School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi, P.R. China.
Department of Chemistry, Johns Hopkins University, Baltimore, MD, United States.
Methods Enzymol. 2024;703:195-213. doi: 10.1016/bs.mie.2024.07.003. Epub 2024 Jul 23.
Nonheme iron enzymes are versatile biocatalysts for a broad range of unique and powerful transformations, such as hydroxylation, chlorination, and epimerization as well as cyclization/ring-opening of organic molecules. Beyond their native biological functions, these enzymes are robust for engineering due to their structural diversity and high evolvability. Based on enzyme promiscuity and directed evolution as well as inspired by synthetic organic chemistry, nonheme iron enzymes can be repurposed to catalyze reactions previously only accessible with synthetic catalysts. To this end, our group has engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for new-to-nature radical transformations. In particular, we have demonstrated that a nonheme iron enzyme, (4-hydroxyphenyl)pyruvate dioxygenase from streptomyces avermitilis (SavHppD), can be repurposed to enable abiological radical-relay process to access C(sp)-H azidation products. This represents the first known instance of enzymatic radical relay azidation reactions. In this chapter, we describe the detailed experimental protocol to convert promiscuous nonheme iron enzymes into efficient and selective biocatalyst for radical relay azidation reactions. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation mutagenesis, protein expression and whole-cell reactions screening in a 96-well plate. These protocol details might be useful to engineer various nonheme iron enzymes for other applications.
非血红素铁酶是一类多功能的生物催化剂，能够催化多种独特而强大的转化反应，如羟化、氯化和差向异构化，以及有机分子的环化/开环反应。除了其天然的生物学功能外，由于其结构多样性和高度可进化性，这些酶在工程应用中也非常强大。基于酶的多功能性和定向进化，以及受合成有机化学的启发，非血红素铁酶可以被重新用于催化以前只能使用合成催化剂进行的反应。为此，我们的团队设计了一系列非血红素铁酶，以利用非天然的自由基接力机制来实现新的自然自由基转化。特别是，我们已经证明，一种非血红素铁酶，来自链霉菌avermitilis 的（4-羟基苯丙酮酸双加氧酶）（SavHppD），可以被重新用于实现非生物的自由基接力过程，从而获得 C(sp)-H 叠氮化物产物。这代表了酶促自由基接力叠氮化反应的首例已知实例。在本章中，我们描述了将多功能非血红素铁酶转化为高效、选择性的自由基接力叠氮化反应生物催化剂的详细实验方案。详细描述了一轮定向进化，包括易错 PCR 产生和处理、点饱和突变、在 96 孔板中进行蛋白表达和全细胞反应筛选。这些方案细节可能对其他应用中各种非血红素铁酶的工程设计有用。
