title: Sequential hydroxylation of vitamin D2 by a genetically engineered CYP105A1.
abstract: Our previous studies revealed that the double variants of CYP105A1- R73A/R84A and R73V/R84A-show high levels of activity with respect to conversion of vitamin D3 to its biologically active form, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study, we found that both the double variants were also capable of converting vitamin D2 to its active form, that is, 1α,25-dihydroxyvitamin D2 (1α,25(OH)2D2), via 25(OH)D2, whereas its 1α-hydroxylation activity toward 25(OH)D2 was much lower than that toward 25(OH)D3. Comparison of the wild type and the double variants revealed that the amino acid substitutions remarkably enhanced both 25- and 26-hydroxylation activity toward vitamin D2. After 25-hydroxylation of vitamin D2, further hydroxylation at C26 may occur frequently without the release of 25(OH)D2 from the substrate-binding pocket. Thus, the double variants of CYP105A1 are quite useful to produce 25,26(OH)2D2 that is one of the metabolites of vitamin D2 detected in human serum.
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Hayashi
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: 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: 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: 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: 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: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004872:Ergocalciferols / Q000737:chemistry* / Q000378:metabolism*; D006900:Hydroxylation / Q000502:physiology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D004872:Ergocalciferols; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C086254:P450SU1 protein, Streptomyces griseolus
keywords: CYP105; Crystal structure; Cytochrome P450; Protein engineering; Sequential hydroxylation; Vitamin D(2)