C-23 Hydroxylation by Arabidopsis CYP90C1 and CYP90D1 Reveals a Novel Shortcut in Brassinosteroid Biosynthesis

• Published in: The Plant cell Vol. 18; no. 11; pp. 3275 - 3288
• Main Authors: Ohnishi, Toshiyuki, Szatmari, Anna-Maria, Watanabe, Bunta, Fujita, Satomi, Bancos, Simona, Koncz, Csaba, Lafos, Marcel, Shibata, Kyomi, Yokota, Takao, Sakata, Kanzo, Szekeres, Miklos, Mizutani, Masaharu
• Format: Journal Article
• Language: English
• Published: United States American Society of Plant Biologists 01.11.2006
• Subjects: Animals; Arabidopsis - drug effects; Arabidopsis - enzymology; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Biosynthesis; Brassinosteroids; Carbon - metabolism; Catalysis - drug effects; Cotyledon - drug effects; Cotyledon - enzymology; Cotyledons; Cytochrome P-450 Enzyme System - deficiency; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Enzymes; Exons - genetics; Gas Chromatography-Mass Spectrometry; Gene Expression - drug effects; Gene Expression Regulation, Enzymologic - drug effects; Gene Expression Regulation, Plant - drug effects; Hydroxylation - drug effects; Hypocotyl - drug effects; Hypocotyl - enzymology; Hypocotyls; Insecta - cytology; Introns - genetics; Kinetics; Mutation - genetics; Oxidation; Phenotype; Phenotypes; Phytosterols - analysis; Phytosterols - biosynthesis; Phytosterols - chemistry; Phytosterols - pharmacology; Plant cells; Plants; RNA, Messenger - genetics; RNA, Messenger - metabolism; Seedlings; Substrate Specificity
• ISSN: 1040-4651; 1532-298X; 1532-298X
• DOI: 10.1105/tpc.106.045443

Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5α-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5α-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.