Purification, crystallization and preliminary X-ray crystallographic analysis of 3-ketosteroid ?1-dehydrogenase from Rhodococcus erythropolis SQ1.

ABSTRACT: 3-Ketosteroid ?(1)-dehydrogenase plays a crucial role in the early steps of steroid degradation by introducing a double bond between the C1 and C2 atoms of the A-ring of its 3-ketosteroid substrates. The 3-ketosteroid ?(1)-dehydrogenase from Rhodococcus erythropolis SQ1, a 56?kDa flavoprotein, was crystallized using the sitting-drop vapour-diffusion method at room temperature. The crystals grew in various buffers over a wide pH range (from pH 5.5 to 10.5), but the best crystallization condition consisted of 2%(v/v) PEG 400, 0.1?M HEPES pH 7.5, 2.0?M ammonium sulfate. A native crystal diffracted X-rays to 2.0?Å resolution. It belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 107.4, b = 131.6, c = 363.2?Å, and contained eight molecules in the asymmetric unit. The initial structure of the enzyme was solved using multi-wavelength anomalous dispersion (MAD) data collected from a Pt-derivatized crystal.

SUBMITTER: Rohman A 

PROVIDER: S-EPMC3374511 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature