TY - JOUR
T1 - I86A/C295A mutant secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus has broadened substrate specificity for aryl ketones
AU - Nealon, Christopher M.
AU - Welsh, Travis P.
AU - Kim, Chang Sup
AU - Phillips, Robert S.
N1 - Publisher Copyright:
© 2016 Elsevier Inc.
PY - 2016/9/15
Y1 - 2016/9/15
N2 - 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.
AB - 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.
KW - Alcohol dehydrogenase
KW - Mutagenesis
KW - Stereospecificity
KW - Substrate specificity
KW - Thermophilic
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U2 - 10.1016/j.abb.2016.08.002
DO - 10.1016/j.abb.2016.08.002
M3 - Article
C2 - 27495738
AN - SCOPUS:84981216602
SN - 0003-9861
VL - 606
SP - 151
EP - 156
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
ER -