Partial purification and characterization of an NAD-dependent 3 beta-hydroxysteroid dehydrogenase from Clostridium innocuum.
In nine strains of Clostridium innocuum, 3 beta-hydroxysteroid-dehydrogenating activities were detected. 3 beta, 7 alpha, 12 alpha-Trihydroxy- and 3 beta-hydroxy-12-keto-5 beta-cholanoic acids were identified as reduction products of the respective 3-keto bile acids by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. One strain was shown to contain a NAD-dependent 3 beta-hydroxysteroid dehydrogenase. Enzyme production was constitutive in the absence of added bile acids. The specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids, with trisubstituted acids being more effective than disubstituted ones. A pH optimum of 10.0 to 10.2 was found after partial purification by DEAE-cellulose chromatography. A molecular weight of about 56,000 was established. 3 beta-hydroxysteroid dehydrogenase activity was also found in the membrane fraction after solubilization with Triton X-100, suggesting that the enzyme was originally membrane bound. The enzyme reduced a 3-keto group in unconjugated and conjugated bile acids, lower Km values being demonstrated with disubstituted than with trisubstituted bile acids. Keto functions at C-7 and C-12 further reduced the Km value. The enzyme was found to be partially heat labile (86% inactivation at 50 degrees C for 10 min).
Institute of Hygiene, University of Mainz, Federal Republic of Germany.
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Edenharder
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Pfützner
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D001647:Bile Acids and Salts; D003013:Clostridium / Q000201:enzymology* / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity
D001647:Bile Acids and Salts; D015096:3-Hydroxysteroid Dehydrogenases