Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.
Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.
Methods Enzymol. 2024;703:167-192. doi: 10.1016/bs.mie.2024.05.013. Epub 2024 Jun 12.
Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.
Rieske 非血红素铁依赖性加氧酶（ROs）是一组用途广泛的酶，传统上与芳香族异生物质的降解有关。此外，ROs 还被发现在天然产物生物合成中发挥关键作用，表现出广泛的催化多样性，通常具有较高的区域和立体选择性。然而，由于其复杂的结构和功能特性，包括多组分组成、辅助因子依赖性和对活性氧的敏感性，ROs 的详细表征具有相当大的挑战性。此外，天然产物生物合成中间体的底物可用性、芳香烃的有限溶解度和自由基介导的反应机制可能会进一步使功能测定复杂化。尽管存在这些挑战，但 ROs 作为药物应用和生物修复的生物催化剂具有巨大的潜力。本文以荧光假单胞菌 IP01 的枯烯双加氧酶（CDO）为例，详细介绍了用于表征芳香烃加氧酶的技术。此外，还描述了表征新型 ROs 的潜在陷阱、预期并发症和建议的解决方案，为未来的 RO 研究和研究该酶类的策略提供了框架。特别是，我们描述了从构建设计到表达条件再到纯化程序，最终通过各种活性测定来获得 CDO 的方法，以及最终的活性测定。
