A Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutant derivative highly active and stereoselective on phenylacetone and benzylacetone

Protein Eng Des Sel. 2007 Feb;20(2):47-55. doi: 10.1093/protein/gzl052. Epub 2007 Feb 5.


The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus 39E (TeSADH) is highly thermostable and solvent-stable, and it is active on a broad range of substrates. These properties make TeSADH an excellent template to engineer an industrial catalyst for chiral chemical synthesis. (S)-1-Phenyl-2-propanol was our target product because it is a precursor to major pharmaceuticals containing secondary alcohol groups. TeSADH has no detectable activity on this alcohol, but it is highly active on 2-butanol. The structural model we used to plan our mutagenesis strategy was based on the substrate's orientation in a horse liver alcohol dehydrogenase*p-bromobenzyl alcohol*NAD(+) ternary complex (PDB entry 1HLD). The W110A TeSADH mutant now uses (S)-1-phenyl-2-propanol, (S)-4-phenyl-2-butanol and the corresponding ketones as substrates. W110A TeSADH's kinetic parameters on these substrates are in the same range as those of TeSADH on 2-butanol, making W110A TeSADH an excellent catalyst. In particular, W110A TeSADH is twice as efficient on benzylacetone as TeSADH is on 2-butanol, and it produces (S)-4-phenyl-2-butanol from benzylacetone with an enantiomeric excess above 99%. W110A TeSADH is optimally active at 87.5 degrees C and remains highly thermostable. W110A TeSADH is active on aryl derivatives of phenylacetone and benzylacetone, making this enzyme a potentially useful catalyst for the chiral synthesis of aryl derivatives of alcohols. As a control in our engineering approach, we used the TbSADH*(S)-2-butanol binary complex (PDB entry 1BXZ) as the template to model a mutation that would make TeSADH active on (S)-1-phenyl-2-propanol. Mutant Y267G TeSADH did not have the substrate specificity predicted in this modeling study. Our results suggest that (S)-2-butanol's orientation in the TbSADH*(S)-2-butanol binary complex does not reflect its orientation in the ternary enzyme-substrate-cofactor complex.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetone / analogs & derivatives*
  • Acetone / chemistry*
  • Acetone / metabolism
  • Alcohol Oxidoreductases / chemistry
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism*
  • Benzyl Compounds / metabolism*
  • Binding Sites
  • Catalysis
  • Molecular Structure
  • Mutation*
  • Oxidation-Reduction
  • Phenylethyl Alcohol / analogs & derivatives
  • Phenylethyl Alcohol / metabolism
  • Stereoisomerism
  • Substrate Specificity
  • Thermoanaerobacter / enzymology*


  • Benzyl Compounds
  • Acetone
  • 1-phenyl-2-propanol
  • Alcohol Oxidoreductases
  • isopropanol dehydrogenase (NADP)
  • Phenylethyl Alcohol
  • 1-phenyl-2-propanone