Avtor/Urednik		Buedefeld, Tomaž; Casetta, Alberto; Lanišnik-Rižner, Tea; Kristan, Katja; Stojan, Jure; Lamba, Doriano
Naslov		Purification, crystallization, X-ray diffraction analysis and phasing of a fungal 17beta-hydroxysteroid dehydrogenase
Tip		članek
Vir		In: Weiner H, Plapp B, Lindahl R, et al, editors. Enzymology and molecular biology of carbonyl metabolism. Vol 12. West Lafayette, Indiana: Purdue university press,
Leto izdaje		2006
Obseg		str. 360-5
Jezik		eng
Abstrakt		17beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17betaHSDcI) is an NADP(H) dependent enzyme that preferentially catalyses oxidoreduction of estrogens and androgens. It bears sequence similarity to fungal ketoreductases, to bacterial 7alpha-HSD and even to human 17beta-HSD types 4 and 8. A structure-based model of 17beta-HSDcI 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 X-ray structural determination. The protein coding sequence of the 17HSDcl gene was cloned into the pGEX expression vector as a fusion protein with glutathione-S-transferase (GST). The recombinant protein was expressed in Escherichia coli, purified to homogeneity by affinity chromatography on Glutathione Sepharose and recovered, following thrombin cleavage, as recombinant 17beta-HSDcI. The purified enzyme was about 95% homogenous, as revealed by SDS PAGE. Activity surveillance at 4°C and 21°C showed adequate protein stability only at the lower temperature. The optimal conditions for crystallization of 17beta-HSDcI (apo form) were established. Crystals appeared as well shaped bi-pyramids, displayed I4 1 22 space group symmetry and diffracted to a resolution of 1.7A. The unit cell parameters were determined to be a = b = 67.17A and c = 266.90A. Phasing was successfully performed by Patterson search techniques. The crystallographic refinement and the modelling of solvent molecules are now in progress.
Deskriptorji		17-HYDROXYSTEROID DEHYDROGENASES ASCOMYCETES CRYSTALLIZATION X-RAY DIFFRACTION RECOMBINANT PROTEINS BLOTTING, WESTERN