Characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenases from Peptostreptococcus productus and Eubacterium aerofaciens.
Peptostreptococcus productus strain b-52 (a human fecal isolate) and Eubacterium aerofaciens ATCC 25986 were found to contain NADP-dependent 7 beta-hydroxysteriod dehydrogenase activity. The enzyme was synthesized constitutively by both organisms, and the enzyme yields were suppressed by the addition of 0.5 mM 7 beta-hydroxy bile acid to the growth medium. Purification of the enzyme by chromatography resulted in preparations with 3.5 (P. productus b-52, on Sephadex G-200) and 1.8 (E. aerofaciens, on Bio-Gel A-1.5 M) times the activity of the crude cell extracts. A pH optimum of 9.8 and a molecular weight of approximately 53,000 were shown for the enzyme of strain b-52, and an optimum pH at 10.5 and a molecular weight of 45,000 was shown for that from strain ATCC 25986. Kinetic studies revealed that both enzyme preparations oxidized the 7 beta-hydroxy group in unconjugated and conjugated bile acids, a lower Km value being demonstrated with free bile acid than with glycine and taurine conjugates. No measureable activity against 3 alpha-, 7 alpha-, or 12 alpha-hydroxy groups was detected in either enzyme preparation. When tested with strain ATCC 25986, little 7 beta-hydroxy-steroid dehydrogenase activity was detected in cells grown in the presence of glucose in excess. The enzyme from strain b-52 was found to be heat labile (90% inactivation at 50 degrees C for 3 min) and highly sensitive to sulfhydryl inhibitors.
D002413:Cations, Divalent / Q000494:pharmacology; D004790:Enzyme Induction; D005051:Eubacterium / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010462:Peptostreptococcus / Q000201:enzymology*; D014580:Ursodeoxycholic Acid / Q000378:metabolism
D002413:Cations, Divalent; D014580:Ursodeoxycholic Acid; D006913:Hydroxysteroid Dehydrogenases; C031932:7 beta-hydroxysteroid dehydrogenase