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Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii

Costello, Colleen A., Payson, Robert A., Menke, Michael A., Larson, Jeffrey L., Brown, Keith A., Tanner, Joseph E., Kaiser, Raymond E., Hershberger, Charles L., Zmijewski, Milton J.
European journal of biochemistry 2000 v.267 no.17 pp. 5493-5501
Escherichia coli, NADP (coenzyme), Pichia pastoris, Zygosaccharomyces rouxii, acetone, cDNA libraries, catalytic activity, complementary DNA, genes, histidine, sequence analysis, substrate specificity
A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8‐kDa ketoreductase was purified more than 300‐fold to > 95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4‐methylenedioxyphenyl acetone of 2.9 mm and a Km for NADPH of 23.5 µm. The enzyme is able to effectively reduce α‐ketolactones, α‐ketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0‐kb ketoreductase gene was cloned and sequenced from a Z. rouxii cDNA library using a degenerate primer to the N‐terminal sequence of the purified protein. Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.