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

DOI: 10.1111/febs.14279 DOI Help

Authors: Emil Hamnevik (Uppsala University) , Thilak Reddy Enugala (Uppsala University) , Dirk Maurer (Uppsala University) , Siphosethu Ntuku (Uppsala University) , Ana Oliveira (Uppsala University) , Doreen Dobritzsch (Uppsala University) , Mikael Widersten (Uppsala University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: The Febs Journal

State: Published (Approved)
Published: September 2017
Diamond Proposal Number(s): 11171

Abstract: 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: (1) 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. (2) 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.

Journal Keywords: alcohol dehydrogenase; biocatalysis; stereoselectivity; directed evolution; crystal structures; enzyme kinetics

Subject Areas: Chemistry


Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography