Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 606, Issue -, Pages 151-156Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2016.08.002
Keywords
Mutagenesis; Substrate specificity; Stereospecificity; Alcohol dehydrogenase; Thermophilic
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Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (SADH) reduces aliphatic ketones according to Prelog's Rule, with binding pockets for small and large substituents. It was shown previously that the I86A mutant SADH reduces acetophenone, which is not a substrate of wild-type SADH, to give the anti-Prelog R-product (Musa, M. M.; Lott, N.; Laivenieks, M.; Watanabe, L; Vieille, C.; Phillips, R. S. ChemCatChem 2009,1, 89-93.). However, I86A SADH did not reduce aryl ketones with substituents larger than fluorine. We have now expanded the small pocket of the active site of I86A SADH by mutation of Cys-295 to alanine to allow reaction of substituted acetophenones. As predicted, the double mutant I86A/ C295A SADH has broadened substrate specificity for meta-substituted, but not pars-substituted, acetophenones. However, the increase of the substrate specificity of I86A/C295A SADH is accompanied by a decrease in the k(cat)/K-m, values of acetophenones, possibly due to the substrates fitting loosely inside the more open active site. Nevertheless, I86A/C295A SADH gives high conversions and very high enantiomeric excess of the anti-Prelog R-alcohols from the tested substrates. (C) 2016 Elsevier Inc. All rights reserved.
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