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

Author:
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.
Source:
European journal of biochemistry 2000 v.267 no.17 pp. 5493-5501
ISSN:
0014-2956
Subject:
Escherichia coli, NADP (coenzyme), Pichia pastoris, Zygosaccharomyces rouxii, acetone, cDNA libraries, catalytic activity, complementary DNA, genes, histidine, sequence analysis, substrate specificity
Abstract:
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.
Agid:
115786