NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenases from bacteroides fragilis

Biochim Biophys Acta. 1975 Mar 28;384(1):12-24. doi: 10.1016/0005-2744(75)90091-1.

Abstract

Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown to contain NAD-dependent 7alpha-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-depedent 7alpha-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particulary rich in both 7alpha-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 Km of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid) by the dehydrogenase revealed a band corresponding to that of synthetic 3alpha-hydroxy, 7-keto-5beta-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7alpha-hydroxysteroid dehydrogenase and commercially available 3alpha-hy-droxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5beta-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 degrees C. No 3 alpha- or 12alpha-oriented oxidoreductase activity was demonstrated in any of the strains examined.

Publication types

  • Comparative Study

MeSH terms

  • Bacteroides / enzymology*
  • Bacteroides / growth & development
  • Chenodeoxycholic Acid / metabolism
  • Escherichia coli / enzymology
  • Hydrogen-Ion Concentration
  • Hydroxysteroid Dehydrogenases / metabolism*
  • Kinetics
  • NAD / pharmacology*
  • NADP / pharmacology*
  • Structure-Activity Relationship
  • Ultracentrifugation

Substances

  • Chenodeoxycholic Acid
  • NAD
  • NADP
  • Hydroxysteroid Dehydrogenases