Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India.
Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India.
Methods Enzymol. 2024;704:39-58. doi: 10.1016/bs.mie.2024.05.014. Epub 2024 Jun 8.
Non-heme iron oxygenases constitute a versatile enzyme family that is crucial for incorporating molecular oxygen into diverse biomolecules. Despite their importance, only a limited number of these enzymes have been structurally and functionally characterized. Surprisingly, there remains a significant gap in understanding how these enzymes utilize a typical architecture and reaction mechanism to catalyze a wide range of reactions. Improving our understanding of these catalysts holds promise for advancing both fundamental enzymology and practical applications. This chapter aims to outline methods for heterologous expression, enzyme preparation, in vitro enzyme assays, and crystallization of biphenyl dioxygenase, phthalate dioxygenase and terephthalate dioxygenase. These enzymes catalyze the dihydroxylation of biphenyl, phthalate and terephthalate molecules, serving as a model for functional and structural analysis of other non-heme iron oxygenases.
非血红素铁加氧酶构成了一个多功能的酶家族，对于将分子氧掺入各种生物分子中至关重要。尽管它们很重要，但只有少数这些酶在结构和功能上得到了表征。令人惊讶的是，对于这些酶如何利用典型的结构和反应机制来催化广泛的反应，我们的理解仍然存在很大的差距。提高我们对这些催化剂的理解有望促进基础酶学和实际应用的发展。本章旨在概述联苯双加氧酶、邻苯二甲酸双加氧酶和对苯二甲酸双加氧酶的异源表达、酶制备、体外酶测定和结晶的方法。这些酶催化联苯、邻苯二甲酸和对苯二甲酸分子的二羟基化，作为其他非血红素铁加氧酶的功能和结构分析的模型。
