12alpha- and 7alpha-hydroxysteroid dehydrogenase activities from Fusobacterium spp.

On screening fecal organisms for hydroxysteroid dehydrogenase activities applicable to bile acid metabolism studies, we have isolated a gram negative "Bacteroides-like" anaerobe which yields both 12alpha- and 7alpha-hydroxysteroid dehydrogenase (HSDH) activities in cell-free preparation. At the optimal harvest time of 36 hours, approximately 4500 units 12alpha-HSDH and 360 units 7alpha-HSDH were produced per 10(10) viable cells. The two enzymes appear to be separate entities in the basis of their stabilities on freezing, and prolonged storage at room temperature and elution volumes on Sephadex G 200. Thin layer chromatography studies on oxidation products confirmed the respective sites of oxidation to be the 12alpha-OH and 7alpha-OH position. No 3alpha-OH oriented activity was measurable. Preliminary kinetic studies of the 12alpha-HSDH revealed a broad pH curve with optimal activity at pH 9.5. Michaelis constants for glycodeoxycholate and NADP were estimated at 1.5 x 10(-4)M and 3.3 x 10(-5)M respectively.