GA3 (ent-kaurene oxidase, KO, CYP701A3; At5g25900) is the sole Arabidopsis ent-kaurene oxidase, a multifunctional cytochrome P450 that catalyses three sequential oxidations of the C-19 (C4-alpha) methyl group of ent-kaur-16-ene via ent-kaurenol and ent-kaurenal to ent-kaurenoic acid, with intermediates retained in the active site and the first hydroxylation rate-limiting. KO is targeted to the outer face of the plastid envelope (organelle proteomics also co-fractionate it with the ER), where it is thought to connect the plastid-localized cyclase steps (GA1/CPS, GA2/KS) to the ER-localized KAO step. It is broadly expressed, most strongly in inflorescences and in the cortex/endodermis of germinating embryos. ga3 nonsense mutants lack KO activity and are gibberellin-responsive dwarfs.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004497 monooxygenase activity | IEA GO_REF:0000002 | MODIFY | Summary: Monooxygenase activity from InterPro P450 domains. Reason: Correct parent; KO (CYP701A3) is a P450 monooxygenase whose specific activity is ent-kaurene oxidase (three successive oxidations of C-19). Proposed replacements: ent-kaurene oxidase activity Supporting Evidence: PMID:9952446 demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid |
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Iron ion binding from InterPro P450 signatures; reflects the heme iron. Reason: The P450 coordinates the heme iron through its proximal cysteine thiolate, so iron binding is not wrong. GO:0020037 heme binding is not a descendant of GO:0005506, so the two terms are not redundant. Iron is a catalytic cofactor rather than the core function (consistent with other ARATH P450 reviews and with magnesium ion binding on the GA1/GA2 terpene cyclases); retained as non-core. Supporting Evidence: PMID:20698828 KO (kaurene oxidase) is a multifunctional cytochrome P450 catalysing three sequential oxidations in gibberellin phytohormone biosynthesis. |
| GO:0005783 endoplasmic reticulum | HDA PMID:16618929 Mapping the Arabidopsis organelle proteome. | KEEP AS NON CORE | Summary: LOPIT organelle proteomics assigned KO to the endoplasmic reticulum. Reason: High-throughput density-gradient data place KO with ER markers, whereas import assays place it on the outer face of the chloroplast envelope; plastid-ER contact sites or dual localization could reconcile these. Retained but not core. |
| GO:0005783 endoplasmic reticulum | HDA PMID:22923678 Putative glycosyltransferases and other plant Golgi apparatu... | KEEP AS NON CORE | Summary: Second LOPIT dataset assigning KO to the ER. Reason: As above; the P450 steps downstream of KO (KAO) are ER-localized, and KO bridges plastid and ER steps. Supporting Evidence: PMID:11722763 These data suggest that the AtKO1 protein links the plastid- and endoplasmic reticulum-located steps of the gibberellin biosynthesis pathway by association with the outer envelope of the plastid. |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | ACCEPT | Summary: AtSubP prediction of chloroplast localization. Reason: The N-terminal region directs GFP to chloroplasts, and the protein associates with the outer envelope; chloroplast is a correct general location. Supporting Evidence: PMID:11722763 The N-terminal portion of the cytochrome P450 protein ent-kaurene oxidase (AtKO1) directs GFP to chloroplasts in tobacco leaf transient assays. |
| GO:0009686 gibberellin biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: KO catalyses the first P450 step of GA biosynthesis (ent-kaurene to ent-kaurenoic acid). Reason: Conserved role of the CYP701 clade. Supporting Evidence: PMID:9671797 We present data showing that the ga3-1 mutant is deficient in ent-kaurene oxidase activity, the first cytochrome P450-mediated step in the gibberellin biosynthetic pathway. |
| GO:0009686 gibberellin biosynthetic process | IDA PMID:9952446 Arabidopsis ent-kaurene oxidase catalyzes three steps of gib... | ACCEPT | Summary: Yeast expressing GA3 converts ent-kaurene to ent-kaurenoic acid. Reason: Direct biochemical evidence. Supporting Evidence: PMID:9952446 demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid |
| GO:0009686 gibberellin biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Pathway mapping. Reason: Consistent with experimental evidence. Supporting Evidence: PMID:9952446 demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid |
