tailieunhanh - Báo cáo khoa học: Three-step hydroxylation of vitamin D3 by a genetically engineered CYP105A1

Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V⁄R84A, has a high ability to convert vitamin D3 to its biologically active form, 1a,25-dihydroxyvitamin D3 [1a,25(OH)2D3 ], suggesting the possibility for R73V⁄R84A to produce 1a,25(OH)2D3 . BecauseActinomycetes, including Streptomyces, exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance | ỊFEBS Journal Three-step hydroxylation of vitamin D3 by a genetically engineered CYP105A1 Enzymes and catalysis Keiko Hayashi1 Kaori Yasuda1 Hiroshi Sugimoto2 Shinichi Ikushiro1 Masaki Kamakura1 Atsushi Kittaka3 Ronald L. Horst4 Tai C. Chen5 Miho Ohta6 Yoshitsugu Shiro2 and Toshiyuki Sakaki1 1 Department of Biotechnology Faculty of Engineering Toyama PrefecturalUniversity Kurokawa Imizu Toyama Japan 2 RIKEN SPring-8 Center Harima Institute Sayo Hyogo Japan 3 Faculty of PharmaceuticalSciences Teikyo University Sagamiko Kanagawa Japan 4 Heartland Assays Inc. Ames IA USA 5 Boston University Schoolof Medicine Boston Massachusetts USA 6 Department of Food and Nutrition Management Studies Faculty of Human Development Soai University Nanko-naka Suminoe-ku Osaka Japan Keywords crystal structure cytochrome P450 electron transport chain protein engineering vitamin D Correspondence T. Sakaki Department of Biotechnology Faculty of Engineering Toyama Prefectural University Kurokawa Imizu Toyama Japan Fax 81 766 56 2498 Tel 81 766 56 7500 E-mail tsakaki@ Database Structural data are available in the Protein Data Bank database under the accession numbers 3CV8 and 3CV9 Received 14 May 2010 revised 1 July 2010 accepted 27 July 2010 doi Our previous studies revealed that the double variant of cytochrome P450 CYP 105A1 R73V R84A has a high ability to convert vitamin D3 to its biologically active form 1a 25-dihydroxyvitamin D3 1a 25 OH 2D3 suggesting the possibility for R73V R84A to produce 1a 25 OH 2D3. Because Actinomycetes including Streptomyces exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance we examined the expression of R73V R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter. As expected the metabolites 25-hydroxyvitamin D3 25 OH D3 and 1a 25 OH 2D3 were detected in the cell culture of the recombinant S. lividans. A large .