title: Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Δ(4)-(5α)-dehydrogenase from Rhodococcus jostii RHA1.
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.
authors.affiliation: Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: van Oosterwijk
authors.forename: Jan
authors.initials: J
authors.lastname: Knol
authors.forename: Lubbert
authors.initials: L
authors.lastname: Dijkhuizen
authors.forename: Robert
authors.initials: R
authors.lastname: van der Geize
authors.forename: Bauke W
authors.initials: BW
authors.lastname: Dijkstra
mesh_terms: D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012240:Rhodococcus / Q000201:enzymology*
chemical_list: D010088:Oxidoreductases
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