Regulation of bile acid 7-dehydroxylase activity by NAD+ and NADH in cell extracts of Eubacterium species V.P.I. 12708

B. A. White, D. A.M. Paone, A. F. Cacciapuoti, R. J. Fricke, E. H. Mosbach, P. B. Hylemon

Research output: Contribution to journalArticlepeer-review

Abstract

The 7α-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 (<0.15 mM) and inhibited at higher concentrations. Reduction of the proposed Δ6-intermediate (3α-hydroxy-5β-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 Δ6-intermediate to lithocholic acid. 7-Dehydroxylase was highly specific for bile acid substrates and required a free C-24 carboxyl group and an unhindered 7α- or 7β-hydroxy group on the B-ring of the steroid nucleus for activity. Bile acid 7α- and 7β-dehydroxylase and Δ6-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 M(r)) 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.

Original languageEnglish (US)
Pages (from-to)20-27
Number of pages8
JournalJournal of Lipid Research
Volume24
Issue number1
StatePublished - 1983
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

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