Avtor/Urednik		Kristan, Katja; Kovačič, Brina; Stojan, Jure; Adamski, Jerzy; Lanišnik-Rižner, Tea
Naslov		Further insight into the structural basis of coenzyme and substrate specificity of 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. 352-9
Jezik		eng
Abstrakt		17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is an NADPH dependent member of the short-chain dehydrogenase reductase (SDR) szuperfamily. From its 3D homology built model, based on the crystal structure of 1,3,8-trihydroxynaphthalene reductase from the fungus Magnaporthe grisea, we have performed a series of structure-function relationship studies. Byconstructing the appropriate mutants, we examined the significance of Ser52 (Ser52Ala) and Asn51 (Asn 51 Arg7Ser52Thr) that are situated in the coenzyme binding pocket, and of Val161 (Val161 Gly) and Tyr212 (Tyr212Ala) that might interfere with the substrate C19 methyl group and this manner prevent binding and conversion of androgens. The replacement of Ser52 by Ala resulted in slightly decreased enzyme activity with coenzymed NADP+ and NADPH. A similar effect was observed for the Asn51Arg/Ser52Thr mutant. The catalytic efficiency of both mutants in the presence of the NAD+/NADH pair of coenzymes remained very low, as was also observed for the wild type. The mutations thus demonstrated the importance of Ser52 and Asn51 for appropriate accommodation of the coenzymes NADPH/NADP+. The replacement of Val161 and Tyr212 in the substrate binding region by smaller residues, Gly and Ala, respectively, resulted in increased initial velocities in the presence of the two androgen pairs tested. Androstenedione and testosterone were the best converted pair of androgens, followed by 5alfa-androstane-3, 17-dione and 5alfa-androstane-17 beta-ol-3-one. These results confirmed that both Tyr212 and Val161 interfere with the substrate C19 methyl group and in this manner impair binding of androgens.
Deskriptorji		COENZYMES SUBSTRATE SPECIFICITY ASCOMYCETES 17-HYDROXYSTEROID DEHYDROGENASES MUTAGENESIS, SITE-DIRECTED BINDING SITES