Characterization of a NADH:flavin oxidoreductase induced by cholic acid in a 7 alpha-dehydroxylating intestinal Eubacterium species.

A NADH:flavin oxidoreductase was partially purified (seven-fold) from an intestinal Eubacterium species V.P.I. 12708 using Bio-Gel A 0.5-M and DEAE-cellulose column chromatography. Enzyme activity was measured spectrophotometrically at 340 nm under anaerobic conditions. A molecular weight of 260 000 was estimated by gel filtration chromatography. The partially purified enzyme preparation exhibited single displacement kinetics with respect to the substrates NADH and FAD. The pH optimum under these conditions was 6.8. NADH:flavin oxidoreductase showed an absolute specificity for NADH as electron donor. However, methylene blue, 2,6-dichlorophenolindophenol, K3Fe(CN)6, menadione, riboflavin, FMN and molecular oxygen served as alternate electron acceptors with varying degrees of efficiency. Acriflavin, rotenone, o-phenanthroline, p-chloromercuribenzoate, dicoumarol and 2,4-dinitrophenol inhibited enzyme activity. Surprisingly, 0.1 mM cholic acid, but not 0.1 mM deoxycholic acid, rapidly induced NADH:flavin oxidoreductase activity in growing cultures.