Purification, crystallization, X-ray diffraction analysis and phasing of a fungal 17β-hydroxysteroid dehydrogenase

17 beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17 beta-HSDcl) is an NADP(H) dependent enzyme that catalyses oxidoreduction of estrogens and androgens. It bears sequence similarity to fungal ketoreductases, to bacterial 7 alpha-HSD and even to human 17 beta-HSD types 4 and 8. A structure-based model of 17 beta-HSDcl with a docked coenzyme NADPH and the substrate androstenedione, was built previously, based on the crystal structure of a homologous fungal ketoreductase, which also belongs to the short-chain dehydrogenase-reductase superfamily. To validate the homology built model and to gain further insight into the structure and function of this model enzyme, we initiated the production and purification of the wild type enzyme on a multi-milligram scale for its Xray structural determination. The protein coding sequence of the 17HSDcl gene was cloned into the pGEX expression vector as a fusion protein in with grutathione-S-transferase (GST). The recombination protein was expressed in Escherichia coli, purified to homogeneity by affinity chromatography on Glutathione Sepharose and recovered, following thrombin cleavage, as recombinant binant 17 beta-HSDcl. The purified enzyme was about 95% homogenous, as revealed by SDS PAGE. Activity surveillance at 4 degrees C and 21 degrees C showed adequate protein stability only at the lower temperature. The optimal conditions for crystallization of 17 beta-HSDcl (apo form) were established. Crystals appeared as well shaped bi-pyramids, displayed 14,22 space group symmetry and diffracted to a resolution of 1.7 angstrom. The unit cell parameters were determined to be a = b = 67.17 angstrom and c = 266.90 angstrom. Phasing was successfully performed by Patterson search techniques. The crystallographic refinement and the modelling of solvent molecules are now in progress.