Open AccessPurification, crystallization and preliminary X‐ray crystallographic analysis of 3‐ketosteroid Δ 1 ‐dehydrogenase from Rhodococcus erythropolis SQ1
Author(s) -
Rohman Ali,
van Oosterwijk Niels,
Dijkstra Bauke W.
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112011025
Subject(s) - ketosteroid , crystallization , rhodococcus , x ray , crystallography , x ray crystallography , chemistry , materials science , physics , diffraction , enzyme , biochemistry , organic chemistry , isomerase , optics
3‐Ketosteroid Δ 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 Δ 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 P 2 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.