Department of Biological Sciences, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, United States.
Methods Enzymol. 2024;704:27-38. doi: 10.1016/bs.mie.2024.05.007. Epub 2024 Jun 27.
Rieske non-heme iron oxygenases are ubiquitously expressed in prokaryotes. These enzymes catalyze a wide variety of reactions, including cis-dihydroxylation, mono-hydroxylation, sulfoxidation, and demethylation. They contain a Rieske-type [2Fe-2S] cluster and an active site with a mono-nuclear iron bound to a 2-His carboxylate triad. Naphthalene 1,2 dioxygenase, a representative of this family, catalyzes the conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. This transformation requires naphthalene, two electrons, and an oxygen molecule. The first structure of the terminal oxygenase component of a Rieske non-heme iron oxygenase to be determined was naphthalene 1,2 dioxygenase (NDO-O). In this article, we describe in detail the methods used to recombinantly express and purify NDO-O in rich and minimal salts media, the crystallization of NDO-O for structure determination by X-ray crystallography, the challenges faced, and the methods used for the preparation of enzyme ligand complexes. The methods used here resulted in the determination of several NDO-O complexes with aromatic substrates, nitric oxide, oxygen molecule, and products, leading to an initial understanding of the mechanism of enzyme catalysis and the molecular determinants of the regio- and stereo-specificity of this class of enzymes.
Rieske 非血红素铁加氧酶广泛存在于原核生物中。这些酶催化多种反应，包括顺式二羟基化、单羟基化、亚磺化和脱甲基化。它们含有 Rieske 型 [2Fe-2S] 簇和一个活性位点，其中单核铁与 2-His 羧酸盐三联体结合。萘 1,2-二加氧酶是该家族的代表，催化萘转化为 (+)-顺式-(1R,2S)-二羟基-1,2-二氢萘。这种转化需要萘、两个电子和一个氧分子。第一个被确定的 Rieske 非血红素铁加氧酶末端加氧酶组分的结构是萘 1,2-二加氧酶（NDO-O）。在本文中，我们详细描述了在丰富和基础盐培养基中重组表达和纯化 NDO-O 的方法、用于 X 射线晶体学结构测定的 NDO-O 的结晶、所面临的挑战以及酶配体复合物的制备方法。这里使用的方法导致了几种 NDO-O 与芳香族底物、一氧化氮、氧分子和产物的复合物的确定，从而初步了解了酶催化的机制和这类酶的区域和立体特异性的分子决定因素。
