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. 2001 Feb 13;98(4):2065-70.
doi: 10.1073/pnas.98.4.2065. Epub 2001 Feb 6.

The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway

C A Helliwell  1 , P M Chandler, A Poole, E S Dennis, W J Peacock
Affiliations

The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway

C A Helliwell et al. Proc Natl Acad Sci U S A. .

Abstract

We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA(12). A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains. Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified. Arabidopsis thaliana has two CYP88A genes, both of which are expressed. Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA(12). Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase.

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Figures

Figure 1
Figure 1
A tree showing the relationship between CYP88A, ent-kaurene oxidase (KO), and brassinosteroid biosynthesis P450 proteins. CYP88A proteins are CYP88A3 (AtKAO1), CYP88A4 (AtKAO2), Grd5 (HvKAO1), Dwarf3 (ZmKAO1) (8), CYP88A2 (CmKAO) (13), and CY88A5 (OsKAO1; GenBank accession no. AP000616). KO sequences are CYP701A3 (AtKO1) (7) and CYP701A1 (CmKO1) (13), and brassinosteroid biosynthesis genes are CYP90B1 (DWF4) (15), CYP90A1 (CPD) (17), and CYP85A1 (DWARF) (16). The tree was produced from a ClustalW alignment by using the KITSCH program on Bionavigator (http://www.bionavigator.com).
Figure 2
Figure 2
Alterations in the Grd5 gene product in different grd5 mutants. Reverse transcription–PCR of endosperm RNA from different grd5 mutants (M362, M574, and M594) and sequence analysis of the products revealed amino acid substitutions at the positions indicated. A frameshift mutation in the M574 sequence causes an immediate termination codon.
Figure 3
Figure 3
Expression of CYP88A3 and CYP88A4 mRNA determined by ribonuclease protection assay. RNA samples prepared from rosette leaf (RL), cauline leaf (CL), inflorescence stem (STEM), stem tip including buds and mature flowers (INFL), and siliques (SIL). All RNA samples were from Arabidopsis ecotype C24. Yeast-negative control for the ribonuclease protection assay also is shown.
Figure 4
Figure 4
Cytochrome P450-mediated steps of the GA biosynthesis pathway showing the steps catalyzed by KO and KAO.
See this image and copyright information in PMC

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