Random mutagenesis of human cytochrome P450 2A6 and screening with indole oxidation products
Random mutagenesis of human cytochrome P450 2A6 and screening with indole oxidation products
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DOI：
10.1006/abbi.2001.2569
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发表时间：
2001-11-01
影响因子：
3.9
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
--
作者：
Nakamura, K;Martin, MV;Guengerich, FP
基于吲哚代谢，在大肠杆菌中筛选底物识别序列 (SRS) 区域中生成的随机文库中的细胞色素 P450 (P450) 2A6 突变体。 SRS 3 和 4 文库产生的菌落至少与野生型 (WT) P450 2A6 一样产生靛蓝，并且一些菌落在重新接种后始终呈更蓝色。一种突变体 F209T 显示吲哚 3-羟基化 < WT，但香豆素 7-羟基化的 k(cat) 为 13 倍 > WT。双突变体 L240C/N297Q 始终产生非常蓝色的菌落。根据产品的可见光谱和 HPLC 判断，五种突变体产生的吲哚色素混合物与 WT 不同。当表达突变体的细菌在 26 种取代吲哚中的每一种存在下生长时，在几种突变体中观察到不同染料形成的多种模式。这种方法对于了解 P450 2A6 功能以及生成新染料和其他产品具有潜在价值。 (C) 2001 年学术出版社。
Cytochrome P450 (P450) 2A6 mutants from randomized libraries generated in the substrate recognition sequence (SRS) regions were screened in Escherichia coli on the basis of indole metabolism. SRS 3 and 4 libraries yielded colonies that produced indigo at least as well as wild-type (WT) P450 2A6, and some colonies were consistently more blue upon replating. One mutant, F209T, showed indole 3-hydroxylation < WT but had a k(cat) for coumarin 7-hydroxylation 13-fold > WT. The double mutant L240C/N297Q consistently produced very blue colonies. Five mutants yielded mixtures of pigments from indole different than WT, as judged by visible spectra and HPLC of products. When bacteria expressing the mutants were grown in the presence of each of 26 substituted indoles, a variety of patterns of formation of different dyes was seen with several of the mutants. This approach has potential value in understanding P450 2A6 function and generating new dyestuffs and other products. (C) 2001 Academic Press.