Relaxation of nonproductive binding and increased rate of coenzyme release in an alcohol dehydrogenase increases turnover with a nonpreferred alcohol enantiomer
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Media type:
E-Article
Title:
Relaxation of nonproductive binding and increased rate of coenzyme release in an alcohol dehydrogenase increases turnover with a nonpreferred alcohol enantiomer
Published in:
The FEBS Journal, 284 (2017) 22, Seite 3895-3914
Language:
English
DOI:
10.1111/febs.14279
ISSN:
1742-4658;
1742-464X
Origination:
Footnote:
Description:
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.DatabaseStructural data are available in the Protein Data Bank with accession codes 5o8q for A2, 5o8h for A2C2, 5o9f for A2C3, and 5o9d for A2C2B1.