Author/Editor     Kristan, Katja
Title     Povezava med strukturo in delovanjem glivne 17beta-hidroksisteroid-dehidrogenaze, modelnega encima naddružine kratkoverižnih dehidrogenaz/reduktaz
Translated title     Structure/function relationship in fungual 17beta-hydroxysteroid dehydrogenase, a model enzyme of the short-chain dehydrogenase/reductase superfamily
Type     monografija
Place     Ljubljana
Publisher     Medicinska fakulteta
Publication year     2005
Volume     str. 147
Language     slo
Abstract     17beta-hydroxysteroid dehydrogenase from the fungus Cochliobulus lunatus (17beta-HSDcI) is currently the only fungal HSD member of the short-chain dehydrogenase/reductase (SDR) superfamily. It is a regiospecific dimeric enzyme that preferentially catalyses reversible oxidoreductions of androgens and estrogens in the presence of the coenzyme NADP(H). A structural model of 17beta-HSDc1 was prepared and this model directed our studies. To elucidate the role of individual amino acids for enzyme activity, we generated variants by site-directed mutagenesis. The mutations demonstrated the importance of Thr200, Thr202, Met204 and Phe205 for the appropriate accommodation of the coenzyme into the active site to enable a hydride transfer, while Arg28 compensates for the two negative charges of the 2'-phosphate group of the coenzyme NADP(H). The enzyme that possesses only one positively charged amino-acid residue is already adapted to NADP(H)-specific oxidoreductions, and so the introduction of an additional basic residue does not significantly improve the activity. The introduction of an acidic residue at position 49 (Tyr49Asp) switched the cofactor preference of the enzyme, and changed the enzyme to an NAD(H)-dependent 17(3-HSDcI. Va1161 and Tyr212 interfere with the substrate C 19 methyl group and in this manner they prevent the binding and conversion of androgens. The results also suggest that the presence of a hydrogen bond between His164 and Tyr212 at the entrance to the active centre has a role in the opening and closing of the active site. (Abstract truncated at 2000 characters)
Descriptors     ASCOMYCETES
17-HYDROXYSTEROID DEHYDROGENASES
SUBSTRATE SPECIFICITY
BINDING SITES
COENZYMES
ENZYME INHIBITORS
ESTROGENS
STRUCTURE-ACTIVITY RELATIONSHIP
X-RAY DIFFRACTION