Structural basis for the 4'-hydroxylation of diclofenac by a microbial cytochrome P450 monooxygenase.
Structural basis for the 4'-hydroxylation of diclofenac by a microbial cytochrome P450 monooxygenase.
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DOI：
10.1007/s00253-014-6148-y
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发表时间：
2015
影响因子：
5
通讯作者：
Fushinobu Shinya
中科院分区：
文献类型：
--
作者：
Xu Lian-Hua;Ikeda Haruo;Liu Ling;Arakawa Takatoshi;Wakagi Takayoshi;Shoun Hirofumi;Fushinobu Shinya
双氯芬酸是一种非甾体抗炎药。通过哺乳动物细胞色素P450酶在4′-和/或5′-位进行羟基化。来自阿维链霉菌的细菌P450酶CYP 105 D 7已被证明催化1-脱氧戊二酸和大豆苷元的羟基化。在这里，我们证明了CYP 105 D 7也催化双氯芬酸在C4′-位的羟基化。光谱分析表明，CYP 105 D 7以轻微的协同方式与双氯芬酸结合，亲和力为65 μM，希尔系数为1.16。以2.2 Å的分辨率测定了CYP 105 D 7与双氯芬酸复合物的晶体结构。CYP 105 D 7的远端口袋含有两个双氯芬酸分子，说明药物识别具有双配体结合模式。一个双氯芬酸分子的二氯苯基环的C3′和C4′原子位于血红素铁附近，表明其位置适合于芳族羟基化，以产生4′-羟基化产物。然而，CYP 105 D 7对双氯芬酸的识别与兔CYP 2C 5的识别完全不同，兔CYP 2C 5将一个双氯芬酸分子与一簇水分子结合。CYP 105 D 7的远端口袋含有四个精氨酸残基，形成底物结合口袋的壁，并且精氨酸残基在CYP 105家族的细菌P450中是保守的。
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 fromStreptomyces 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. A spectroscopic analysis showed that CYP105D7 binds diclofenac in a slightly cooperative manner with an affinity of 65 μM and a Hill coefficient of 1.16. The crystal structure of CYP105D7 in complex with diclofenac was determined at 2.2 Å 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.