title: Production of an active form of vitamin D2by genetically engineered CYP105A1.
abstract: Our previous studies revealed that CYP105A1 can convert vitamin D3(VD3) to its active form, 1α,25-dihydroxyvitamin D3(1,25D3). Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity; the activity of double variants R73A/R84A and R73A/R84V was more than 100-fold higher than that of the wild type of CYP105A1. In contrast, these variants had a low ability to convert vitamin D2(VD2) to 1α,25-dihydroxyvitamin D2(1,25D2), whereas they catalyzed the sequential hydroxylation at positions C25 and C26 to produce 25,26D2. A comparison of the docking models of 25D2 and 25D3 into the substrate-binding pocket of R73A/R84A suggests that the side chain of the Met239 inhibits the binding of 25D2 for 1α-hydroxylation. Therefore, the Met239 residue of R73A/R84A was substituted for Ala. As expected, the triple variant R73A/R84A/M239A showed a 22-fold higher 1α-hydroxylation activity towards 25D2. To the best of our knowledge, this is the first report on the generation of microbial cytochrome P450 that converts VD2 to 1,25D2 via 25D2.
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Yasuda
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Yogo
authors.affiliation: RIKEN Spring-8 Center, Harima Institute, Sayo, Hyogo 679-5148, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sugimoto
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Mano
authors.affiliation: Division of Food Science and Technology, Graduate School of Agriculture, Kyoto University, KitashirakawaOiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Teisuke
authors.initials: T
authors.lastname: Takita
authors.affiliation: Department of Food and Nutrition Management Studies, Faculty of Human Development, Soai University, 4-4-1 Nanko-naka, Suminoe-ku, Osaka 559-0033, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Ohta
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Kamakura
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Ikushiro
authors.affiliation: Division of Food Science and Technology, Graduate School of Agriculture, Kyoto University, KitashirakawaOiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
authors.affiliation: RIKEN Spring-8 Center, Harima Institute, Sayo, Hyogo 679-5148, Japan.
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Shiro
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan. Electronic address: tsakaki@pu-toyama.ac.jp.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Sakaki
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004872:Ergocalciferols / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006900:Hydroxylation; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D000072417:Protein Domains; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004872:Ergocalciferols; D011994:Recombinant Proteins; C003564:1,25-dihydroxyergocalciferol; D003577:Cytochrome P-450 Enzyme System; C086254:P450SU1 protein, Streptomyces griseolus
keywords: Active form of vitamin D(2); CYP105; Crystal structure; Cytochrome P450; Protein engineering