CYP83A1 (REF2; (methylsulfanyl)alkanaldoxime N-monooxygenase) is an endoplasmic reticulum-anchored cytochrome P450 monooxygenase that catalyzes the second step of core structure formation in methionine-derived (aliphatic) glucosinolate biosynthesis. It oxidizes the omega-(methylthio)alkanaldoximes produced by CYP79F1/CYP79F2 to a reactive aci-nitro/nitrile-oxide intermediate that is captured by glutathione, yielding S-alkylthiohydroximate glutathione conjugates (EC 1.14.14.43), which are subsequently processed by GGP1, the C-S lyase SUR1, UGT74 S-glucosyltransferases and sulfotransferases. CYP83A1 efficiently metabolizes aliphatic oximes of both short and long chain length, and has lower-affinity activity on aromatic (phenylalanine-, tyrosine- and tryptophan-derived) aldoximes; its paralog CYP83B1 is the physiological enzyme for indole-3-acetaldoxime, and the two are non-redundant under normal conditions. ref2/cyp83a1 mutants have reduced levels of all aliphatic glucosinolates and increased indole glucosinolates. The reduced sinapate esters and syringyl lignin of ref2 mutants are attributed to inhibition of phenylpropanoid O-methyltransferases by accumulating aliphatic aldoximes, an indirect metabolic consequence rather than a direct role in phenylpropanoid metabolism.
Definition: Catalysis of the reaction: an (E)-omega-(methylsulfanyl)-alkanal oxime + glutathione + reduced [NADPH--hemoprotein reductase] + O2 = an S-[(1E)-1-(hydroxyimino)-omega-(methylsulfanyl)alkyl]-L-glutathione + oxidized [NADPH--hemoprotein reductase] + 2 H2O + H+. The cytochrome P450 oxidizes the aldoxime to a reactive aci-nitro/nitrile-oxide species that is conjugated to glutathione; a step of aliphatic glucosinolate biosynthesis.
Justification: CYP83A1 has a directly demonstrated, well-defined reaction (EC 1.14.14.43; RHEA:51992) with no corresponding GO term. The only existing IDA term (GO:0016709) is both generic and placed on the NAD(P)H-donor branch, whereas the enzyme uses the flavoprotein NADPH-cytochrome P450 reductase, as for the sibling CYP79F activity GO:0120526, which GO places under GO:0016712. A specific term would give the aliphatic glucosinolate core pathway a complete set of reaction-level MF terms.
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004497 monooxygenase activity | IEA GO_REF:0000002 | MODIFY | Summary: CYP83A1 oxygenates aldoximes during glucosinolate core formation. Reason: Refine the broad oxidation claim to the specifically supported proposed (methylsulfanyl)alkanaldoxime N-monooxygenase activity. CYP83A1 uses electron supply through a P450 reductase; its aliphatic-oxime preference is established by recombinant assays, while aromatic oximes are less efficiently accepted. Proposed replacements: (methylsulfanyl)alkanaldoxime N-monooxygenase activity Supporting Evidence: PMID:12970475 we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1, whereas CYP83B1 metabolizes these substrates with very low efficiency. |
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: Heme iron supports the CYP83A1 P450 catalytic reaction. Reason: This is an intrinsic cofactor-binding feature of the aldoxime oxygenase, not a peripheral role. Supporting Evidence: file:ARATH/CYP83A1/CYP83A1-uniprot.txt Name=heme; Xref=ChEBI:CHEBI:30413; |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (ER membrane, single-pass; by similarity). CYP83A1 is an N-terminally anchored microsomal P450 whose activity was characterized in microsomes co-expressing NADPH-cytochrome P450 reductase. Reason: Consistent with the conserved microsomal P450 architecture and with the requirement for the ER-resident reductase partner; the functionally relevant location. Supporting Evidence: PMID:12970475 Microsomes of yeast ( Saccharomyces cerevisiae ) cells expressing recombinant CYP83A1 and CYP83B1 in the yeast strain WAT11 co-expressing the Arabidopsis NADPH:cytochrome P450 reductase ATR1 |
| GO:0005829 cytosol | HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati... | MARK AS OVER ANNOTATED | Summary: The microsome experiment does not independently establish cytosol localization. Reason: Full-text inspection of PMID:28887381 shows that washed microsomes were profiled by chromatography and sucrose gradients, while subcellular labels were taken from SUBAcon and prior GFP data. The authors explicitly report that no organelle occupied a single gradient cluster. Detection or membrane association is meaningful, but assigning CYP83A1 specifically to cytosol exceeds the spatial resolution of this experiment. This is not a claim that the detected protein is absent or that all additional localization is impossible; the unsupported compartment assignment, rather than the proteomic observation, is marked as over-annotated. Supporting Evidence: PMID:28887381 The proteins with known localizations had scattered distributions across the entire gradient and no organelle fell in a single cluster. PMID:28887381 We used SUBAcon localization predictions to analyze oligomerization as a function of subcellular localization (Fig. 7C). |
| GO:0009625 response to insect | IEP PMID:23144921 Water stress and aphid feeding differentially influence meta... | KEEP AS NON CORE | Summary: CYP83A1 transcript levels decreased after aphid infestation. This is an expression response of a biosynthetic gene. Reason: Valid expression-pattern observation; aliphatic glucosinolates are anti-herbivore compounds, but CYP83A1 does not execute a response process itself. Supporting Evidence: PMID:23144921 transcript levels of biosynthetic genes MAM1, CYP79F1, CYP83A1, and UGT74C1 decreased after infestation with either aphid species |
