title: His164 regulates accessibility to the active site in fungal 17beta-hydroxysteroid dehydrogenase.
abstract: 17beta-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is an NADPH-dependent member of the short-chain dehydrogenase/ reductase superfamily. To study the catalytic properties of this enzyme, we prepared several specific mutations of 17beta-HSDcl (Tyr167Phe, His164Trp/Gly, Tyr212Ala). Wild-type 17beta-HSDcl and the 17beta-HSDcl mutants were evaluated by chromatographic, kinetic and thermodynamic means. The Tyr167Phe mutation resulted in a complete loss of enzyme activity, while substitution of His164 with Trp and Gly both resulted in higher specificity number (V/K) for the steroid substrates, which are mainly a consequence of easier accessibility of steroid substrates to the active-site hollow under optimized conditions. The Tyr212Ala mutant showed increased activity in the oxidative direction, which appears to be a consequence of increased NADPH dissociation. The kinetic characterizations and thermodynamic analyses also suggest that His164 and Tyr212 in 17beta-HSDcl have a role in the opening and closing of the active site of this enzyme and in the discrimination between oxidized and reduced coenzyme.
authors.affiliation: Institute of Biochemistry, Medical Faculty, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia.
authors.forename: Katja
authors.initials: K
authors.lastname: Kristan
authors.forename: Jerzy
authors.initials: J
authors.lastname: Adamski
authors.forename: Tea Lanisnik
authors.initials: TL
authors.lastname: Rizner
authors.forename: Jure
authors.initials: J
authors.lastname: Stojan
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D001203:Ascomycota / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013816:Thermodynamics
chemical_list: D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase