Relaxation of nonproductive binding and increased rate of coenzyme release in an alcohol dehydrogenase increases turnover with a nonpreferred alcohol enantiomer

FEBS J. 2017 Nov;284(22):3895-3914. doi: 10.1111/febs.14279. Epub 2017 Oct 20.


Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (a) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (b) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate-binding modes.

Database: Structural data are available in the Protein Data Bank with accession codes 5o8q for A2, 5o8h for A2C2, 5o9f for A2C3, and 5o9d for A2C2B1.

Keywords: alcohol dehydrogenase; biocatalysis; crystal structures; directed evolution; stereoselectivity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Dehydrogenase / chemistry*
  • Alcohol Dehydrogenase / genetics
  • Alcohol Dehydrogenase / metabolism*
  • Binding Sites
  • Catalysis
  • Catalytic Domain
  • Coenzymes / metabolism*
  • Kinetics
  • Mutagenesis, Site-Directed
  • Mutation / genetics
  • Oxidation-Reduction
  • Phenylethyl Alcohol / metabolism*
  • Protein Conformation
  • Rhodococcus / enzymology*
  • Stereoisomerism
  • Substrate Specificity


  • Coenzymes
  • Alcohol Dehydrogenase
  • Phenylethyl Alcohol