeliana rco1, and cao-1), and phenylpropanoid cleavers
      (Pseudomonas isoeugenol monooxygenase) - so the carotenoid label over-propagates to
      the stilbenoid clade.
    propagation_review:
      root_cause: PROPAGATION_BAD
      failure_modes:
        - FUNCTIONAL_DIVERGENCE
      source_entities:
        - source_id: PANTHER:PTN001631894
          source_label: PTHR10543:SF89 ancestral node (IBA source for GO:0010436)
          source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
          comment: >-
            Node/subfamily annotated with carotenoid dioxygenase activity; valid for the
            carotenoid-cleaving members but should not transfer to the stilbenoid-cleaving
            clade containing cao-1. A separate stilbenoid-cleaving node should be recognized
            and annotated with stilbenoid alpha,beta-dioxygenase activity instead.
        - source_id: UniProtKB:P9WPR5
          source_label: M. tuberculosis carotenoid cleavage oxygenase (IBA with/from)
          source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
          comment: >-
            A genuine carotenoid/apocarotenoid cleaver used as an experimental anchor for the
            IBA; its activity does not extend to cao-1, which cleaves stilbenoids not carotenoids.
    supported_by:
      - reference_id: PMID:23893079
        supporting_text: >-
          we tested CAO-1 activity with carotenoid substrates that were, however, not converted
      - reference_id: PMID:23893079
        supporting_text: >-
          Here, we show that CAO-1 is not a carotenoid or apocarotenoid cleavage enzyme.
      - reference_id: file:NEUCR/cao-1/cao-1-hypotheses/function-hypothesis-go-0010436/openscientist.md
        supporting_text: >-
          Verdict: REFUTED (over-annotated)
- term:
    id: GO:0016121
    label: carotene catabolic process
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: involved_in
  review:
    summary: >-
      Phylogenetic (IBA) propagation placing CAO-1 in carotene catabolism. The catabolic
      framing is correct in spirit (CAO-1 is a catabolic double-bond-cleaving oxygenase),
      but the substrate class is wrong: CAO-1 acts on stilbenoids, not carotenes. The
      biologically supported process is stilbene catabolism (degradation of the phytoalexin
      resveratrol).
    action: MODIFY
    reason: >-
      Wrong substrate class for an otherwise reasonable "oxidative catabolic cleavage"
      annotation. Replace with GO:0046272 (stilbene catabolic process), which matches the
      demonstrated resveratrol/piceatannol cleavage activity and the resveratrol-inducible
      expression.
    proposed_replacement_terms:
      - id: GO:0046272
        label: stilbene catabolic process
    propagation_review:
      root_cause: PROPAGATION_BAD
      failure_modes:
        - FUNCTIONAL_DIVERGENCE
      source_entities:
        - source_id: PANTHER:PTN001631894
          source_label: PTHR10543:SF89 ancestral node (IBA source for GO:0016121)
          source_status: SUPPORTS_SOURCE_BUT_NOT_TARGET
          comment: >-
            Carotene catabolic process propagated from the carotenoid-cleaving members of the
            heterogeneous SF89 subfamily; cao-1's catabolic role is on stilbenes, not carotenes.
    supported_by:
      - reference_id: PMID:23893079
        supporting_text: >-
          it efficiently cleaved resveratrol and its derivative piceatannol
      - reference_id: PMID:23893079
        supporting_text: >-
          adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light,
          a key regulator of carotenoid metabolism, did not alter them
- 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) annotation of dioxygenase activity (incorporation of two oxygen
      atoms). This is correct and consistent with the experimentally demonstrated
      stilbenoid dioxygenase activity; it is the most specific correct MF term currently
      available in GO for this enzyme.
    action: ACCEPT
    reason: >-
      Accurate general dioxygenase MF term, corroborated by the IDA annotations of the same
      term from PMID:23893079 and PMID:28493664. Represents the core catalytic function.
    supported_by:
      - reference_id: PMID:28493664
        supporting_text: >-
          The enzymatic activity of CAO1 and NOV2 was assessed as previously described using
          either resveratrol or piceatannol as a substrate.
