Characterization of NADP-dependent 7β-hydroxysteroid dehydrogenases from Peptostreptococcus productus and Eubacterium aerofaciens

Abstract

P. productus strain b-52 (a human fecal isolate) and E. aerofaciens ATCC 25986 were found to contain NADP-dependent 7β-hydroxysteroid dehydrogenase activity. The enzyme was synthesized constitutively by both organisms, and the enzyme yields were suppressed by the addition of 0.5 mM 7β-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 7β-hydroxy group in unconjugated and conjugated bile acids, a lower K(m) value being demonstrated with free bile acid than with glycine and taurine conjugates. No measurable activity against 3α-, 7α-, or 12α-hydroxy groups was detected in either enzyme preparation. When tested with strain ATCC 25986, little 7β-hydroxysteroid 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°C for 3 min) and highly sensitive to sulfhydryl inhibitors.