title: Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol.
abstract: Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C28- and C29-Δ(24(28))-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ(24(25))intermediate. In this study, we characterized theArDWF1gene from an expression sequence tag library ofAjuga reptansvar.atropurpureahairy roots. The gene was functionally expressed in the yeast T21 strain. Thein vivoandin vitrostudy of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-(2)H3,28-(13)C)-24-methylenecholesterol, followed by(13)C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ(24(25))intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes,Oryza sativa DWF1(OsDWF1) was similarly expressed in yeast. Anin vivoassay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in theAjugahairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.
authors.affiliation: From the Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tsukagoshi
authors.affiliation: the Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan, and.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: the 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: the 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: From the Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ohyama
authors.affiliation: From the Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan, fujimoto.y.aa@gmail.com.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Fujimoto
mesh_terms: D031328:Ajuga / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010802:Phylogeny; D010840:Phytosterols / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D010840:Phytosterols; D010940:Plant Proteins; C021273:campesterol; D002784:Cholesterol; D010088:Oxidoreductases; C002537:24-methylenecholesterol
keywords: biosynthesis; enzyme mechanism; plant biochemistry; reductase; sterol