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

Eight related alcohol dehydrogenases, which had been originally isolated by laboratory evolution of ADH-A from Rhodococcus ruber DSM44541 for modified substrate scopes, were together with their parent wild-type subjected to biochemical characterization of possible activities with a panel of chiral alcohols and pro-chiral ketones. Determinations of rates of catalyzed alcohol oxidations and ketone reductions, and of cofactor release, pointed out to the role of a W295A substitution as being decisive in steering enantioselectivity in the oxidation of arylated 1-methyl substituted alcohols. Molecular dynamics simulations of enzyme-substrate interactions in the Michaelis complexes of wild-type ADH-A and a Y294F/W295A double mutant could rationalize the experimentally observed shift in enantioselectivity and differences in catalytic activity with 4-phenyl-2-butanol. Finally, we present herein evidence for apparent interdependency between substrate/product and the cofactor in the ternary complex, which directly affects the NADH dissociation rates; therefore, this substrate-coenzyme crosstalk plays a direct role in determining the turnover rates.