title: Extracellular 3beta-hydroxysteroid oxidase of Mycobacterium vaccae VKM Ac-1815D.
abstract: Extracellular 3beta-hydroxysteroid oxidase (SO) has been isolated from cell-free cultivation broth at the growth of Mycobacterium vaccae VKM Ac-1815D on glycerol-mineral medium in the presence of sitosterol. The enzyme is responsible for the transformation of 3beta-hydroxy-5-ene- to 3-keto-4-ene-moiety of steroids including dehydrogenation of 3beta-hydroxy function followed by delta5-->delta4 isomerization. 6-Hydroxy-4-sitosten-3-one and 6-hydroxy-4-androsten-3,17-dione were revealed among the metabolites at the incubation of the enzyme preparations with sitosterol and dehydroepiandrosterone (DHEA), respectively. The enzyme was strongly NADH or NADPH dependent. SO has been purified over 300-fold using cultivation broth concentration on hollow fibers followed by fractionation by ammonium sulphate, column chromatography on DEAE-Toyopearl, hydroxyapatite Bio-Gel HTP and double gel-filtration on Bio-Gel A 0.5 M. SDS-electrophoresis gave a molecular mass estimate of 62 +/- 4 kDa. The purified SO obeyed Michaelis-Menten kinetics, double reciprocal plots kinetics revealed Km value towards DHEA 5 x 10(-4) M. Along with SO activity, 17-hydroxysteroid dehydrogenase (17-OH SDH) and 3-ketosteroid-1(2)-dehydrogenase (1(2)-SDH) activities were detected in cell-free cultivation broth. The extracellular steroid transforming activities of C-17-ketosteroid producing mycobacteria were hitherto unreported.
authors.affiliation: Laboratory of Microbial Transformation of Organic Compounds, GK Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow Region.
authors.forename: V M
authors.initials: VM
authors.lastname: Nikolayeva
authors.forename: O V
authors.initials: OV
authors.lastname: Egorova
authors.forename: D V
authors.initials: DV
authors.lastname: Dovbnya
authors.forename: M V
authors.initials: MV
authors.lastname: Donova
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000735:Androstenedione / Q000378:metabolism; D002473:Cell Wall / Q000201:enzymology; D002474:Cell-Free System; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003687:Dehydroepiandrosterone / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D009161:Mycobacterium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D012855:Sitosterols / Q000378:metabolism; D013256:Steroids / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: D012855:Sitosterols; D013256:Steroids; D009243:NAD; D013739:Testosterone; D000735:Androstenedione; D003687:Dehydroepiandrosterone; D009249:NADP; C025473:gamma-sitosterol; D006859:Hydrogen; D010088:Oxidoreductases; D015096:3-Hydroxysteroid Dehydrogenases; D006913:Hydroxysteroid Dehydrogenases; C035507:3(17)-hydroxysteroid dehydrogenase; C022656:3-oxosteroid delta(1) dehydrogenase; D005990:Glycerol