- term:
    id: GO:0016116
    label: carotenoid metabolic process
  evidence_type: IDA
  original_reference_id: PMID:23893079
  qualifier: involved_in
  negated: true
  review:
    summary: >-
      NOT annotation asserting that CAO-1 is not involved in carotenoid metabolism. This is
      strongly supported: heterologously expressed CAO-1 failed to convert beta-carotene or
      any carotenoid/apocarotenoid substrate, and cao-1 expression is light-independent,
      unlike carotenoid-pathway genes.
    action: ACCEPT
    reason: >-
      A correct and valuable negative annotation that corrects the family-derived carotenoid
      assumption. Directly supported by IDA in PMID:23893079.
    supported_by:
      - reference_id: PMID:23893079
        supporting_text: >-
          we tested CAO-1 activity with carotenoid substrates that were, however, not converted
      - reference_id: PMID:23893079
        supporting_text: >-
          Here, we show that CAO-1 is not a carotenoid or apocarotenoid cleavage enzyme.
- term:
    id: GO:0016702
    label: oxidoreductase activity, acting on single donors with incorporation of
      molecular oxygen, incorporation of two atoms of oxygen
  evidence_type: IDA
  original_reference_id: PMID:23893079
  qualifier: enables
  review:
    summary: >-
      Direct experimental (IDA) annotation of dioxygenase activity based on in vitro and in
      vivo assays showing CAO-1 cleaves the interphenyl double bond of resveratrol and
      piceatannol with incorporation of molecular oxygen. Best available MF term for this
      stilbenoid cleavage activity.
    action: ACCEPT
    reason: >-
      Core molecular function directly demonstrated in PMID:23893079. No more specific GO MF
      term (e.g. a stilbene/resveratrol cleavage dioxygenase) currently exists.
    supported_by:
      - reference_id: PMID:23893079
        supporting_text: >-
          it efficiently cleaved resveratrol and its derivative piceatannol
- term:
    id: GO:0016702
    label: oxidoreductase activity, acting on single donors with incorporation of
      molecular oxygen, incorporation of two atoms of oxygen
  evidence_type: IDA
  original_reference_id: PMID:28493664
  qualifier: enables
  review:
    summary: >-
      Direct experimental (IDA) annotation of dioxygenase activity from the structural and
      spectroscopic study, which characterized CAO-1 as a stilbenoid-cleaving CCO with a
      non-heme Fe(II) center and confirmed resveratrol/piceatannol cleavage activity.
    action: ACCEPT
    reason: >-
      Core catalytic function, independently confirmed structurally and biochemically in
      PMID:28493664. Duplicate term id relative to the PMID:23893079 IDA annotation but from
      a distinct reference/experiment.
    supported_by:
      - reference_id: PMID:28493664
        supporting_text: >-
          The crystal structure of a fungal stilbenoid-cleaving CCO, CAO1, reveals strong
          similarity between its iron center and those of carotenoid-cleaving CCOs, but with a
          markedly different substrate-binding cleft
- term:
    id: GO:0005506
    label: iron ion binding
  evidence_type: IDA
  original_reference_id: PMID:28493664
  qualifier: enables
  review:
    summary: >-
      Direct experimental (IDA) annotation of iron binding. Crystallography and spectroscopy
      show a mononuclear non-heme Fe(II) center coordinated by four histidines (the
      conserved CCO four-His motif), essential for catalysis.
    action: ACCEPT
    reason: >-
      Well-supported cofactor-binding function; the catalytic Fe(II) center is defined
      crystallographically (Fe ligands His197, His248, His313, His510 in UniProt).
    supported_by:
      - reference_id: PMID:28493664
        supporting_text: >-
          three to four His-derived imidazole units bound to the iron center in each enzyme,
          consistent with the known four-His coordination motif of CCOs
- term:
    id: GO:1905594
    label: resveratrol binding
  evidence_type: IDA
  original_reference_id: PMID:28493664
  qualifier: enables
  review:
    summary: >-
      Direct experimental (IDA) annotation of resveratrol binding, based on co-crystal
      structures of CAO-1 with resveratrol (and piceatannol) occupying the active-site cleft.
