title: C-23 hydroxylation by Arabidopsis CYP90C1 and CYP90D1 reveals a novel shortcut in brassinosteroid biosynthesis.
abstract: 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-5alpha-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-5alpha-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Ohnishi
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Szatmari
authors.forename: Bunta
authors.initials: B
authors.lastname: Watanabe
authors.forename: Satomi
authors.initials: S
authors.lastname: Fujita
authors.forename: Simona
authors.initials: S
authors.lastname: Bancos
authors.forename: Csaba
authors.initials: C
authors.lastname: Koncz
authors.forename: Marcel
authors.initials: M
authors.lastname: Lafos
authors.forename: Kyomi
authors.initials: K
authors.lastname: Shibata
authors.forename: Takao
authors.initials: T
authors.lastname: Yokota
authors.forename: Kanzo
authors.initials: K
authors.lastname: Sakata
authors.forename: Miklos
authors.initials: M
authors.lastname: Szekeres
authors.forename: Masaharu
authors.initials: M
authors.lastname: Mizutani
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D018548:Cotyledon / Q000187:drug effects / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D015870:Gene Expression / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D006900:Hydroxylation / Q000187:drug effects; D018546:Hypocotyl / Q000187:drug effects / Q000201:enzymology; D007313:Insecta / Q000166:cytology; D007438:Introns / Q000235:genetics; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010840:Phytosterols / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D010840:Phytosterols; D012333:RNA, Messenger; D002244:Carbon; D003577:Cytochrome P-450 Enzyme System; C500774:cytochrome P-450 90D1, Arabidopsis; C113876:ROT3 protein, Arabidopsis