Characterization of delta 4-3-ketosteroid-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase in cell extracts of Clostridium innocuum.
Cell extracts prepared anaerobically from Clostridium innocuum and Clostridium paraputrificum reduced delta 4-3-ketosteroids to 3 beta 5 beta and 3 alpha 5 beta derivatives, respectively. delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) from both organisms required NADH for activity. 5 beta-Reductase from C. innocuum had a pH optimum of 5.0. The substrate concentration at half-maximal reaction velocity was 4.2 microM, and a specific activity of 17 nmol product formed/h per mg protein was determined using 4-pregnen-3,20-dione (progesterone) as a substrate. delta 4-3-Ketosteroid-5 beta-reductase from C. innocuum reduced progesterone and testosterone, but not 4-cholesten-3-one, to corresponding 3-keto-5 beta derivatives. A relative molecular (Mr) weight of 80 000 was estimated for 5 beta-reductase using HPLC-gel filtration chromatography. 3 beta-Hydroxysteroid dehydrogenase in cell extracts of C. innocuum was oxygen sensitive and required NADH for activity. An Mr of 80 000 was estimated for 3 beta-hydroxysteroid dehydrogenase. However, 5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase activities were separated using an HPLC-DEAE chromatography technique.
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D002783:Cholestenones / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D003013:Clostridium / Q000201:enzymology*; D003067:Coenzymes / Q000494:pharmacology; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification*; D011374:Progesterone / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D002783:Cholestenones; D003067:Coenzymes; D013739:Testosterone; D011374:Progesterone; C000475:cholest-4-en-3-one; D002784:Cholesterol; D010088:Oxidoreductases; D015096:3-Hydroxysteroid Dehydrogenases; C021936:3-oxo-5 beta-steroid delta 4-dehydrogenase