title: Functional characterization of CYP71D443, a cytochrome P450 catalyzing C-22 hydroxylation in the 20-hydroxyecdysone biosynthesis of Ajuga hairy roots.
abstract: 20-Hydroxyecdysone (20HE), a molting hormone of insects, is also distributed among a variety of plant families. 20HE is thought to play a role in protecting plants from insect herbivores. In insects, biosynthesis of 20HE from cholesterol proceeds via 7-dehydrocholesterol and 3β,14α-dihydroxy-5β-cholest-7-en-6-one (5β-ketodiol), the latter being converted to 20HE through sequential hydroxylation catalyzed by four P450 enzymes, which have been cloned and identified. In contrast, little is known about plant 20HE biosynthesis, and no biosynthetic 20HE gene has been reported thus far. We recently proposed involvement of 3β-hydroxy-5β-cholestan-6-one (5β-ketone) in 20HE biosynthesis in the hairy roots of Ajuga reptans var. atropurpurea (Lamiaceae). In this study, an Ajuga EST library was generated from the hairy roots and P450 genes were deduced from the library. Five genes with a high expression level (CYP71D443, CYP76AH19, CYP76AH20, CYP76AH21 and CYP716D27) were screened for a possible involvement in 20HE biosynthesis. As a result, CYP71D443 was shown to have C-22 hydroxylation activity for the 5β-ketone substrate using a yeast expression system. The hydroxylated product, 22-hydroxy-5β-ketone, had a 22R configuration in agreement with that of 20HE. Furthermore, labeling experiments indicated that (22R)-22-hydroxy-5β-ketone was converted to 20HE in Ajuga hairy roots. Based on the present results, a possible 20HE biosynthetic pathway in Ajuga plants involved CYP71D443 is proposed.
authors.affiliation: Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro, Tokyo 152-8551, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tsukagoshi
authors.affiliation: Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro, Tokyo 152-8551, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ohyama
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Seki
authors.affiliation: Department of Applied Biological Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan.
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Akashi
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Muranaka
authors.affiliation: Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro, Tokyo 152-8551, Japan. Electronic address: fujimoto.y.aa@gmail.com.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Fujimoto
mesh_terms: D031328:Ajuga / Q000737:chemistry* / Q000235:genetics; D002778:Cholestanones / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D003684:Dehydrocholesterols / Q000378:metabolism; D026461:Ecdysteroids / Q000737:chemistry; D004441:Ecdysterone / Q000096:biosynthesis* / Q000378:metabolism*; D019686:Lamiaceae / Q000737:chemistry* / Q000378:metabolism; D015394:Molecular Structure; D018517:Plant Roots / Q000737:chemistry
chemical_list: C000597999:3beta-hydroxy-5beta-cholestan-6-one; D002778:Cholestanones; D003684:Dehydrocholesterols; D026461:Ecdysteroids; D004441:Ecdysterone; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; C016705:7-dehydrocholesterol; C569463:CYP1A1 protein, human; D019363:Cytochrome P-450 CYP1A1
keywords: 20-Hydroxyecdysone; Biosynthesis; Bugle weed Ajuga reptans; CYP71D; Cytochrome P450; Lamiaceae; Phytoecdysteroid