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Relaxation of nonproductive binding and increased rate of coenzyme release in an alcohol dehydrogenase increases turnover with a nonpreferred alcohol enantiomer
Author(s) -
Hamnevik Emil,
Enugala Thilak Reddy,
Maurer Dirk,
Ntuku Siphosethu,
Oliveira Ana,
Dobritzsch Doreen,
Widersten Mikael
Publication year - 2017
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14279
Subject(s) - alcohol dehydrogenase , stereochemistry , chemistry , stereoselectivity , enantiomer , active site , alcohol , saturated mutagenesis , mutagenesis , alcohol oxidation , enzyme , biochemistry , catalysis , mutant , gene
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.

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