High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.
High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.
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DOI：
10.1016/s0956-5663(01)00285-8
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发表时间：
2002
影响因子：
12.6
通讯作者：
G. Tsotsou;A. Cass;G. Gilardi
中科院分区：
文献类型：
--
作者：
G. Tsotsou;A. Cass;G. Gilardi
描述了一种快速鉴定药物代谢酶细胞色素P450潜在底物化合物的方法。该策略是基于在实验条件下自发裂解的大肠杆菌细胞中表达的酶在底物周转期间检测NAD（P）H氧化的降解产物。该方法的性能进行了测试的野生型细胞色素P450 BM 3，花生四烯酸（AA）和月桂酸（LA），和两个基板与环境意义，阴离子表面活性剂十二烷基硫酸钠（SDS），和溶剂1，1，2，2-四氯乙烷（TCE）的两个已知的基板。在没有添加底物的情况下，表达细胞色素P450 BM 3的细胞给出的最小背景信号仅为存在饱和底物时信号的3%。对照实验已经证明，该方法通过P450 BM 3的催化周转特异性地检测NADPH氧化。该测定已被改编成微量滴定板格式，并用于筛选一系列呋咱衍生物作为潜在的底物。三种衍生物被确定为底物。该方法给出了一个显着不同的信号为两个异构体呋咱衍生物。用纯化的酶验证和确认细胞裂解物上发现的所有结果。该策略为自动化高通量筛选药理学和生物技术感兴趣的分子的NAD（P）H-连接的酶活性以及NAD（P）H-依赖性生物催化剂的随机突变体库开辟了道路。
A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts.