Regulation of bile acid 7-dehydroxylase activity by NAD⁺ and NADH in cell extracts of Eubacterium species V.P.I. 12708

The 7α-dehydroxylation of primary bile acids by Eubacterium sp. V.P.I. 12708 required a cell extract prepared from a cholic acid-induced culture and NAD⁺. NADH (0.5 mM) inhibited bile acid 7-dehydroxylase activity more than 50% when added to reaction mixtures containing NAD⁺ (0.5 mM). Saturation kinetics and double reciprocal plots of NADH inhibition were consistent with negative cooperativity. 7-Dehydroxylase activity was modulated by the molar ratio of NAD⁺-NADH with maximal activity at a NAD⁺ mole fraction of 0.75 to 0.85. NADH stimulated 7-dehydroxylase activity (30% to 50%) at low concentration (<0.15 mM) and inhibited at higher concentrations. Reduction of the proposed Δ⁶-intermediate (3α-hydroxy-5β-6-cholen-24-oic acid) to lithocholic acid required a cell extract from a cholic acid-induced culture and was stimulated by the addition of NAD⁺. Reduced flavin nucleotides stimulated (32% to 62%) and NADH (0.5 mM) inhibited (78%) the reduction of the Δ⁶-intermediate to lithocholic acid. 7-Dehydroxylase was highly specific for bile acid substrates and required a free C-24 carboxyl group and an unhindered 7α- or 7β-hydroxy group on the B-ring of the steroid nucleus for activity. Bile acid 7α- and 7β-dehydroxylase and Δ⁶-reductase activities all co-eluted from an anaerobic high performance liquid chromatography gel filtration column. However, approximately 80% to 96% of the total units of activity were lost. A substantial portion (20% to 30%) of the total activity was recovered when material from low molecular weight (8,000 to 14,000 M(r)) eluting fractions was added back to fractions containing enzyme activity. These studies show that 7-dehydroxylase is highly specific for substrates and its activity may be regulated by the NAD⁺-NADH ratio in the bacterial cell.