title: I86A/C295A mutant secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus has broadened substrate specificity for aryl ketones.
abstract: 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. ChemCatChem2009, 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 para-substituted, acetophenones. However, the increase of the substrate specificity of I86A/C295A SADH is accompanied by a decrease in the kcat/Km 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.
authors: Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
authors: Christopher M
authors: CM
authors: Nealon
authors: Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
authors: Travis P
authors: TP
authors: Welsh
authors: Department of Chemical and Biological Engineering, Hanbat National University, Daejeon 34158, South Korea.
authors: Chang Sup
authors: CS
authors: Kim
authors: Department of Chemistry, University of Georgia, Athens, GA 30602, USA; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA. Electronic address: plp@uga.edu.
authors: Robert S
authors: RS
authors: Phillips
mesh_terms: D000409:Alanine / Q000737:chemistry; D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000438:Alcohols / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain; D002849:Chromatography, Gas; D007659:Ketones / Q000737:chemistry*; D007700:Kinetics; D008968:Molecular Conformation; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D013237:Stereoisomerism; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology / Q000235:genetics*
chemical_list: D000438:Alcohols; D001426:Bacterial Proteins; D007659:Ketones; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; C025192:isopropanol dehydrogenase (NADP); D000409:Alanine
keywords: Alcohol dehydrogenase; Mutagenesis; Stereospecificity; Substrate specificity; Thermophilic