Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Delta(4)-(5 alpha)-dehydrogenase from Rhodococcus jostii RHA1

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 Delta(4)-(5 alpha)-dehydrogenase [Delta(4)-(5 alpha)-KSTD] from R. jostii RHA1 was cloned and overexpressed in Escherichia coli. His-tagged Delta(4)-(5 alpha)-KSTD enzyme was purified by Ni2+-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 angstrom. Data were collected to a resolution of 1.6 angstrom