title: Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens.
abstract: A gene encoding an NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH) from Collinsella aerofaciens DSM 3979 (ATCC 25986, formerly Eubacterium aerofaciens) was identified and cloned in this study. Sequence comparison of the translated amino acid sequence suggests that the enzyme belongs to the short-chain dehydrogenase superfamily. This enzyme was expressed in Escherichia coli with a yield of 330 mg (5,828 U) per liter of culture. The enzyme catalyzes both the oxidation of ursodeoxycholic acid (UDA) forming 7-keto-lithocholic acid (KLA) and the reduction of KLA forming UDA acid in the presence of NADP(+) or NADPH, respectively. In the presence of NADPH, 7β-HSDH can also reduce dehydrocholic acid. SDS-PAGE and gel filtration of the expressed and purified enzyme revealed a dimeric nature of 7β-HSDH with a size of 30 kDa for each subunit. If used for the oxidation of UDA, its pH optimum is between 9 and 10 whereas for the reduction of KLA and dehydrocholic acid it shows an optimum between pH 4 to 6. Usage of the enzyme for the biotransformation of KLA in a 0.5-g scale showed that this 7β-HSDH is a useful biocatalyst for producing UDA from suitable precursors in a preparative scale.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany. liuluo02@gmail.com
authors.forename: Luo
authors.initials: L
authors.lastname: Liu
authors.forename: Arno
authors.initials: A
authors.lastname: Aigner
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
mesh_terms: D039903:Actinobacteria / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D010802:Phylogeny; D016415:Sequence Alignment; D013379:Substrate Specificity; D014580:Ursodeoxycholic Acid / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009249:NADP; D014580:Ursodeoxycholic Acid; D006913:Hydroxysteroid Dehydrogenases; C031932:7 beta-hydroxysteroid dehydrogenase