Improving the Activity of Cytochrome P450 BM-3 Catalyzing Indole Hydroxylation by Directed Evolution
Improving the Activity of Cytochrome P450 BM-3 Catalyzing Indole Hydroxylation by Directed Evolution
复制标题
DOI：
10.1007/s12010-013-0353-5
复制
发表时间：
2013-07
影响因子：
3
通讯作者：
Pengpai Zhang;H. Sheng;Mei Lehe;Yinlin Lei;Zhihua Jin;Guixiang Hu
中科院分区：
文献类型：
--
作者：
Pengpai Zhang;H. Sheng;Mei Lehe;Yinlin Lei;Zhihua Jin;Guixiang Hu
细胞色素P450 BM-3（A74 G/F87 V/L188 Q）可催化吲哚生成靛蓝。为了进一步提高这种能力，用易错PCR对P450 BM-3（A74 G/F87 V/L188 Q）的血红素结构域进行随机诱变。从易错文库中筛选出一个突变体V445 A，该突变体对吲哚的比活性最高。V445 A的动力学参数也得到了很大的改善。与亲本酶相比，V445 A的转化率（kcat）提高了7.5倍，Km值降低了9.2%。因此，V445 A的催化效率（kcat/Km）比亲本酶提高到8.2倍。此外，通过在Val 445位置的饱和诱变证实丙氨酸是最佳的氨基酸取代。三维结构分析也被用来合理化的突变对酶的性质的影响。本研究表明，随机突变是有效的，以确定突变体的潜在价值的工业和增加我们的洞察力P450 BM-3。
Cytochrome P450 BM-3 (A74G/F87V/L188Q) could catalyze indole to produce indigo. To further improve this capability, random mutagenesis was performed on the heme domain of P450 BM-3 (A74G/F87V/L188Q) with error-prone PCR. A single mutant V445A was selected out from the error-prone library and exhibited the highest specific activity toward indole among the mutants obtained. The kinetic parameters of V445A were also highly improved. Compared with the parent enzyme, the turnover rate (kcat) of V445A was increased by 7.5 times, while itsKmvalue decreased by 9.2 %. Consequently, the catalytic efficiency (kcat/Km) of V445A was raised to 8.2 times than that of the parent enzyme. Moreover, alanine was confirmed as the best amino acid substitution by saturated mutagenesis in Val445 position. Three-dimensional structure analysis was also used to rationalize the effect on the enzyme properties of the mutation. This study showed that random mutagenesis was efficient to identify mutants with potential values in industry and increased our insight into P450 BM-3.