title: Cloning, expression, and biochemical characterization of a novel NADP+-dependent 7α-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids.
abstract: A gene encoding a novel 7α-specific NADP+-dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60°C, but the enzyme is inactivated at this temperature with a half-life time of 5min. Contrary to other known 7α-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition. In order to demonstrate the applicability of this enzyme, a small-scale biotransformation of the bile acid chenodeoxycholic acid (CDCA) into 7-ketolithocholic acid (7-KLCA) was carried out with simultaneous regeneration of NADP+using an NADPH oxidase that resulted in a complete conversion (<99%). Furthermore, by a structure-based site-directed mutagenesis, cofactor specificity of the 7α-HSDH from Clostridium difficile was altered to accept NAD(H). This mutant was biochemically characterized and compared to the wild-type.
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich Heine University of Düsseldorf, Research Centre Jülich, Wilhelm-Johnen-Straße, 52426 Jülich, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Bakonyi
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich Heine University of Düsseldorf, Research Centre Jülich, Wilhelm-Johnen-Straße, 52426 Jülich, Germany. Electronic address: werner.hummel@uni-bielefeld.de.
authors.forename: Werner
authors.initials: W
authors.lastname: Hummel
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001647:Bile Acids and Salts / Q000378:metabolism*; D001711:Biotransformation; D003001:Cloning, Molecular; D016360:Clostridioides difficile / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D001647:Bile Acids and Salts; D009243:NAD; D009249:NADP; D006913:Hydroxysteroid Dehydrogenases; C025376:7 alpha-hydroxysteroid dehydrogenase
keywords: 7-ketolithocholic acid; 7α-hydroxysteroid dehydrogenase; Bile acids; Clostridium difficile; Cofactor specificity; NAD(P)H oxidase