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

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.

Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-diol and Its Corresponding Acyloin

“…In this conformation, the imidazole side chain flips away from the substrate-binding site, enlarging it. We have observed the same F43H substitution when searching for ADH-A variants with improved activities toward other alcohols . Interestingly, this substitution “restores” a hydrogen-bonding chain that has been proposed to be part of the catalytic machinery in the related horse liver ADH − (Figure A).…”

“…We have observed the same F43H substitution when searching for ADH-A variants with improved activities toward other alcohols. 23 Interestingly, this substitution "restores" a hydrogen-bonding chain that has been proposed to be part of the catalytic machinery in the related horse liver ADH 24−27 (Figure 2A). The significance of this change in enzyme−cofactor interactions is unclear but does obviously facilitate a higher rate of catalytic turnover in the case studied here.…”

“… a See the Supporting Information for a description of the estimation of k 3 and k 5 . Example tracks recorded during the pre-steady state or pre-equilibrium phases and the model fittings to the observed transient rates are shown in Figures S3 and S4. b Data from ref . c Data from ref . …”

Stereo- and Regioselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases

“…(Figure 1A) in the presence of saturating levels of NAD + or NADH as described previously. 22 Curve fitting and model discrimination was performed using programs in the SIMFIT package (http://www.simfit.org.uk/). The steady state parameters k cat and K M were determined after fitting the Michaelis-Menten equation to the experimental data using MMFIT.…”

The Role of Substrate-Coenzyme Crosstalk in Determining Turnover Rates in Rhodococcus ruber Alcohol Dehydrogenase

“…We have previously studied and engineered alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 − (Figure ) for the purpose of generating new enzyme forms with predesigned catalytic properties regarding substrate scope and selectivity. − …”