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Updated: Jul 19, 2026

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae
Published on: February 16, 2015
His164 regulates accessibility to the active site in fungal 17beta-hydroxysteroid dehydrogenase
Katja Kristan1, Jerzy Adamski, Tea Lanisnik Rizner
1Institute of Biochemistry, Medical Faculty, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia.
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.
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