NAD- and NADP-dependent 7α-hydroxysteroid dehydrogenases from Bacteroides fragilis

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Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown to contain NAD-dependent 7α-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-dependent 7α-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particularly rich in both 7α-hydroxysteroid dehydrogenase activities was selected for further study. Measurement of activity as a function of pH revealed a fairly sharp optimal activity range of 9.5–10.0 for the NAD-dependent enzyme and a broad flat optimal range of 7.0–9.0 for the NADP-dependent enzyme. Michaelis constants for trihydroxy-bile acids for the NAD-dependent enzyme were in the range of 0.32–0.34 mM, whereas dihydroxy-bile acids gave a K m of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3α,7α-dihydroxy-5β-cholanoic acid (chenodeoxycholic acid )by the dehydrogenase revealed a band corresponding to that of synthetic 3α-hydroxy, 7-keto-5β-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7α-hydroxysteroid dehydrogenase and commercially available 3α-hydroxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5β-cholanoic acid. Neither of these two oxidation products could be distinguished from those by the Escherichia coli dehydrogenase oxidation previously reported. Disc-gel electrophoresis of a cell-free lyophilized preparation indicated one active band for NAD-dependent activity of mobility similar to that for the NADP-dependent E. coli enzyme. The NADP-dependent dehydrogenase was unstable and rapidly lost activity after polyacylamide disc-gel electrophoresis, ultracentrifugation, freezing on refrigeration at 4°C. No 3α- or 12α-oriented oxidoreductase activity was demonstrated in any of the strains examined.