Tailoring the substrate specificity of secondary alcohol dehydrogenase

Abstract

Our research with the thermophilic secondary-alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus has provided novel information regarding the physical basis of enzyme substrate specificity and stereospecificity. We demonstrated that oxidation of secondary alcohols catalyzed by T. ethanolicus SADH exhibits temperature-dependent enantiospecificity. In other studies, we found that the structure of co-factor analogs also significantly affects the stereochemistry of the SADH reaction. More recently, we demonstrated that pH can also have a modest effect on SADH enantiospecificity. Organic solvents have also been shown by others to affect the stereochemistry of SADH reactions. We designed and prepared S39T and C295A mutant forms of SADH by site-directed mutagenesis, and we evaluated the effects of the mutations by analysis of the temperature dependence of the enantiomeric ratio (E) for simple chiral alcohols such as 2-butanol. This procedure allows for the determination of the differential Eyring parameters (ΔΔH‡and ΔΔS‡) for the reaction. We demonstrated that this technique is a sensitive method for analysis of the effects of mutation on enzyme stereospecificity. S39T and C295A SADH exhibit significant changes in substrate specificity and stereospecificity consistent with the changes in the volume of the alkyl-binding pockets. Thus, it is possible to alter the substrate specificity and stereospecificity of alcohol dehydrogenase by changing either the reaction medium or the protein structure.Key words: alcohol dehydrogenase, substrate specificity, stereospecificity, temperature dependence, site-directed mutagenesis.