Chiral alcohols are highly desirable pharmaceutical building blocks, but their synthesis from carbonyl compounds by chemical and industrial methods can be hazardous, difficult, and costly. One potential strategy to safe, easy, and cost-effective synthesis of these chiral alcohols is the reduction of keto-esters by aldo-keto reductases (AKRs). However, it is often laborious and time consuming identifying the ester/enzyme combination that produces the desired stereochemistry required for a particular medical compound. Our work seeks to explore and characterize the stereoselectivity of a model AKR, YDL124w, by identifying amino acid residues that are important for substrate binding and orientation. For our analysis we have targeted residues located in the reductase's Substrate Specificity Loop A (Loop A) for Alanine-scanning mutagenesis. Wildtype and mutant proteins were expressed in bacteria as GST-chimeras, and the resulting crude lysates were used to characterize the reduction of several keto-ester substrates. In all substrates tested, mutant S123A3 exhibited a reduced ability to convert the carbonyl substrate into the chiral alcohol, while other Loop A mutant lysates worked as well as wildtype lysates. In addition, GST-YDL124w wild type enzyme was purified using Glutathione-agarose resin and it was determined that its Km for ethyl-4-chloroacetoacetate was 850 �M. Subsequently, the Alanine-scanning mutants were purified using glutathione-agarose and their ability to reduce ethyl-4-chloroacetoacetate was determined. Only mutant S123A3 exhibited any difference from the wildtype enzyme in its ability to reduce ethyl-4-chloroacetoacetate, which was consistent with the crude lysate data. Collectively, this data suggest that the residues altered in the S123A3 mutant play an important part in substrate binding and may also play an important role in enzyme stereoselectivity.

Identification of residues that specify substrate binding in the aldo-keto reductase, YDL124w

243rd National Meeting (2012)

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