Sherrod J A, Hylemon P B
Biochim Biophys Acta. 1977 Feb 23;486(2):351-8. doi: 10.1016/0005-2760(77)90031-5.
A NAD-dependent 7alpha-hydroxysteroid dehydrogenase was purified 18-fold over the activity in crude cell extracts prepared from Bacteroides thetaiotaomicron NCTC 10852 using Bio-Gel A 1.5-M column chromatography. A molecular weight of 320 000 was estimated for the partially purified intact enzyme. Substrate saturation kinetics were performed using the 18-fold purified enzyme and the lowest Km values were obtained for 3alpha,7alpha-dihydroxy bile acid and bile salt substrates including chenodeoxycholic acid (Km 0.048 mM), glycochenodeoxycholic acid (Km 0.083 mM) and taurochenodeoxycholic acid (Km 0.059 mM). In contrast, 3alpha,7alpha,12alpha-trihydroxy bile acid and bile salts had higher Km values, i.e. cholic acid (Km 0.22 mM), glycoholic acid Km 0.32 mM) and taurocholic acid Km 0.26 mM). NAD had a Km value of 0.20 mM. The possible physiological significance of 7alpha-hydroxy bile acid oxidation to intestinal bacteroides strains was accessed by determining the rate of conversion of [14C]-cholic acid to 7-ketodeoxy[14C]cholic acid by whole cell suspensions under different incubation conditions. The rate of biotransformation of bile acid to keto-bile acid incubated anaerobically under N2 gas increased markedly when potential electron acceptors such as fumarate (10 mM) or menadione (4 mM) was added exogenously. These results suggest that bile acid oxidation reactions may be linked to energy-generating systems in this bacterium.
使用Bio-Gel A 1.5-M柱色谱法，从嗜热栖热放线菌NCTC 10852制备的粗细胞提取物中，将一种NAD依赖性7α-羟基类固醇脱氢酶纯化至活性提高了18倍。估计部分纯化的完整酶的分子量为320 000。使用纯化了18倍的酶进行底物饱和动力学研究，对于3α,7α-二羟基胆汁酸和胆汁盐底物，包括鹅去氧胆酸（Km 0.048 mM）、甘氨鹅去氧胆酸（Km 0.083 mM）和牛磺鹅去氧胆酸（Km 0.059 mM），获得了最低的Km值。相比之下，3α,7α,12α-三羟基胆汁酸和胆汁盐的Km值较高，即胆酸（Km 0.22 mM）、甘氨胆酸（Km 0.32 mM）和牛磺胆酸（Km 0.26 mM）。NAD的Km值为0.20 mM。通过测定在不同培养条件下全细胞悬浮液中[14C] - 胆酸向7-酮脱氧[14C]胆酸的转化速率，探讨了7α-羟基胆汁酸氧化对肠道拟杆菌菌株的可能生理意义。当外源添加潜在的电子受体如富马酸盐（10 mM）或甲萘醌（4 mM）时，在N2气体下厌氧培养时胆汁酸向酮胆汁酸的生物转化速率显著增加。这些结果表明，胆汁酸氧化反应可能与该细菌中的能量产生系统有关。
