Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant
Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant
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DOI：
10.1016/s0167-4838(00)00268-5
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发表时间：
2001-02-09
期刊：
影响因子：
--
通讯作者：
Schmid, RD
中科院分区：
文献类型：
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作者：
Li, QS;Schwaneberg, U;Schmid, RD
通过合理进化工程化来自巨大芽孢杆菌的细胞色素P-450 BM-3的单突变体F87 A，以实现对中等链长底物（C8-C10）的改善的羟基化活性。理性进化结合了理性设计和定向进化，克服了这些方法单独应用时的缺点。基于该酶的X射线结构，通过建模确定了8个突变位点（P25，V26，R47，Y51，S72，A74，L188和M354）。使用基于ω-对硝基苯氧基羧酸（pNCA）的光谱测定筛选通过每个单个位点的位点特异性随机化诱变产生的亚文库。将对较短链长底物显示活性的突变体组合，并再次筛选这些组合文库中具有甚至更好催化性质的突变体。使用这种方法，获得了具有五个突变（V26 T、R47 F、A74 G、L188 K和F87 A）的P-450 BM-3变体，其有效水解8-pNCA。该突变体对ω-对硝基苯氧基癸酸（10-pNCA）和ω-对硝基苯氧基十二烷酸（12-pNCA）的催化效率与野生型P-450 BM-3相当。(C)2001 Elsevier Science B. V.保留所有权利。
The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium, was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega -p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega -p-nitrophenoxydecanoic acid (10-pNCA) and omega -p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3. (C) 2001 Elsevier Science B.V. All rights reserved.