Rational design of novel mutants of fungal 17beta-hydroxysteroid dehydrogenase.
Reduction of 17-ketosteroids is a biocatalytic process of economic significance for the production of steroid drugs. This reaction can be catalyzed by different microbial 17beta-hydroxysteroid dehydrogenases (17beta-HSD), like the 17beta-HSD activity of Saccharomyces cerevisiae, Pichia faranosa and Mycobacterium sp., and by purified 3beta,17beta-HSD from Pseudomonas testosteroni. In addition to the bacterial 3beta,17beta-HSD the 17beta-HSD of the filamentous fungus Cochliobolus lunatus is the only microbial 17beta-HSD that has been expressed as a recombinant protein and fully characterized. On the basis of its modeled 3D structure, we selected several positions for the replacement of amino acids by site-directed mutagenesis to change substrate specificity, alter coenzyme requirements, and improve overall catalytic activity. Replacement of Val161 and Tyr212 in the substrate-binding region by Gly and Ala, respectively, increased the initial rates for the conversion of androstenedione to testosterone. Replacement of Tyr49 within the coenzyme binding site by Asp changed the coenzyme specificity of the enzyme. This latter mutant can convert the steroids not only in the presence of NADP(+) and NADPH, but also in the presence of NADH and NAD(+). The replacement of His164, located in the non-flexible part of the 'lid' covering the active center resulted in a conformation of the enzyme that possessed a higher catalytic activity.
Kristan
Katja
K
Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia.
Stojan
Jure
J
Adamski
Jerzy
J
Lanisnik Rizner
Tea
T
D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D003067:Coenzymes; D005658:Fungi / Q000201:enzymology*; D005818:Genetic Engineering / Q000379:methods*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D003067:Coenzymes; D050505:Mutant Proteins; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase