Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii.
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 microM. The enzyme is able to effectively reduce alpha-ketolactones, alpha-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.
Costello
C A
CA
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.
Payson
R A
RA
Menke
M A
MA
Larson
J L
JL
Brown
K A
KA
Tanner
J E
JE
Kaiser
R E
RE
Hershberger
C L
CL
Zmijewski
M J
MJ
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D020068:Zygosaccharomyces / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C414752:3,4-methylenedioxyphenyl acetone reductase