Purification and characterization of 7 beta-hydroxysteroid dehydrogenase from Ruminococcus sp. of human intestine.
7 beta-Hydroxysteroid dehydrogenase (7 beta-HSD) was produced by Ruminococcus sp. PO1-3 obtained from among human intestinal bacteria. The enzyme was purified from a crude extract by ammonium sulfate fractionation, and Butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A and Octyl-Sepharose chromatographies. The purified enzyme was obtained as a single band on polyacrylamide gel electrophoresis with enzyme activity staining and as one band corresponding to a molecular weight of 30,000 on SDS-polyacrylamide gel electrophoresis. On gel filtration, its apparent molecular weight was estimated to be 60,000. The enzyme had a sulfhydryl group(s) in its active site. Substrate specificity studies revealed that the enzyme showed absolute specificity for the beta-configuration of a hydroxyl group at the 7 position of bile acids, and required NADP+ and NADPH as cosubstrates. The Km values for ursodeoxycholic acid, 7-k etolithocholic acid, NADP+, and NADPH were 5.0, 8.5, 7.7, and 24 microM, respectively.
D001426:Bacterial Proteins / Q000032:analysis; D001438:Bacteroidaceae / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005243:Feces / Q000382:microbiology*; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000032:analysis / Q000302:isolation & purification*; D007422:Intestines / Q000382:microbiology*; D008970:Molecular Weight; D013379:Substrate Specificity
D001426:Bacterial Proteins; D006913:Hydroxysteroid Dehydrogenases; C031932:7 beta-hydroxysteroid dehydrogenase