title: Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols.
abstract: Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K(m)) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C(27-29) sterols. Cholesterol (C27 sterol) is the best substrate, followed by CR (C28 sterol), whereas sitosterol (C29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C27/C28/C29 sterols and C27/C28/C29 BRs.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Fujita
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Ohnishi
authors.forename: Bunta
authors.initials: B
authors.lastname: Watanabe
authors.forename: Takao
authors.initials: T
authors.lastname: Yokota
authors.forename: Suguru
authors.initials: S
authors.lastname: Takatsuto
authors.forename: Shozo
authors.initials: S
authors.lastname: Fujioka
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yoshida
authors.forename: Kanzo
authors.initials: K
authors.lastname: Sakata
authors.forename: Masaharu
authors.initials: M
authors.lastname: Mizutani
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004926:Escherichia coli; D015870:Gene Expression; D006900:Hydroxylation; D010840:Phytosterols / Q000378:metabolism*; D013053:Spectrophotometry; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013379:Substrate Specificity; D014169:Transformation, Bacterial
chemical_list: C111451:AT3G50660 protein, Arabidopsis; D029681:Arabidopsis Proteins; D010840:Phytosterols; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases