White B A, Paone D A, Cacciapuoti A F, Fricke R J, Mosbach E H, Hylemon P B
J Lipid Res. 1983 Jan;24(1):20-7.
The 7 alpha-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 (less than 0.15 mM) and inhibited at higher concentrations. Reduction of the proposed delta 6-intermediate (3 alpha-hydroxy-5 beta-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 delta 6-intermediate to lithocholic acid. 7-Dehydroxylase was highly specific for bile acid substrates and required a free C-24 carboxyl group and an unhindered 7 alpha- or 7 beta-hydroxy group on the B-ring of the steroid nucleus for activity. Bile acid 7 alpha- and 7 beta-dehydroxylase and delta 6-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 Mr) 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.
真杆菌属V.P.I. 12708对初级胆汁酸的7α-脱羟基作用需要用胆酸诱导培养物制备的细胞提取物和NAD⁺。当向含有NAD⁺（0.5 mM）的反应混合物中添加NADH（0.5 mM）时，其对胆汁酸7-脱羟基酶活性的抑制超过50%。NADH抑制作用的饱和动力学和双倒数图与负协同性一致。7-脱羟基酶活性受NAD⁺-NADH摩尔比的调节，在NAD⁺摩尔分数为0.75至0.85时活性最高。低浓度（小于0.15 mM）的NADH刺激7-脱羟基酶活性（30%至50%），而高浓度时则抑制。将推测的δ6-中间体（3α-羟基-5β-6-胆烯-24-酸）还原为石胆酸需要胆酸诱导培养物的细胞提取物，并受NAD⁺添加的刺激。还原型黄素核苷酸刺激（32%至62%），而NADH（0.5 mM）抑制（78%）δ6-中间体还原为石胆酸。7-脱羟基酶对胆汁酸底物具有高度特异性，并且需要类固醇核B环上有一个游离的C-24羧基和一个不受阻碍的7α-或7β-羟基才能发挥活性。胆汁酸7α-和7β-脱羟基酶以及δ6-还原酶活性都从厌氧高效液相色谱凝胶过滤柱上共同洗脱下来。然而，总活性单位大约损失了80%至96%。当将低分子量（8000至14000 Mr）洗脱级分中的物质重新添加到含有酶活性的级分中时，总活性的很大一部分（20%至30%）得以恢复。这些研究表明，7-脱羟基酶对底物具有高度特异性，其活性可能受细菌细胞中NAD⁺-NADH比例的调节。
