Jump to Main Content
Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomycesârouxii
- 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 Zygosaccharomycesârouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8âkDa ketoreductase was purified more than 300âfold to >â95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4âmethylenedioxyphenyl acetone of 2.9âmm and a Km for NADPH of 23.5âÂµm. The enzyme is able to effectively reduce Î±âketolactones, Î±âketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0âkb ketoreductase gene was cloned and sequenced from a Z.ârouxii cDNA library using a degenerate primer to the Nâterminal sequence of the purified protein. Furthermore, it was expressed in both Escherichiaâcoli and Pichiaâpastoris 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.