caa a22 4500 605500150 CHVBK 20210128100554.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00253-014-6148-y doi (NATIONALLICENCE)springer-10.1007/s00253-014-6148-y Structural basis for the 4′-hydroxylation of diclofenac by a microbial cytochrome P450 monooxygenase [Elektronische Daten] [Lian-Hua Xu, Haruo Ikeda, Ling Liu, Takatoshi Arakawa, Takayoshi Wakagi, Hirofumi Shoun, Shinya Fushinobu] Diclofenac is a nonsteroidal anti-inflammatory drug. It undergoes hydroxylation by mammalian cytochrome P450 enzymes at 4′- and/or 5′-positions. A bacterial P450 enzyme, CYP105D7 from Streptomyces avermitilis, has been shown to catalyze hydroxylation of 1-deoxypentalenic acid and an isoflavone daidzein. Here, we demonstrated that CYP105D7 also catalyzes hydroxylation of diclofenac at the C4′-position. Aspectroscopic analysis showed that CYP105D7 binds diclofenac in a slightly cooperative manner with an affinity of 65μM and a Hill coefficient of 1.16. The crystal structure of CYP105D7 in complex with diclofenac was determined at 2.2Å resolution. The distal pocket of CYP105D7 contains two diclofenac molecules, illustrating drug recognition with a double-ligand-binding mode. The C3′ and C4′ atoms of the dichlorophenyl ring of one diclofenac molecule are positioned near the heme iron, suggesting that it is positioned appropriately for aromatic hydroxylation to yield the 4′-hydroxylated product. However, recognition of diclofenac by CYP105D7 was completely different from that of rabbit CYP2C5, which binds one diclofenac molecule with a cluster of water molecules. The distal pocket of CYP105D7 contains four arginine residues, forming a wall of the substrate-binding pocket, and the arginine residues are conserved in bacterial P450s in the CYP105 family. Springer-Verlag Berlin Heidelberg, 2014 Diclofenac nationallicence Cytochrome P450 nationallicence X-ray crystallography nationallicence Streptomyces avermitilis nationallicence Xenobiotics nationallicence Xu Lian-Hua Ocean College, Zhejiang University, 866 Yuhangtang Road, 310058, Hangzhou, Zhejiang, China aut Ikeda Haruo Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, 252-0373, Sagamihara, Kanagawa, Japan aut Liu Ling Ocean College, Zhejiang University, 866 Yuhangtang Road, 310058, Hangzhou, Zhejiang, China aut Arakawa Takatoshi Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan aut Wakagi Takayoshi Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan aut Shoun Hirofumi Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan aut Fushinobu Shinya Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/7(2015-04-01), 3081-3091 0175-7598 99:7<3081 2015 99 253 https://doi.org/10.1007/s00253-014-6148-y text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6148-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Xu Lian-Hua Ocean College, Zhejiang University, 866 Yuhangtang Road, 310058, Hangzhou, Zhejiang, China aut NATIONALLICENCE 700 1- Ikeda Haruo Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, 252-0373, Sagamihara, Kanagawa, Japan aut NATIONALLICENCE 700 1- Liu Ling Ocean College, Zhejiang University, 866 Yuhangtang Road, 310058, Hangzhou, Zhejiang, China aut NATIONALLICENCE 700 1- Arakawa Takatoshi Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan aut NATIONALLICENCE 700 1- Wakagi Takayoshi Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan aut NATIONALLICENCE 700 1- Shoun Hirofumi Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan aut NATIONALLICENCE 700 1- Fushinobu Shinya Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/7(2015-04-01), 3081-3091 0175-7598 99:7<3081 2015 99 253