Miyanaga Akimasa, Takayanagi Ryuichi, Furuya Takashi, Kawamata Ayano, Itagaki Tomohiro, Iwabuchi Yoshiharu, Kanoh Naoki, Kudo Fumitaka, Eguchi Tadashi
Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan.
Chembiochem. 2017 Nov 2;18(21):2179-2187. doi: 10.1002/cbic.201700429. Epub 2017 Sep 18.
GfsF is a multifunctional P450 monooxygenase that catalyzes epoxidation and subsequent hydroxylation in the biosynthesis of macrolide polyketide FD-891. Here, we describe the biochemical and structural analysis of GfsF. To obtain the structural basis of a dual-function reaction, we determined the crystal structure of ligand-free GfsF, which revealed GfsF to have a predominantly hydrophobic substrate binding pocket. The docking models, in conjunction with the results of the enzymatic assay with substrate analogues and site-directed mutagenesis suggested two distinct substrate binding modes for epoxidation and hydroxylation reactions, which explained how GfsF regulates the order of two oxidative reactions. These findings provide new insights into the reaction mechanism of multifunctional P450 monooxygenases.
GfsF是一种多功能细胞色素P450单加氧酶，在大环内酯聚酮化合物FD - 891的生物合成中催化环氧化及随后的羟基化反应。在此，我们描述了GfsF的生化和结构分析。为获得双功能反应的结构基础，我们测定了无配体GfsF的晶体结构，结果显示GfsF具有一个主要为疏水性的底物结合口袋。对接模型，结合与底物类似物的酶活性测定结果和定点诱变，提示了环氧化和羟基化反应两种不同的底物结合模式，这解释了GfsF如何调控两个氧化反应的顺序。这些发现为多功能细胞色素P450单加氧酶的反应机制提供了新的见解。
