Mutation of Serine-39 to Threonine in Thermostable Secondary Alcohol Dehydrogenase from <i>Thermoanaerobacter ethanolicus</i> Changes Enantiospecificity

Abstract

The substrate specificity of wild-type and Ser39 → Thr (S39T) secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus was examined. The S39T mutation increases activity for 2-propanol without any significant effect on NADP+ binding. There is no significant effect of the mutation on the primary and secondary alcohol specificity of SADH. However, an effect on the enantiospecificity of SADH by the S39T mutation is demonstrated. Throughout the temperature range from 15 to 55 °C, wild-type SADH exhibits a preference for (S)-2-pentanol. In contrast, a temperature-dependent reversal of enantiospecificity is observed for 2-butanol, with a racemic temperature of 297 K. Throughout the same range of temperatures, S39T SADH exhibits higher enantiospecificity for the (R)-enantiomers of both 2-butanol and 2-pentanol. Examination of individual kcat/Km values for each enantiomer of the chiral alcohols reveals that the effect of the mutation is to decrease (S)-2-butanol specificity, and to preferentially enhance (R)-2-pentanol specificity relative to (S)-2-pentanol. These results are the first step toward expanding the synthetic utility of SADH to allow efficient preparation of a range of (R)-alcohols.