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

2016 ◽  
Vol 606 ◽  
pp. 151-156 ◽  
Author(s):  
Christopher M. Nealon ◽  
Travis P. Welsh ◽  
Chang Sup Kim ◽  
Robert S. Phillips
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Secondary Alcohol ◽  
Thermoanaerobacter Ethanolicus ◽  
Secondary Alcohol Dehydrogenase ◽  
Aryl Ketones

scholarly journals Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme

1996 ◽  
Vol 316 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Douglas S. BURDETTE ◽  
Claire VIEILLE ◽  
J. Gregory ZEIKUS
Keyword(s):  
Alcohol Dehydrogenase ◽  
Secondary Alcohol ◽  
Primary Alcohol ◽  
Recombinant Enzyme ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Arrhenius Plots ◽  
Thermoanaerobacter Ethanolicus ◽  
Secondary Alcohol Dehydrogenase

The adhB gene encoding Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (S-ADH) was cloned, sequenced and expressed in Escherichia coli. The 1056 bp gene encodes a homotetrameric recombinant enzyme consisting of 37.7 kDa subunits. The purified recombinant enzyme is optimally active above 90 °C with a half-life of approx. 1.7 h at 90 °C. An NADP(H)-dependent enzyme, the recombinant S-ADH has 1400-fold greater catalytic efficiency in propan-2-ol oxidation than in ethanol oxidation. The enzyme was inactivated by chemical modification with dithionitrobenzoate (DTNB) and diethylpyrocarbonate, indicating that Cys and His residues are involved in catalysis. Zinc was the only metal enhancing S-ADH reactivation after DTNB modification, implicating the involvement of bound zinc in catalysis. Arrhenius plots for the oxidation of propan-2-ol by the native and recombinant S-ADHs were linear from 25 to 90 °C when the enzymes were incubated at 55 °C before assay. Discontinuities in the Arrhenius plots for propan-2-ol and ethanol oxidations were observed, however, when the enzymes were preincubated at 0 or 25 °C. The observed Arrhenius discontinuity therefore resulted from a temperature-dependent, catalytically significant S-ADH structural change. Hydrophobic cluster analysis comparisons of both mesophilic and thermophilic S-ADH and primary- versus S-ADH amino acid sequences were performed. These comparisons predicted that specific proline residues might contribute to S-ADH thermostability and thermophilicity, and that the catalytic Zn ligands are different in primary-alcohol dehydrogenases (two Cys and a His) and S-ADHs (Cys, His, and Asp).

Racemization of enantiopure secondary alcohols by Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase

2013 ◽  
Vol 11 (17) ◽  
pp. 2911 ◽  
Author(s):  
Musa M. Musa ◽  
Robert S. Phillips ◽  
Maris Laivenieks ◽  
Claire Vieille ◽  
Masateru Takahashi ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Secondary Alcohol ◽  
Secondary Alcohols ◽  
Thermoanaerobacter Ethanolicus ◽  
Secondary Alcohol Dehydrogenase
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