Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway, New Jersey 07065, USA.
Department of Chemistry, University of Chicago, 5735 S. Ellis Avenue, Chicago, Illinois 60637, USA.
Nat Chem. 2018 Mar;10(3):318-324. doi: 10.1038/nchem.2927. Epub 2018 Jan 22.
Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.
随机诱变有可能在不要求详细了解蛋白质结构的情况下优化蛋白质催化剂的效率和选择性；然而，引入合成金属辅因子会使酶文库的表达和筛选复杂化，并且必须消除来自游离辅因子的活性。在这里，我们报告了一个通过随机诱变创建和筛选人工金属酶 (ArM) 文库的有效平台，我们使用该平台来进化具有高度选择性的二钌环丙烷酶。易错 PCR 和组合密码子诱变使随机突变的多重分析成为可能，包括在远离假定的 ArM 活性位点的位点，这些位点很难通过靶向诱变方法来识别。鉴定出对每个环丙烷产物对映体的选择性都显著提高的变体，并且观察到比通过靶向诱变获得的先前报道的 ArM 环丙烷酶更高的活性。这种提高的选择性扩展到其他二钌催化的转化，包括 N-H、S-H 和 Si-H 插入，表明为一种反应进化的 ArM 可以作为进化其他催化剂的起点。
