Targeted mutation of the yeast reductase, YDL124w, affects enzyme stereoselectivity

BIOL 101

Crystal Archer1, Davis Erik1, Ruchita Patel, Brent D. Feske, feskebre@mail.armstrong.edu2, and Scott C. Mateer, mateersc@mail.armstrong.edu1. (1) Department of Biology, Armstrong Atlantic State University, 11935 Abercorn Street, Savannah, GA 31419, (2) Department of Chemistry and Physics, Armstrong Atlantic State University, 11935 Abercorn St, Savannah, GA 31419
The enantioselective reduction of ketones to produce homochiral alcohols is a popular strategy for the synthesis of many pharmaceutical compounds. In particular, yeast reductases have been used to generate specific chiral alcohols from β-keto esters. However, the asymmetric molecular mechanism utilized by these reductases is not clearly understood. Here we describe our efforts to identify regions of the reductase, YDL124w, that regulate its stereoselectivity. Several YDL124w mutants were generated, and expressed as Glutathione-S-Transferase (GST) fusion proteins in E. coli. The ability of these mutants to reduce beta-keto esters were then evaluated using a heterologous whole-cell system. One point mutant, I63-A63, and two double point mutants, E62I63-N62V63 and S123P124-C123A124, were found to have altered stereoselectivity profiles. What role these mutations play in stereoselectivity will be discussed. Clearly continued use of this mutagenes strategy will identify other amino acid residues that affect reductase stereoselectivity and therefore, provide valuable insight into the molecular mechanisms that influence enzyme stereoselectivity.
 

Frontiers in Chemical Biology
5:00 PM-7:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster

Division of Biological Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007