Structure-guided manipulation of the regioselectivity of the cyclosporine A hydroxylase CYP-sb21 from Sebekia benihana  

作　　者：Fengwei Li Li Ma Xingwang Zhang Jingfei Chen Feifei Qi Yinyue Huang Zepeng Qu Lishan Yao Wei Zhang Eung-Soo Kim Shengying Li 

机构地区：[1]State Key Laboratory of Microbial Technology,Shandong University,Qingdao,Shandong,266237,China [2]Shandong Provincial Key Laboratory of Synthetic Biology,CAS Key Laboratory of Biofuels,Qingdao Institute of Bioenergy and Bioprocess Technology,Chinese Academy of Sciences,Qingdao,Shandong,266101,China [3]Department of Biological Engineering,Inha University,Incheon,22212,South Korea [4]Laboratory for Marine Biology and Biotechnology,Qingdao National Laboratory for Marine Science and Technology,Qingdao,Shandong,266237,China

出　　处：《Synthetic and Systems Biotechnology》2020年第3期236-243,共8页合成和系统生物技术（英文）

基　　金：This work was supported by the National Key Research and Development Program of China(2019YFA0905100 to S.L.);the National Natural Science Foundation of China(31800664 to F.L.,31872729 to S.L.and 31600045 to L.M.);General Support from China Postdoctoral Science Foundation(Grant No.2017M622293 to F.L.);Shandong Provincial Natural Science Foundation,China(ZR2019ZD22 to S.L.and ZR2016CQ05 to L.M.);the Applied Basic Research Programs of Science and Technology of Qingdao(17-1-1-60-jch to L.M.);National Research Foundation of Korea(No.NRF-2017R1A2A2A05069859 to E.-S.K.).

摘　　要：The cytochrome P450 enzyme CYP-sb21 from the rare actinomycete Sebekia benihana is capable of hydroxylating the immunosuppressive drug molecule cyclosporine A(CsA)primarily at the 4th N-methyl leucine(MeLeu4),giving rise toγ-hydroxy-N-methyl-L-Leu4-CsA(CsA-4-OH).This oxidative modification of CsA leads to dramatically reduced immunosuppressive activity while retaining the hair growth-promoting side-effect,thus demonstrating great application potential in both pharmaceutical and cosmetic industries.However,this P450 enzyme also hydroxylates CsA at the unwanted position of the 9th N-methyl leucine(MeLeu9),indicating that the regioselectivity needs to be improved for the development of CsA-4-OH into a commercial hair growth stimulator.Herein,we report the crystal structure of CYP-sb21 in its substrate-free form at 1.85Å.Together with sequence and 3D structure comparisons,Autodock-based substrate docking,molecular dynamics(MD)simulation,and site-directed mutagenesis,we identified a number of key residues including R294,E264,and M179 that can improve catalytic efficiency or change the regioselectivity of CYP-sb21 towards CsA,setting the stage for better enzymatic preparation of CsA-4-OH.This study also provides new insights into the substrate recognition and binding mechanism of P450 enzymes that accommodate bulky substrates.

关 键 词：Cytochrome P450 monooxygenase Crystal structure Cyclosporine A Regioselectivity Hair growth stimulator 

分 类 号：Q814[生物学—生物工程]