Purification, cloning, and overexpression of an alcohol dehydrogenase from Nocardia globerula reducing aliphatic ketones and bulky ketoesters.
For the huge amount of chiral chemicals and precursors that can potentially be produced by biocatalysis, there is a tremendous need of enzymes with new substrate spectra, higher enantioselectivity, and increased activity. In this paper, a highly active alcohol dehydrogenase is presented isolated from Nocardia globerula that shows a unique substrate spectrum toward different prochiral aliphatic ketones and bulky ketoesters as well as thioesters. For example, the enzyme reduced ethyl 4-chloro-3-oxo butanoate with an ee >99% to (S)-4-chloro-3-hydroxy butanoate. Very interesting is also the fact that 3-oxobutanoic acid tert-butylthioester is reduced with 49.4% of the maximal activity while the corresponding tert-butyloxyester is not reduced at all. Furthermore, it has to be mentioned that acetophenone, a standard substrate for many known alcohol dehydrogenases, is not reduced by this enzyme. The enzyme was purified from wild-type N. globerula cells, and the corresponding 915-bp-long gene was determined, cloned, expressed in Escherichia coli, and applied in biotransformations. The N. globerula alcohol dehydrogenase is a tetramer of about 135 kDa in size as determined from gel filtration. Its sequence is related to several hypothetical 3-hydroxyacyl-CoA dehydrogenases whose sequences were derived by whole-genome sequencing from bacterial sources as well as known mammalian 3-hydroxyacyl-CoA dehydrogenases and ss-hydroxyacyl-CoA dehydrogenases from different clostridiae.
Parkot
Julia
J
Institut für Molekulare Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, Forschungzentrum Jülich, Stetternicher Forst, 52426, Jülich, Germany.
Gröger
Harald
H
Hummel
Werner
W
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D055162:Biocatalysis; D001711:Biotransformation; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004952:Esters / Q000378:metabolism; D007659:Ketones / Q000378:metabolism*; D008969:Molecular Sequence Data; D009615:Nocardia / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004952:Esters; D007659:Ketones; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase