来自约氏红球菌RHA1的3-酮甾体Δ(4)-(5α)-脱氢酶的克隆、过表达、纯化、结晶及初步X射线分析。
Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Δ(4)-(5α)-dehydrogenase from Rhodococcus jostii RHA1.
作者信息
van Oosterwijk Niels, Knol Jan, Dijkhuizen Lubbert, van der Geize Robert, Dijkstra Bauke W
机构信息
Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
出版信息
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Oct 1;67(Pt 10):1269-73. doi: 10.1107/S1744309111028727. Epub 2011 Sep 30.
Abstract
3-Ketosteroid dehydrogenases are flavoproteins which play key roles in steroid ring degradation. The enzymes are abundantly present in actinobacteria, including the catabolic powerhouse Rhodococcus jostii and the pathogenic species R. equi and Mycobacterium tuberculosis. The gene for 3-ketosteroid Δ(4)-(5α)-dehydrogenase [Δ(4)-(5α)-KSTD] from R. jostii RHA1 was cloned and overexpressed in Escherichia coli. His-tagged Δ(4)-(5α)-KSTD enzyme was purified by Ni(2+)-NTA affinity chromatography, anion-exchange chromatography and size-exclusion chromatography and was crystallized using the hanging-drop vapour-diffusion method. Seeding greatly improved the number of crystals obtained. The crystals belonged to space group C222(1), with unit-cell parameters a = 99.2, b = 114.3, c = 110.2 Å. Data were collected to a resolution of 1.6 Å.
摘要
3-酮甾体脱氢酶是黄素蛋白，在甾体环降解中起关键作用。这些酶大量存在于放线菌中，包括分解代谢能力强的约氏红球菌以及致病物种马红球菌和结核分枝杆菌。从约氏红球菌RHA1中克隆了3-酮甾体Δ(4)-(5α)-脱氢酶[Δ(4)-(5α)-KSTD]基因，并在大肠杆菌中进行了过表达。带有His标签的Δ(4)-(5α)-KSTD酶通过Ni(2+)-NTA亲和层析、阴离子交换层析和尺寸排阻层析进行纯化，并采用悬滴气相扩散法进行结晶。晶种大大提高了获得的晶体数量。晶体属于空间群C222(1)，晶胞参数a = 99.2、b = 114.3、c = 110.2 Å。数据收集分辨率为1.6 Å。
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