      For CAO-1 resveratrol is the catalytic substrate, not merely a binding ligand. GO is
      obsoleting GO:1905594 resveratrol binding (go-ontology issues #32321/#32333, Jul 2026,
      "not clearly defined and usage has been inconsistent") and reannotating CAO-1 to a
      catalytic-activity term (go-annotation issue #6483).
    action: MODIFY
    reason: >-
      Resveratrol is CAO-1's substrate, so the binding term should be replaced by the catalytic
      activity. GO has now created exactly this term - GO:7770086 "resveratrol dioxygenase
      activity" (def "trans-resveratrol + O2 = 3,5-dihydroxybenzaldehyde + 4-hydroxybenzaldehyde";
      parent GO:0016702; RHEA:73735) - which independently matches this review's proposed term and
      REMOVE-of-carotenoid reasoning (the new term "deliberately avoids placement under carotenoid
      dioxygenase activity"). PROVENANCE: the term comes from go-ontology PR #32332
      (https://github.com/geneontology/go-ontology/pull/32332), merged 2026-07-17, and now resolves
      live in QuickGO
      (https://www.ebi.ac.uk/QuickGO/services/ontology/go/terms/GO%3A7770086) as non-obsolete with
      the definition and RHEA:73735 / PMID:28493664 xrefs quoted above. It is not yet present in the
      oaklib ontology snapshot this repo validates against, so the structured replacement below uses
      the validated parent GO:0016702; the intended replacement is GO:7770086 and should be
      substituted once the snapshot includes it.
    proposed_replacement_terms:
      - id: GO:0016702
        label: oxidoreductase activity, acting on single donors with incorporation of
          molecular oxygen, incorporation of two atoms of oxygen
    supported_by:
      - reference_id: PMID:28493664
        supporting_text: >-
          Crystals of Co-CAO1 in complex with resveratrol and piceatannol were obtained
core_functions:
- description: >-
    Mononuclear non-heme Fe(II) dioxygenase that oxidatively cleaves the interphenyl
    Calpha-Cbeta double bond of the hydroxystilbene resveratrol (and piceatannol),
    incorporating both atoms of molecular oxygen to yield aromatic aldehydes. GO:0016702 is used
    here as the most specific MF term available in the ontology snapshot this repo validates
    against; the specific term GO:7770086 (resveratrol dioxygenase activity, RHEA:73735) was added
    by go-ontology PR #32332 (merged 2026-07-17) and is live in QuickGO, but is not yet in that
    snapshot; a hydroxystilbene grouping is proposed as its parent (see
    proposed_new_terms). Iron and resveratrol (substrate) binding are integral to this activity.
  molecular_function:
    id: GO:0016702
    label: oxidoreductase activity, acting on single donors with incorporation of
      molecular oxygen, incorporation of two atoms of oxygen
  directly_involved_in:
    - id: GO:0046272
      label: stilbene catabolic process
  supported_by:
    - reference_id: PMID:23893079
      supporting_text: >-
        it efficiently cleaved resveratrol and its derivative piceatannol
    - reference_id: PMID:28493664
      supporting_text: >-
        The crystal structure of a fungal stilbenoid-cleaving CCO, CAO1, reveals strong
        similarity between its iron center and those of carotenoid-cleaving CCOs, but with a
        markedly different substrate-binding cleft
proposed_new_terms:
- proposed_name: hydroxystilbene alpha,beta-dioxygenase activity
  proposed_definition: >-
    Catalysis of the oxidative cleavage of the interphenyl Calpha-Cbeta double bond of a
    hydroxystilbene (a stilbenoid bearing free phenolic hydroxyl group(s)), incorporating both
    atoms of molecular oxygen to yield two aromatic aldehydes. Example substrates include
    resveratrol and piceatannol (RHEA:73735, RHEA:73815) and lignostilbene (RHEA:21340).
  justification: >-
    Proposed as a grouping (substrate-class) molecular-function term that sits under GO:0016702 and
    is the PARENT of the existing/added leaf terms GO:0050054 (lignostilbene alpha,beta-dioxygenase
    activity, EC 1.13.11.43) and GO:7770086 (resveratrol dioxygenase activity, RHEA:73735, added Jul
    2026 in go-ontology issue #32332). It captures the evolved chemistry of the fungal/bacterial
    stilbenoid-cleaving CCO subfamily (interphenyl double-bond scission of hydroxystilbenes) at the
    grain a gene product is plausibly selected for, filling the gap between the generic GO:0016702 and
    the single-reaction leaves. Rhea deliberately models only fully-specified leaf reactions and has
    no generic "a hydroxystilbene + O2" reaction, so GO molecular function is the appropriate layer
    for this grouping - mirroring the carotenoid side of the same family, where GO:0010436 (carotenoid
    dioxygenase activity) already groups the specific carotenoid-cleaving activities.
    Scope note (fixing an earlier over-broad draft): the class is HYDROXYstilbene, not all stilbenoids
    (CHEBI:26776) - family members require free hydroxyls (CAO-1 does not cleave non-hydroxylated
    trans-stilbene, pinosylvin lacking the 4'-OH, or fully methoxylated/glycosylated stilbenes), so a
    "stilbenoid"-scoped term would over-claim their specificity.
    Annotation grain: CAO-1 itself is best annotated to the demonstrated leaf activities (GO:7770086
    resveratrol dioxygenase, plus a piceatannol counterpart), while this grouping is the right grain
    for the family/subfamily node (IBA propagation) and would organize the currently-flat set of
    stilbenoid leaf terms. A narrower intermediate ("4'-hydroxystilbene alpha,beta-dioxygenase",
    covering just resveratrol + piceatannol but not lignostilbene) is the tightest fit to CAO-1's own
    range if an enzyme-level grouping is also wanted.
    Nomenclature note: "hydroxystilbene alpha,beta-dioxygenase activity" is a descriptive name coined
    here, not established terminology. The literature calls this class stilbene cleavage oxygenases
    (SCOs) / lignostilbene alpha,beta-dioxygenases (LSDs, EC 1.13.11.43); by the lignostilbene
    precedent the conventional grouping name would be "stilbene alpha,beta-dioxygenase activity"
    (a suitable exact_synonym). The class is chemistry-defined (alpha,beta / interphenyl double-bond
    scission of stilbenes); per-member substrate specificity varies (CAO-1 needs several free
    hydroxyls, bacterial LsdA cleaves even 4-hydroxystilbene) and should be captured by the leaf terms,
    not the grouping. There is substantial structural/mechanistic literature on the class - CAO-1
    co-crystals with resveratrol/piceatannol (PMID:28493664), NOV1 structure + mechanism
    (PMID:27911781), and bacterial LSD residue-level SAR (PMID:31292192). A structural analysis of the
    CAO-1 co-crystals in this repo (file:NEUCR/cao-1/cao-1-bioinformatics/RESULTS.md) shows a
    two-ring-anchor model (4'-OH -> Tyr133/Lys164; 3/5-OH -> Glu383; scissile alkene ~4.6 A over the
    metal) that retrospectively explains the whole empirical panel - so the specificity is now
    structure-explained, though not yet a validated predictor (only the two substrates were
    co-crystallized; no docking/energetics were run).
  proposed_parent:
    id: GO:0016702
    label: oxidoreductase activity, acting on single donors with incorporation of
      molecular oxygen, incorporation of two atoms of oxygen
  proposed_mappings:
    - predicate: skos:narrowMatch
      target_term:
        id: RHEA:73735
        label: trans-resveratrol + O2 = 3,5-dihydroxybenzaldehyde + 4-hydroxybenzaldehyde
    - predicate: skos:narrowMatch
      target_term:
        id: RHEA:73815
        label: piceatannol + O2 = 3,5-dihydroxybenzaldehyde + 3,4-dihydroxybenzaldehyde
    - predicate: skos:narrowMatch
      target_term:
        id: RHEA:21340
        label: lignostilbene + O2 = 2 vanillin
  supported_by:
    - reference_id: PMID:23893079
      supporting_text: >-
        it efficiently cleaved resveratrol and its derivative piceatannol
suggested_questions:
- question: >-
    What is the natural physiological role and selective advantage of resveratrol/stilbene
    cleavage for Neurospora crassa, a primary colonizer of burned vegetation - detoxification
    of plant phytoalexins, competition with resveratrol-producing microbes, or catabolism of
    lignin-derived biphenolics?
suggested_experiments:
- description: >-
    Profile the stilbenoid content (resveratrol, piceatannol, and 4'-hydroxystilbene derivatives)
    of the specific burned host substrates Neurospora colonizes in the wild - especially grasses/
    sugarcane, which accumulate both resveratrol and piceatannol - and test each as a CAO-1
    substrate, to identify the ecologically relevant natural substrate rather than the convenient
    laboratory one.
- description: >-
    Untargeted metabolomics of wild-type versus delta-cao-1 mycelia grown with and without
    resveratrol/piceatannol (and candidate lignin-derived stilbenoids) to identify the full in vivo
    substrate range and downstream fate of the aldehyde cleavage products.
