Systematic Investigation of Saccharomyces cerevisiae Enzymes Catalyzing Carbonyl Reductions

• Published in: Journal of the American Chemical Society Vol. 126; no. 40; pp. 12827 - 12832
• Main Authors: KALUZNA, Iwona A., MATSUDA, Tomoko, SEWELL, Aileen K., STEWART, Jon D.
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 13.10.2004
• Subjects: Alcohol Oxidoreductases - biosynthesis; Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Biological and medical sciences; Catalysis; Escherichia coli - enzymology; Escherichia coli - genetics; Esters - chemistry; Esters - metabolism; Fundamental and applied biological sciences. Psychology; Glutathione Transferase - biosynthesis; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Ketones - chemistry; Ketones - metabolism; Mechanisms. Catalysis. Electron transfer. Models; Molecular biophysics; Oxidation-Reduction; Physical chemistry in biology; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - biosynthesis; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Stereoisomerism; Substrate Specificity; Chemical reduction; Catalytic reaction; Enzyme; Catalyst selectivity; Aldehyde reductase; Enzymatic catalysis; Oxidoreductases; (S)-3-Hydroxyacid ester dehydrogenase; Ketoester; Catalyst activity
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0469479

Eighteen key reductases from baker's yeast (Saccharomyces cerevisiae) have been overproduced in Escherichia coli as glutathione S-transferase fusion proteins. A representative set of alpha- and beta-keto esters was tested as substrates (11 total) for each purified fusion protein. The stereoselectivities of beta-keto ester reductions depended both on the identity of the enzyme and the substrate structure, and some reductases yielded both L- and D-alcohols with high stereoselectivities. While alpha-keto esters were generally reduced with lower enantioselectivities, it was possible in all but one case to identify pairs of yeast reductases that delivered both alcohol antipodes in optically pure form. Taken together, the results demonstrate not only that individual yeast reductases can be used to supply important chiral building blocks, but that GST-fusion proteins allow rapid identification of synthetically useful biocatalysts (along with their corresponding genes).