| Summary |
The intestinal bacterium, Bacteroidesfragilis, expresses a 7a-hydroxysteroid dehydrogenase (7a-HSDH) which catalyzes the oxidation of bile acids with a 7a-hydroxy group to the corresponding 7-keto-bile acid. The objectives of this research project were to purify the B. fragilis 7a-HSDH and to characterize its structural and functional properties. The enzyme purification was performed in six steps: soluble protein extraction; heat treatment, ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography, and anion-exchange chromatography. Thin layer chromatographic analysis confirmed that the purified enzyme catalyzed the conversion of chenodeoxycholic acid to 7-keto-lithocholic acid. Gel-filtration and SDSPAGE analyses indicated that the native enzyme exists as a 110 kDa tetramer with subunits of 27.4 kDa. The optimum pH was 8.5 in the forward reaction and 6.5 in the reverse reaction. The enzyme was thermostable, retaining 90% activity after one hour at 65°C. For the oxidative reaction at pH 8.5, substrate Km values were 0,10 mM for NAD, 0.05 mM for CDCA, and 0.16 mM for cholic acid. For the reverse reaction at pH 6.5, Km values were 0.02 mM for NADH and 0.53 mM for 3a-hydroxy-7-keto-5P-cholanic acid. The enzyme was inhibited by the sulfhydryl reactive reagents, p-hydroxymercuribenzoate and mercury chloride. No metal ion requirement was apparent. N-terminal amino acid sequence analysis suggests that the 7a-HSDH was related to the short-chain dehydrogenase family, which includes several hydroxysteroid dehydrogenases from other bacterial genera. |