| GO:0009707 chloroplast outer membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Chloroplast outer membrane localization. Reason: Consistent with chloroplast import assays. Supporting Evidence: PMID:11722763 Chloroplast import assays with 35S-labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. |
| GO:0009707 chloroplast outer membrane | IDA PMID:11722763 A plastid envelope location of Arabidopsis ent-kaurene oxida... | ACCEPT | Summary: 35S-labelled AtKO1 is targeted to the outer face of the chloroplast envelope. Reason: Direct experimental evidence; used as the core-function location. Supporting Evidence: PMID:11722763 Chloroplast import assays with 35S-labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. PMID:11722763 These data suggest that the AtKO1 protein links the plastid- and endoplasmic reticulum-located steps of the gibberellin biosynthesis pathway by association with the outer envelope of the plastid. |
| GO:0009707 chloroplast outer membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping. Reason: Consistent with experimental evidence. Supporting Evidence: PMID:11722763 Chloroplast import assays with 35S-labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. |
| GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen | IEA GO_REF:0000002 | MODIFY | Summary: Generic P450 oxidoreductase activity (InterPro2GO). Reason: Correct parent; use the specific activity. Proposed replacements: ent-kaurene oxidase activity Supporting Evidence: PMID:9952446 demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid |
| 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 | IBA GO_REF:0000033 | MODIFY | Summary: Oxidoreductase acting on paired donors with NAD(P)H as one donor (P450 monooxygenase), propagated in the KO clade. Reason: Correct but general; ent-kaurene oxidase activity is experimentally established for this protein. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN001547377 SUPPORTS TRANSFER KO clade node; the general P450 monooxygenase term is correct but the specific ent-kaurene oxidase term is available. UniProtKB:Q0DBF4 SUPPORTS TRANSFER Rice KO-clade donor; supports the general oxidoreductase activity. Proposed replacements: ent-kaurene oxidase activity Supporting Evidence: PMID:9952446 demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid |
| GO:0019825 oxygen binding | IMP PMID:9671797 Cloning of the Arabidopsis ent-kaurene oxidase gene GA3. | MODIFY | Summary: Oxygen binding inferred from the ga3 mutant (TIGR legacy annotation). Reason: O2 binding is a mechanistic feature of every P450 and is not what the mutant analysis showed; the ga3 mutant lacks ent-kaurene oxidase activity, so the informative term is the enzyme activity. Proposed replacements: ent-kaurene oxidase activity Supporting Evidence: PMID:9671797 We present data showing that the ga3-1 mutant is deficient in ent-kaurene oxidase activity, the first cytochrome P450-mediated step in the gibberellin biosynthetic pathway. |
| GO:0020037 heme binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Heme binding (P450 cofactor). Reason: Correct cofactor binding for a cytochrome P450; non-core. Supporting Evidence: PMID:20698828 KO (kaurene oxidase) is a multifunctional cytochrome P450 catalysing three sequential oxidations in gibberellin phytohormone biosynthesis. |
| GO:0052615 ent-kaurene oxidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: EC/Rhea mapping (RHEA:32323). Reason: Matches experimental evidence. Supporting Evidence: PMID:9952446 demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid |
| GO:0052615 ent-kaurene oxidase activity | IMP PMID:9671797 Cloning of the Arabidopsis ent-kaurene oxidase gene GA3. | ACCEPT | Summary: ga3-1 lacks ent-kaurene oxidase activity; nonsense alleles in the P450 and genomic complementation identify GA3 as KO. Reason: Core activity. Supporting Evidence: PMID:9671797 We present data showing that the ga3-1 mutant is deficient in ent-kaurene oxidase activity, the first cytochrome P450-mediated step in the gibberellin biosynthetic pathway. PMID:9671797 A genomic clone spanning the P450 locus complemented the ga3-2 mutant. |
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Download this section (compressed HTML)Q: Is KO located on the plastid outer envelope, the ER, or at plastid-ER contact sites in vivo, and how is hydrophobic ent-kaurene delivered from the stroma to the enzyme?
Experiment: Image endogenous-promoter KO-fluorescent protein fusions alongside plastid envelope and ER markers at high resolution, and test whether KO co-purifies with plastid-ER contact-site proteins.
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