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Rational design of novel mutants of fungal 17β-hydroxysteroid dehydrogenase

Paper ID Volume ID Publish Year Pages File Format Full-Text
25259 43563 2007 8 PDF Available
Title
Rational design of novel mutants of fungal 17β-hydroxysteroid dehydrogenase
Abstract

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 17β-hydroxysteroid dehydrogenases (17β-HSD), like the 17β-HSD activity of Saccharomyces cerevisiae, Pichia faranosa and Mycobacterium sp., and by purified 3β,17β-HSD from Pseudomonas testosteroni. In addition to the bacterial 3β,17β-HSD the 17β-HSD of the filamentous fungus Cochliobolus lunatus is the only microbial 17β-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.

Keywords
Biotransformation of steroids; 17β-Hydroxysteroid dehydrogenase; Fungi; Cochliobolus lunatus; Site-directed mutagenesis; Enzyme engineering
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Rational design of novel mutants of fungal 17β-hydroxysteroid dehydrogenase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 129, Issue 1, 30 March 2007, Pages 123–130
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us