Author/Editor     Kristan, Katja
Title     Pomen posameznih aminokislinskih ostankov pri delovanju 17beta-hidroksisteroid-dehidrogenaze iz glive Cochliobolus lunatus - priprava in karakterizacija usmerjeno mutiranih proteinov
Type     monografija
Place     Ljubljana
Publisher     Medicinska fakulteta
Publication year     2003
Volume     str. 58
Language     slo
Abstract     17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDc1) is currently the only fungal HSD member of the short-chain dehydrogenase/reductase superfamily. It is an NADP(H) dependent enzyme that preferentially catalyses reversible oxidoreductions of androgens and estrogens. Recently, a structural model of 17beta-HSDcI was prepared and this model directed our studies. We investigated the significance of some amino acid residues for acid-base catalysis, residues that stabilize the nicotinamide ring of the coenzyrne, and those, important for coenzyme specificity. To elucidate the role of individual amino acids, we generated variants by site-directed mutagenesis. The results demonstrated the importance of Tyr167 for acid-base catalysis since the mutation Tyr167Phe resulted in a complete loss of activity. His164, which is situated nearby, ;was substituted for Gly and Trp and both mutants clearly convert the substrates with higher rates than the wild type. We suggest that His164 does not participate in catalysis, but is probably a steric hindrance toward substrate and/or coenzyme. Amino acid residues, which interact with the nicotinamide ring are essential for an appropriate accommodation of coenzyme in the active site. In the model structure of 17betaHSDcI Thr202 is within H-bond distance to the amide moiety of NADPH. We elucidated its importance with two mutants, Thr202I1e and Thr202Va1. The first resulted in an inactive enzyme, while the second retained a very low activity, as observed by TLC analysis after one-hour incubation. The mutation Met204G1y decreased the activity of the enzyme, thus neighboring Met204 might sterically orient the coenzyme into the right position. We suggest the same for Phe205, since the Phe205G1y variant was fully inactive. In NADP(H)-preferring enzymes the two negative charges of the 2'-phosphate group are compensated by two positively charged residues. (Abstract truncated at 2000 characters).
Descriptors     ASCOMYCETES
17-HYDROXYSTEROID DEHYDROGENASES
AMINO ACIDS
MUTAGENESIS, SITE-DIRECTED
COENZYMES
BLOTTING, WESTERN
CHROMATOGRAPHY, THIN LAYER
BACTERIAL PROTEINS
ANTIBODIES, MONOCLONAL
BINDING SITES
NADP
PLASMIDS
POLYMERASE CHAIN REACTION
TRANSFORMATION, GENETIC
ELECTROPHORESIS, POLYACRYLAMIDE GEL
ESCHERICHIA COLI