title: Purification, cloning, and overexpression of an alcohol dehydrogenase from Nocardia globerula reducing aliphatic ketones and bulky ketoesters.
abstract: 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.
authors.affiliation: Institut für Molekulare Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, Forschungzentrum Jülich, Stetternicher Forst, 52426, Jülich, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Parkot
authors.forename: Harald
authors.initials: H
authors.lastname: Gröger
authors.forename: Werner
authors.initials: W
authors.lastname: Hummel
mesh_terms: 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
chemical_list: D004269:DNA, Bacterial; D004952:Esters; D007659:Ketones; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase