title: Purification and characterization of a novel 17 alpha-hydroxysteroid dehydrogenase from an intestinal Eubacterium sp. VPI 12708.
abstract: A novel steroid-inducible 17 alpha-hydroxysteroid dehydrogenase (17 alpha-HSDH) has been purified over 850-fold from an intestinal Eubacterium sp. VPI 12708. The purified protein has a subunit molecular mass of 42,000 daltons and a native molecular weight (M(r)) of 160,000 as estimated by gel filtration chromatography. Enzyme activity was induced by growth in the presence of androstenedione or cholic acid, but not deoxycholic acid. Enzymatic activity required anaerobic conditions and was highly specific for NADP+ and the 17 alpha-hydroxy group of C-19 steroids. Estimated Km values were 31 microM and 70 microM for androstenedione and epitestosterone, respectively. Vmax values were estimated to be 3250 nmol/min per mg protein and 1800 nmol/min per mg protein for the reductive and oxidative reactions, respectively. The pH optima for both the oxidative and reductive reactions ranged between 5.5 and 7.0. Treatment with EDTA completely inactivated 17 alpha-HSDH activity but activity was partially restored by the addition of either Mg2+ (1 mM) or Zn2+ (10 mM). The N-terminal amino acid sequence analysis of purified enzyme suggests that 17 alpha-HSDH may belong to a disulfide reductase gene family.
authors.affiliation: Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0678.
authors.forename: P
authors.initials: P
authors.lastname: de Prada
authors.forename: K D
authors.initials: KD
authors.lastname: Setchell
authors.forename: P B
authors.initials: PB
authors.lastname: Hylemon
mesh_terms: D000595:Amino Acid Sequence; D005051:Eubacterium / Q000201:enzymology*; D006913:Hydroxysteroid Dehydrogenases / Q000096:biosynthesis / Q000302:isolation & purification*; D007422:Intestines / Q000382:microbiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
chemical_list: D006913:Hydroxysteroid Dehydrogenases; C035507:3(17)-hydroxysteroid dehydrogenase