| GO:0016491 oxidoreductase activity | IBA GO_REF:0000033 | MODIFY | Summary: CYP83A1 oxygenates aldoximes during glucosinolate core formation. Reason: Refine the broad oxidation claim to the specifically supported proposed (methylsulfanyl)alkanaldoxime N-monooxygenase activity. CYP83A1 uses electron supply through a P450 reductase; its aliphatic-oxime preference is established by recombinant assays, while aromatic oximes are less efficiently accepted. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN009217258 UNRESOLVED Proximate PAINT node traced from the original annotation. Its full ancestral assertion history has not been reconstructed in this audit; the target-specific assessment is grounded in the primary evidence explained in review.reason, not donor counts or self-inclusion. Proposed replacements: (methylsulfanyl)alkanaldoxime N-monooxygenase activity Supporting Evidence: PMID:11553739 Instead, CYP83A1 catalyzes the initial conversion of aldoximes to thiohydroximates in the synthesis of glucosinolates not derived from tryptophan. |
| GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen | IEA GO_REF:0000002 | MODIFY | Summary: CYP83A1 oxygenates aldoximes during glucosinolate core formation. Reason: Refine the broad oxidation claim to the specifically supported proposed (methylsulfanyl)alkanaldoxime N-monooxygenase activity. CYP83A1 uses electron supply through a P450 reductase; its aliphatic-oxime preference is established by recombinant assays, while aromatic oximes are less efficiently accepted. Proposed replacements: (methylsulfanyl)alkanaldoxime N-monooxygenase activity Supporting Evidence: PMID:12970475 we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1 |
| GO:0016709 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of one atom of oxygen | IDA PMID:12970475 CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzyme... | MODIFY | Summary: CYP83A1 oxygenates aldoximes during glucosinolate core formation. Reason: Refine the broad oxidation claim to the specifically supported proposed (methylsulfanyl)alkanaldoxime N-monooxygenase activity. CYP83A1 uses electron supply through a P450 reductase; its aliphatic-oxime preference is established by recombinant assays, while aromatic oximes are less efficiently accepted. Proposed replacements: (methylsulfanyl)alkanaldoxime N-monooxygenase activity Supporting Evidence: PMID:12970475 When CYP83A1 was incubated with 4-methylthiobutanaldoxime in the presence of [ 35 S]-glutathione as the nucleophile, an NADPH-dependent product was formed PMID:12970475 The product is most likely the S -alkylthiohydroximate designated S -(4-methylthiobutylhydroximoyl)-glutathione, analogous to the conjugates produced when using Cys as sulfur donor. PMID:12970475 The K m values of recombinant CYP83A1 for aliphatic oximes were estimated to be in the range of 20 to 150 |
| GO:0019761 glucosinolate biosynthetic process | IDA PMID:11553739 The involvement of two p450 enzymes, CYP83B1 and CYP83A1, in... | ACCEPT | Summary: Recombinant CYP83A1 converts aldoximes to thiohydroximate adducts in the presence of NADPH and a thiol donor; kinetic comparison with CYP83B1 shows CYP83A1 acts in glucosinolate synthesis from non-tryptophan aldoximes. CYP83A1 performs a catalytic step of the pathway. Reason: Direct biochemical evidence that the enzyme catalyzes a step of glucosinolate biosynthesis, corroborated genetically by the ref2 aliphatic glucosinolate deficiency. Supporting Evidence: PMID:11553739 Instead, CYP83A1 catalyzes the initial conversion of aldoximes to thiohydroximates in the synthesis of glucosinolates not derived from tryptophan. PMID:12509530 These results show that CYP83A1 is involved in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates |
| GO:0020037 heme binding | IEA GO_REF:0000002 | ACCEPT | Summary: Heme iron supports the CYP83A1 P450 catalytic reaction. Reason: This is an intrinsic cofactor-binding feature of the aldoxime oxygenase, not a peripheral role. Supporting Evidence: file:ARATH/CYP83A1/CYP83A1-uniprot.txt Name=heme; Xref=ChEBI:CHEBI:30413; |
Loading supporting content…
Download this section (compressed HTML)Q: Does CYP83A1 release an aci-nitro compound or a nitrile oxide, and is glutathione conjugation spontaneous in vivo or catalyzed by specific GSTs (e.g. GSTF family members)?
Q: Which aliphatic aldoxime(s) accumulating in ref2 inhibit caffeic acid O-methyltransferase, and is this cross-talk relevant to lignin engineering in glucosinolate-containing oilseeds?
Experiment: Reconstitute CYP83A1 with and without candidate GSTFs in N. benthamiana transient expression of the aliphatic core pathway and quantify desulfoglucosinolate output and by-products.
Hypothesis: GST-catalyzed glutathione conjugation is required for efficient channelling of the CYP83A1 product in planta.
Type: pathway reconstitution
Experiment: Test whether blocking aldoxime formation (cyp79f1 cyp79f2) suppresses the sinapoylmalate and syringyl lignin defects of ref2.
Hypothesis: Accumulating aliphatic aldoximes, not glucosinolate deficiency, cause the ref2 phenylpropanoid phenotype.
Type: genetic epistasis
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)