I86A/C295A mutant secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus has broadened substrate specificity for aryl ketones

Christopher M. Nealon, Travis P. Welsh, Chang Sup Kim, Robert S. Phillips

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

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. 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 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.

Original languageEnglish (US)
Pages (from-to)151-156
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume606
DOIs
StatePublished - Sep 15 2016
Externally publishedYes

Keywords

  • Alcohol dehydrogenase
  • Mutagenesis
  • Stereospecificity
  • Substrate specificity
  • Thermophilic

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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