Controlling substrate specificity and stereospecificity of alcohol dehydrogenases through mutagenesis
Nealon, Christopher Michael
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This dissertation includes six chapters. Chapter 1 is an introduction and chapter 2 is a literature review that has been reprinted with permission from a published article. Chapter 3, chapter 4, and chapter 5 will be submitted for publication. Chapter 6 is the conclusion. Chapter 3 of this dissertation discusses the enantioselective asymmetric reductions and kinetic assays of the I86A and I86A/C295A mutants of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (TeSADH). The expansion of the active site small pocket allowed for ring-substituted acetophenones and heterocycles to react with the new TeSADH mutants to yield the anti-Prelog product, the R-alcohol. This broadened substrate specificity came at a cost of the specific activity, though the enantiomeric excess was >99%. Chapter 4 of this dissertation utilizes the previously aforementioned TeSADH mutants for kinetic resolutions of racemic 1-arylethanols with the goal of converting R-alcohols into ketones, and leaving behind the unreacted S-alcohols. Due to the high enantiomeric excess of the forward reaction, this is a creative route to generate the other alcohol isomer. Chapter 5 of this dissertation involves the design and study of two new TeSADH mutants with the goal of expanding the active site small pocket in comparison to wild-type TeSADH. The two residues of interest, Met-151 and Thr-153, were near to each other. By mutation of each of these residues into an Alanine, the enantioselective asymmetric reductions and kinetic assays mostly yielded results that fell short from the wild-type TeSADH. Interestingly, the Met-151 residue isn’t close enough to the Zn to be considered a Zn ligand, though the kinetic assay of M151A with acetone showed noticeable lag. The Met-151 residue is closer to the Asp-150, which could mean that the M151A mutant is unable to support the Asp-150 and keep it in place on the Zn.