Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450 BM3-catalyzed hydroxylation of small alkanes
Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450 BM3-catalyzed hydroxylation of small alkanes
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DOI：
10.1093/protein/gzs004
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发表时间：
2012-04-01
影响因子：
2.4
通讯作者：
Arnold, Frances H.
中科院分区：
文献类型：
--
作者：
Chen, Mike M. Y.;Snow, Christopher D.;Arnold, Frances H.
采用组合位点饱和突变（CSSM）和基于c轨道和CRAM计算设计算法的两个文库（最多10个活性位点残基）三种半理性方法，对细胞色素P450 BM3进行了去甲基化二甲醚和羟化丙烷和乙烷的修饰。与随机诱变文库和针对相同残基的单个位点饱和文库相比，这些小文库（3431028个变体）在功能分数和最大活性方面都是丰富的。尽管CSSM、C-orbit和CRAM文库的平均氨基酸取代水平分别为2.6、5和7.5，但至少有75个文库成员折叠正确。丙烷羟基化和乙烷羟基化的P450 BM3变异体使用了所有三种诱变方法，只有两个氨基酸取代。使用CRAM算法设计的文库旨在减小结合袋的大小，产生了更多数量的活性变体和支持最大数量催化周转率的变体。最活跃的变体E32在36次耦合下支持16800次丙烷周转，其活性可与使用随机和位点饱和诱变方法进行1012轮定向进化后获得的变体相匹敌。在本研究中，没有一个变异实现了丙烷羟基化的完全再专门化（包括93偶联），这是以前通过多轮诱变和筛选获得的。然而，这些半理性方法允许在序列空间中跳跃到具有所需函数的变体。
Three semi-rational approaches, combinatorial site-saturation mutagenesis (CSSM) using a reduced amino acid set and two libraries based on C-orbit and CRAM computational design algorithms targeting up to 10 active site residues, were used to engineer cytochrome P450 BM3 to demethylate dimethyl ether and hydroxylate propane and ethane. These small libraries (3431028 variants) were all enriched with respect to the fraction functional and maximal activities compared with a random mutagenesis library and individual site-saturation libraries targeting the same residues. Despite high average amino acid substitution levels of 2.6, 5 and 7.5, the CSSM, C-orbit and CRAM libraries had at least 75 of library members properly folded. Propane- and ethane-hydroxylating P450 BM3 variants were identified using all three mutagenesis approaches, with as few as two amino acid substitutions. The library designed using the CRAM algorithm, which sought to reduce the size of the binding pocket, produced both a higher number of active variants and variants supporting the greatest number of catalytic turnovers. The most active variant E32 supports 16 800 propane turnovers at 36 coupling, which rivals the activity of variants obtained after 1012 rounds of directed evolution using random and site-saturation mutagenesis. None of the variants in this study achieved the complete re-specialization for propane hydroxylation (including 93 coupling) previously obtained via multiple rounds of mutagenesis and screening. However, these semi-rational approaches allowed for large jumps in sequence space to variants with the desired functions.