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

^a Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
^b Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary
^c Max Planck-Institut für Züchtungsforschung, D-50829 Koeln, Germany
^d Department of Biosciences, Teikyo University, Utsunomiya, 320-8551, Japan

² To whom correspondence should be addressed. E-mail mizutani{at}scl.kyoto-u.ac.jp; fax 81-774-38-3229.

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-5-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-5-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.

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