Arabidopsis CYP90B1 catalyses the early C‐22 hydroxylation of C27, C28 and C29 sterols

• Published in: The Plant journal : for cell and molecular biology 2006-03, Vol.45 (5), p.765-774
• Main Authors: Fujita, Satomi, Ohnishi, Toshiyuki, Watanabe, Bunta, Yokota, Takao, Takatsuto, Suguru, Fujioka, Shozo, Yoshida, Shigeo, Sakata, Kanzo, Mizutani, Masaharu
• Format: Article
• Language: English
• Subjects: Arabidopsis - enzymology; Arabidopsis Proteins - metabolism; Biological and medical sciences; Botany; brassinosteroid biosynthesis; campesterol; CYP90B1; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; DWF4; Enzymes; Escherichia coli; Flowers & plants; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic recombination; Hydroxylation; Mutation; Phytosterols - metabolism; Plant growth. Development of the storage organs; Plant physiology and development; Spectrophotometry; Steroid Hydroxylases - metabolism; Substrate Specificity; Transformation, Bacterial; Vegetative apparatus, growth and morphogenesis. Senescence
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2005.02639.x

Summary 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 (kcat/Km) 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‐5α 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 C27–29 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.