NCU06296 encodes an uncharacterised 616-residue member of the class B flavin-containing monooxygenase (FMO) family. PANTHER places it in a Pezizomycotina-specific subfamily (PTHR23023:SF292) of a fungal FMO clade that is the sister group of the entire animal FMO clade, so animal FMO1-5 are co-orthologs of the fungal lineage rather than close relatives. The protein retains the N-terminal FAD-binding GxGxxG motif and the core of the FMO-identifying motif, consistent with FAD-dependent, NAD(P)H- and O2-dependent oxygenation of an organic substrate, but its second (NADPH) dinucleotide motif and the FATGY motif are diverged and it lacks the C-terminal membrane anchor of mammalian FMOs. No substrate, product, pathway, phenotype or subcellular location has been determined experimentally for this protein or for any member of its fungal subfamily.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004497 monooxygenase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT placed monooxygenase activity at the PTHR23023 root node (PTN009058586), so it is inherited by every descendant, including the fungal SF292 clade that contains NCU06296. The target is unambiguously inside that tree, and its sequence retains the FAD-binding motif and the core of the FMO motif, so there is no target-specific sign of loss of catalytic function. Reason: The IBA rests on a family-root node placement with many experimentally characterised donors (mammalian, nematode, fly and plant FMOs). Generic monooxygenase activity is as precise as the evidence allows, because no member of the fungal clade has a known substrate. The donor substrate specificities (N- and S-oxygenation, glucosinolate S-oxygenation) were deliberately not propagated to the root and are not transferred here. Supporting Evidence: file:NEUCR/NCU06296/NCU06296-bioinformatics/RESULTS.md The IBA "monooxygenase activity" on Q7SAD4 lists PANTHER:PTN009058586, the family root. file:NEUCR/NCU06296/NCU06296-bioinformatics/RESULTS.md the FAD-binding motif and the core of the FMO motif are retained, consistent with a flavin-dependent monooxygenase. file:NEUCR/NCU06296/NCU06296-uniprot.txt DR Pfam; PF00743; FMO-like; 1. |
| GO:0004499 N,N-dimethylaniline monooxygenase activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: GO:0004499 is the specific reaction of the mammalian xenobiotic FMOs (N-oxygenation of N,N-dimethylaniline, EC 1.14.13.8). It is assigned here by an InterPro2GO mapping of the family-wide IPR020946 signature, which also covers FMO-family enzymes with unrelated substrates. NCU06296 belongs to a fungal clade that is only co-orthologous to the animal FMOs, has no characterised member, and has diverged at the NADPH-binding and FATGY motifs. Reason: PAINT curators placed only generic monooxygenase activity at the ancestral nodes above NCU06296; the PANTHER GO-slim annotations on every node of its path are generic, and no substrate-specific activity was propagated even though mammalian donors carry experimental N-oxygenation annotations. That omission is a deliberate phylogenetic judgment. Nothing specific to NCU06296 or its subfamily supports N,N-dimethylaniline as the substrate, so the specific reaction term over-states what is known; the supported claim is the monooxygenase activity that the gene already carries. Supporting Evidence: file:NEUCR/NCU06296/NCU06296-bioinformatics/RESULTS.md no substrate-specific activity (e.g. GO:0004499) is placed on any ancestor. file:NEUCR/NCU06296/NCU06296-bioinformatics/RESULTS.md No member of SF292 or of the fungal clade has experimental GO annotation |
| GO:0050660 flavin adenine dinucleotide binding | IEA GO_REF:0000002 | ACCEPT | Summary: FAD is the prosthetic group of class B FMOs. NCU06296 conserves the N-terminal Rossmann GxGxxG FAD-binding motif (residues 10-15, GSGLFG) that aligns to the FAD motif of human FMO3, and it matches the FMO-like Pfam domain and the FAD/NAD(P)-binding domain superfamily. Reason: Family membership plus a conserved FAD-binding motif at the expected position support FAD binding; this cofactor binding is intrinsic to the predicted monooxygenase activity. Supporting Evidence: file:NEUCR/NCU06296/NCU06296-bioinformatics/RESULTS.md FAD-binding Rossmann motif GxGxxG is conserved at residues 10-15 (GSGLFG), aligned to FMO3 GAGVSG (9-14). file:NEUCR/NCU06296/NCU06296-uniprot.txt DR InterPro; IPR036188; FAD/NAD-bd_sf. |
| GO:0050661 NADP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Characterised class B FMOs use NADPH as the reductant and bind NADP(H) through a second Rossmann-type dinucleotide motif. In NCU06296 that second GxGxxG motif is not conserved, so the nicotinamide cofactor and its preference (NADPH versus NADH) cannot be read from the sequence, although the protein retains the FMO-like domain architecture in which the NADP-binding domain resides. Reason: The annotation is plausible from family membership (characterised FMO-family enzymes use NADPH) but is weaker than FAD binding because the canonical NADPH-binding motif is diverged. It is retained as a non-core cofactor-binding statement; biochemical measurement of NADPH versus NADH turnover would be needed to confirm it. Supporting Evidence: file:NEUCR/NCU06296/NCU06296-bioinformatics/RESULTS.md The canonical NADPH-binding dinucleotide motif and FATGY are diverged, so NADPH (versus NADH) preference cannot be read from the sequence. file:NEUCR/NCU06296/NCU06296-uniprot.txt DR InterPro; IPR020946; Flavin_mOase-like. |
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Download this section (compressed HTML)Q: What is the physiological substrate of the Pezizomycotina SF292 FMO subfamily (NCU06296, its in-paralog NCU08749, Aspergillus fumigatus AFUA_6G01900), and does it oxygenate a nitrogen or sulfur heteroatom as mammalian FMOs do?
Q: Does the diverged second dinucleotide-binding motif of NCU06296 alter its nicotinamide cofactor preference from NADPH to NADH?
Experiment: Express and purify NCU06296, confirm FAD occupancy spectroscopically, compare NADPH and NADH oxidation with oxygen consumption and H2O2 release to measure coupling, and screen fungal metabolites and model N- and S-heteroatom substrates by LC-MS for oxygenated products.
Type: biochemical assay
Experiment: Compare untargeted metabolomes of NCU06296, NCU08749 and double deletion strains with wild type and complemented strains to find a substrate that accumulates in the absence of the enzyme; determine localization with an endogenous fluorescent fusion.
Type: genetic and metabolomic analysis
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