CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.
CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.
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DOI：
10.1002/cbic.202000735
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发表时间：
2021-03-16
期刊：
影响因子：
--
通讯作者：
Schallmey A
中科院分区：
文献类型：
--
作者：
Bracco P;Wijma HJ;Nicolai B;Buitrago JAR;Klünemann T;Vila A;Schrepfer P;Blankenfeldt W;Janssen DB;Schallmey A
来自Nocardia farcinica的CYP 154 C5是一种P450单加氧酶，能够在16α位以高区域和立体选择性羟基化一系列类固醇。利用蛋白质工程和底物修饰的基础上的晶体结构的CYP 154 C5，改变了区域选择性的酶在类固醇羟基化已经实现。因此，突变体CYP 154 C5 F92 A对黄体酮的转化除了16α-羟基化之外，还导致形成相应的21-羟基化产物11-脱氧皮质酮。使用MD模拟，这种改变的区域选择性似乎是由于突变体F92 A的活性位点中类固醇的替代结合模式。分子动力学模拟进一步表明，水进入活性位点导致该突变体中更高的解偶联。此外，在5α-雄甾烷-3-酮的转化中观察到野生型CYP 154 C5的排他性15α-羟基化，在C17处缺乏氧官能团。总的来说，我们的数据提供了宝贵的洞察这种细胞色素P450单加氧酶类固醇羟基化的结构-功能关系。 选择性变化：利用基于关键残基和功能基团的底物和蛋白质工程，改变了细菌细胞色素P450单加氧酶CYP 154 C5在不同类固醇羟基化中的区域选择性。除了16α-羟基化外，还形成了在15α和21位含有羟基的类固醇产物。
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio‐ and stereoselectivity at the 16α‐position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21‐hydroxylated product 11‐deoxycorticosterone in addition to 16α‐hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α‐hydroxylation was observed for wild‐type CYP154C5 in the conversion of 5α‐androstan‐3‐one, lacking an oxy‐functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Changes in selectivity: Using substrate and protein engineering based on key residues and functional groups, the regioselectivity of the bacterial cytochrome P450 monooxygenase CYP154C5 in the hydroxylation of different steroids was altered. Apart from 16α‐hydroxylation, steroid products containing hydroxy groups in 15α and 21 position were formed.
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DOI：
10.1107/s090744491003982x
发表时间：
2011-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Evans PR
通讯作者：
Evans PR
影响因子：
3
作者：
Krieger E;Vriend G
通讯作者：
Vriend G
DOI：
10.1107/s090744491200145x
发表时间：
2012-03-01
影响因子：
2.2
作者：
Bell, Stephen G.;Yang, Wen;Wong, Luet-Lok
通讯作者：
Wong, Luet-Lok
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.9
作者：
Krieger, E;Darden, T;Vriend, G
通讯作者：
Vriend, G