- description: >-
    Competition/co-culture assays of wild-type versus delta-cao-1 Neurospora against
    resveratrol-producing microbes (e.g. Aspergillus, Penicillium, Mucor) to test whether CAO-1
    provides a fitness advantage by degrading a competitor-derived stilbenoid.
- description: >-
    Determine subcellular localization of CAO-1 (e.g. fluorescent fusion or fractionation) to
    test the inference from PMID:23893079 that resveratrol cleavage occurs intracellularly
    (cytoplasm) rather than via secretion.
knowledge_gaps:
- gap_statement: >-
    The natural physiological substrate and biological role of CAO-1 in Neurospora crassa are
    undetermined. The in vitro substrates resveratrol and piceatannol are plant stilbenoids of
    uncertain ecological relevance to this fungus, and it is unknown whether the selected substrate
    is a 4'-hydroxylated hydroxystilbene of plant origin, one produced by a competing microbe, or a
    lignin-derived biphenolic encountered in its burned-vegetation niche.
  boundary: >-
    Known: CAO-1 cleaves the interphenyl double bond of resveratrol and piceatannol in vitro. Its
    specificity is empirical (a 7-compound substrate panel, PMID:23893079): the authors' stated
    requirement is "a minimal number of unmodified hydroxyl groups" - not a single position. Both
    substrates are polyhydroxylated (>=3 free OH) and bear a free 4'-OH, whereas the non-substrates
    have too few free hydroxyls (trans-stilbene, 4-monohydroxystilbene, pinosylvin) or have them
    blocked (trismethoxy-resveratrol; a 4'-methoxy glucoside). Note 4-monohydroxystilbene has a 4'-OH
    yet is not cleaved, so a free 4'-OH is necessary-ish but not sufficient. CAO-1 is also more
    restrictive than bacterial family members (e.g. LsdA reportedly cleaves 4-hydroxystilbene). Unknown:
    the endogenous substrate and the fitness role.
  gap_kind:
    - BIOLOGY
  dark_aspect: BP_DARK
  significance: >-
    Determines whether the enzyme's evolved (selected-effect) function is resveratrol-specific or a
    broader hydroxystilbene catabolism, and thus the correct grain for its process annotation. The
    requirement for several free/unmodified hydroxyls rules out conifer pinosylvin and methoxylated
    lignin phenolics and points to angiosperm polyhydroxystilbenes - resveratrol and especially
    piceatannol.
    Ecologically, Neurospora is a post-fire pioneer of burned biomass, and among its burned hosts
    grasses/sugarcane accumulate both resveratrol and piceatannol, making a burned stilbenoid-
    producing host (or a resveratrol-producing microbial competitor) the leading candidate source.
  provenance:
    - reference_id: PMID:23893079
      supporting_text: >-
        mutants were not impaired by the presence of resveratrol, a phytoalexin
        active against different fungi, which did not significantly affect the growth and
        development of wild-type Neurospora
- gap_statement: >-
    The retinal-forming (beta-carotene cleaving) enzyme of Neurospora crassa is unidentified. The
    hypothesis that motivated the study of cao-1 - that it supplies retinal, the chromophore of the
    NOP-1 rhodopsin - was disproven, and its paralog CAO-2 cleaves torulene (not beta-carotene to
    retinal); N. crassa also lacks a clear ortholog of the retinal-forming CarX of Fusarium fujikuroi.
    It remains unknown which enzyme (if any) makes retinal in N. crassa, and whether the physiological
    NOP-1 chromophore is retinal at all or an apocarotenal such as CAO-2's product beta-apo-4'-carotenal.
  boundary: >-
    Known: cao-1 does not cleave carotenoids and is excluded as the retinal source; NOP-1 binds retinal
    in vitro. Unknown: the identity of any retinal-forming enzyme in N. crassa and the true
    physiological NOP-1 chromophore.
  gap_kind:
    - BIOLOGY
  dark_aspect: BP_DARK
  significance: >-
    This is the "negative space" that generated the original cao-1 mis-annotation: assuming a carotenoid/
    retinal role for a CCO-family member. Identifying the retinal (or alternative-chromophore) source
    would explain NOP-1 rhodopsin function and close the loop on the carotenoid-pathway assumption.
  provenance:
    - reference_id: PMID:23893079
      supporting_text: >-
        our results demonstrate that this enzyme is not CAO-1
    - reference_id: PMID:23893079
      supporting_text: >-
        the physiological NOP-1 chromophore may be a nonretinal molecule
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: LOW
    correctness: MISCITED
    review_notes: >-
      The phylogenetic (IBA) inferences derived carotenoid dioxygenase activity and carotene
      catabolic process, both of which are experimentally refuted for CAO-1. Useful only as
      the source of the annotations being corrected here.
