Affordable Access

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

Authors
  • Costello, C A
  • Payson, R A
  • Menke, M A
  • Larson, J L
  • Brown, K A
  • Tanner, J E
  • Kaiser, R E
  • Hershberger, C L
  • Zmijewski, M J
Type
Published Article
Journal
European journal of biochemistry / FEBS
Publication Date
Sep 01, 2000
Volume
267
Issue
17
Pages
5493–5501
Identifiers
PMID: 10951208
Source
Medline
License
Unknown

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 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.

Report this publication

Statistics

Seen <100 times