Structure-Functional Analysis of Human Cytochrome P450 2C8 Using Directed Evolution

被引:6
作者
Lee, Rowoon [1 ]
Kim, Vitchan [1 ]
Chun, Youngjin [2 ]
Kim, Donghak [1 ]
机构
[1] Konkuk Univ, Dept Biol Sci, Seoul 05029, South Korea
[2] Chung Ang Univ, Coll Pharm, Seoul 06974, South Korea
基金
新加坡国家研究基金会;
关键词
P450; directed evolution; luciferin; paclitaxel; arachidonic acid; mass spectrometry; ARACHIDONIC-ACID; BINDING; EXPRESSION; ENZYMES; P4502C8; STEPS; 2A6; 1A2;
D O I
10.3390/pharmaceutics13091429
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
The human genome includes four cytochrome P450 2C subfamily enzymes, and CYP2C8 has generated research interest because it is subject to drug-drug interactions and various polymorphic outcomes. To address the structure-functional complexity of CYP2C8, its catalytic activity was studied using a directed evolution analysis. Consecutive rounds of random mutagenesis and screening using 6-methoxy-luciferin produced two mutants, which displayed highly increased luciferase activity. Wild-type and selected mutants were expressed on a large scale and purified. The expression levels of the D349Y and D349Y/V237A mutants were similar to 310 and 460 nmol per liter of culture, respectively. The steady-state kinetic analysis of paclitaxel 6 alpha-hydroxylation showed that the mutants exhibited a 5-7-fold increase in k(cat) values and a 3-5-fold increase in catalytic efficiencies (k(cat)/K-M). In arachidonic acid epoxidation, two mutants exhibited a 30-150-fold increase in k(cat) values and a 40-110-fold increase in catalytic efficiencies. The binding titration analyses of paclitaxel and arachidonic acid showed that the V237A mutation had a lower K-d value, indicating a tighter substrate-binding affinity. The structural analysis of CYP2C8 indicated that the D349Y mutation was close enough to the putative binding domain of the redox partner; the increase in catalytic activity could be partially attributed to the enhancement of the P450 coupling efficiency or electron transfer.
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页数:12
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