Yasuda Kaori, Yogo Yuya, Sugimoto Hiroshi, Mano Hiroki, Takita Teisuke, Ohta Miho, Kamakura Masaki, Ikushiro Shinichi, Yasukawa Kiyoshi, Shiro Yoshitsugu, Sakaki Toshiyuki
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
RIKEN Spring-8 Center, Harima Institute, Sayo, Hyogo 679-5148, Japan.
Biochem Biophys Res Commun. 2017 Apr 29;486(2):336-341. doi: 10.1016/j.bbrc.2017.03.040. Epub 2017 Mar 14.
Our previous studies revealed that CYP105A1 can convert vitamin D (VD3) to its active form, 1α,25-dihydroxyvitamin D (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 D (VD2) to 1α,25-dihydroxyvitamin D (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.
我们之前的研究表明，CYP105A1可以将维生素D（VD3）转化为其活性形式1α,25 - 二羟基维生素D（1,25D3）。基于其晶体结构对CYP105A1进行定点诱变可显著提高其活性；双变体R73A/R84A和R73A/R84V的活性比野生型CYP105A1高出100多倍。相比之下，这些变体将维生素D（VD2）转化为1α,25 - 二羟基维生素D（1,25D2）的能力较低，而它们催化在C25和C26位的顺序羟基化反应生成25,26D2。将25D2和25D3对接模型与R73A/R84A的底物结合口袋进行比较表明，Met239的侧链抑制了25D2与1α - 羟基化反应的结合。因此，将R73A/R84A的Met239残基替换为Ala。正如预期的那样，三变体R73A/R84A/M239A对25D2的1α - 羟基化活性提高了22倍。据我们所知，这是关于生成通过25D2将VD2转化为1,25D2的微生物细胞色素P450的首次报道。
