Department of Chemistry, University of Michigan, Ann Arbor, MI, United States.
Department of Chemistry, University of Michigan, Ann Arbor, MI, United States.
Methods Enzymol. 2024;703:215-242. doi: 10.1016/bs.mie.2024.05.015. Epub 2024 Jun 18.
The Rieske non-heme iron oxygenases (Rieske oxygenases) comprise a class of metalloenzymes that are involved in the biosynthesis of complex natural products and the biodegradation of aromatic pollutants. Despite this desirable catalytic repertoire, industrial implementation of Rieske oxygenases has been hindered by the multicomponent nature of these enzymes and their requirement for expensive reducing equivalents in the form of a reduced nicotinamide adenine dinucleotide cosubstrate (NAD(P)H). Fortunately, however, some Rieske oxygenases co-occur with accessory proteins, that through a downstream reaction, recycle the needed NAD(P)H for catalysis. As these pathways and accessory proteins are attractive for bioremediation applications and enzyme engineering campaigns, herein, we describe methods for assembling Rieske oxygenase pathways in vitro. Further, using the TsaMBCD pathway as a model system, in this chapter, we provide enzymatic, spectroscopic, and crystallographic methods that can be adapted to explore both Rieske oxygenases and their co-occurring accessory proteins.
Rieske 非血红素铁加氧酶（ Rieske oxygenases ）属于一类金属酶，参与复杂天然产物的生物合成和芳香族污染物的生物降解。尽管具有这种理想的催化作用，但由于这些酶的多组分性质及其需要以还原型烟酰胺腺嘌呤二核苷酸辅酶（NAD(P)H）的形式提供昂贵的还原当量， Rieske 加氧酶在工业上的应用受到了阻碍。然而，幸运的是，一些 Rieske 加氧酶与辅助蛋白共同存在，通过下游反应循环利用催化所需的 NAD(P)H。由于这些途径和辅助蛋白在生物修复应用和酶工程活动中具有吸引力，本文描述了体外组装 Rieske 加氧酶途径的方法。此外，使用 TsaMBCD 途径作为模型系统，本文提供了可用于探索 Rieske 加氧酶及其共同存在的辅助蛋白的酶学、光谱学和晶体学方法。