- id: PMID:23893079
  title: The oxygenase CAO-1 of Neurospora crassa is a resveratrol cleavage enzyme.
  findings:
  - statement: >-
      CAO-1 does not cleave beta-carotene or any carotenoid/apocarotenoid tested; it is not a
      carotenoid cleavage enzyme.
    supporting_text: >-
      Here, we show that CAO-1 is not a carotenoid or apocarotenoid cleavage enzyme.
  - statement: >-
      CAO-1 cleaves the interphenyl double bond of resveratrol and piceatannol; expression is
      induced by resveratrol and is light-independent.
    supporting_text: >-
      adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light,
      a key regulator of carotenoid metabolism, did not alter them
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Primary functional characterization establishing CAO-1 as a resveratrol/stilbene cleavage
      oxygenase and explicitly excluding carotenoid activity. PMC full text verified.
- id: file:NEUCR/cao-1/cao-1-hypotheses/function-hypothesis-go-0010436/openscientist.md
  title: OpenScientist blinded function-assignment report for cao-1 (carotenoid dioxygenase
    activity, GO:0010436)
  findings:
  - statement: >-
      An independent, blinded OpenScientist run neutrally tested whether CAO-1 has carotenoid
      dioxygenase activity and concluded it is refuted/over-annotated, recommending removal of the
      carotenoid IBA terms and retention of the experimentally supported dioxygenase MF - concurring
      with this review's REMOVE decision. It also attributed the error to CCO/RPE65 (PANTHER
      PTHR10543) family-level IBA propagation.
    supporting_text: >-
      Verdict: REFUTED (over-annotated)
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Blinded computational hypothesis assessment (3-iteration run) used as independent corroboration
      of the manual REMOVE decision, not as primary evidence - the underlying facts rest on the cached
      primary literature (PMID:23893079, PMID:28493664). The report cites two orthologue papers not in
      this review (PMID:21073977 U. maydis Rco1; PMID:30115012 a Pseudomonas stilbene cleavage
      oxygenase structure) as leads on the stilbenoid-cleaving subclade; these are not yet verified/
      cached and are not asserted here as annotations.
- id: file:NEUCR/cao-1/cao-1-bioinformatics/RESULTS.md
  title: Structure-based analysis of CAO-1 stilbenoid substrate specificity (co-crystal
    H-bond network)
  findings:
  - statement: >-
      A reproducible analysis of the CAO-1 co-crystal structures (5U90 resveratrol, 5U97 piceatannol)
      recovers the UniProt binding residues (Tyr133/Lys164, Glu383) and a two-ring-anchor recognition
      model (4'-OH -> Tyr133/Lys164; 3/5-OH -> Glu383; scissile alkene ~4.6 A from the metal) that
      explains all seven compounds of the empirical substrate panel - notably why a free 4'-OH is
      necessary but not sufficient (4-hydroxystilbene has it but lacks the second, ring-B anchor).
    supporting_text: >-
      The two-anchor model accounts for
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Own bioinformatics analysis (this repo). A retrospective structural rationalization, not a
      validated predictor - only the two substrates were co-crystallized; non-substrate losses are
      inferred, and no docking/energetics were computed. Advances the specificity basis from
      "structure-consistent" to "structure-explained".
- id: PMID:28493664
  title: Structure and Spectroscopy of Alkene-Cleaving Dioxygenases Containing an
    Atypically Coordinated Non-Heme Iron Center.
  findings:
  - statement: >-
      Crystal structure of CAO-1 shows a non-heme Fe center (four-His CCO motif) with a
      stilbenoid-adapted substrate-binding cleft; resveratrol and piceatannol bind in the
      active site.
    supporting_text: >-
      The crystal structure of a fungal stilbenoid-cleaving CCO, CAO1, reveals strong
      similarity between its iron center and those of carotenoid-cleaving CCOs, but with a
      markedly different substrate-binding cleft
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Structural/spectroscopic study providing crystallographic support for iron binding,
      resveratrol binding, and stilbenoid dioxygenase activity. PMC full text verified.