Bile acid induction of 7a- and 76-hydroxysteroid dehydrogenases in Clostridium limosum

When grown in the presence of bile acids, two strains of Clostridium limosum were found to contain significant amounts of NADP-dependent 7a/7/3-hydroxysteroid dehydrogenase and NAD-dependent 7a-hydroxysteroid dehydrogenase which were active against conjugated and unconjugated bile acids. No measurable activity could be found when deoxycholic acid (3a, 12a-dihydroxy-5/3-cholan-24-oic acid) was used as substrate. No 78-hydroxysteroid dehydrogenase activity and only a trace of 7a-hydroxysteroid dehydrogenase activity could be demonstrated when bile acid was deleted from the growth medium. If bile acid was added after the time of inoculation, the amounts of 7a/7@-hydroxysteroid dehydrogenase were greatly reduced. Enzyme enhancement was blocked by addition of rifampicin. The 7a/78-hydroxysteroid dehydrogenase components had pH optima of approximately 10.5. Both the 7a/7/3-hydroxysteroid dehydrogenase activities were heat-labile, with the 78-component being the more stable of the two. When ranked according to the level of enzymes induced, the order in increasing bile acid induction power on an equimolar scale (0.4 mM) was: 7-ketodeoxycholic acid, cholic acid, chenodeoxycholic acid, and deoxycholic acid. Both 7-ketolithocholic acid and ursodeoxycholic acid were ineffective as enzyme inducers. Optimal induction was achieved with high concentrations of cholic acid (5 mM) and a harvest time of 24 hr. Addition of ursodeoxycholic acid to medium containing optimal concentrations of deoxycholic acid suppressed enzyme induction. With regard to the NADP-dependent 7a-hydroxysteroid dehydrogenase activity, a dihydroxy bile acid had a higher apparent K,,, value than a trihydroxy bile acid; chenodeoxycholic acid (0.42 mM) versus cholic acid (0.14 mM). In contrast, with the NADP-dependent 78-hydroxysteroid dehydrogenase activity, a trihydroxy bile acid had a higher K, value; ursocholic acid (0.076 mM) versus ursodeoxycholic acid (0.018 mM). The NAD-dependent 7a-hydroxysteroid dehydrogenase activity with cholic acid as substrate had the highest apparent K,,, value of 0.62 mM. Suthcrland, J. D., and C. N. Williams. Bile acid induction of 7aand 78hydroxysteroid dehydrogenases in Clostridium limosum. J. Lipid RCS. 1985. 26: 344-350. Supplementary key w o r d s bile acids enzyme induction A number of anaerobes have been shown to participate in the formation of urso bile acids from primary bile acids. These include Clostridium absonum (1, 2 ) , certain lecthinase-lipase-negative clostridia (3, 4), Peptostreptococcus productur ( 5 , S) , and Eubacterium aemfuiens (6-8). Clostridium absonum and the unidentified clostridia are examples of single organisms that are capable of 7ahydroxyl group epimerization in pure culture (intraspecies epimerization, (9)). The 7ar/7P-hydroxysteroid dehydrogenases responsible for the epimerization by C. absonum have been demonstrated and studied (10, 11). In contrast I? pmductus and E. aemfmiens contain only a 7phydroxysteroid dehydrogenase (6-8) and epimerization (5, 8) in whole cell cultures (interspecies epimerization, (9)) depends on co-culturing either of these organisms with a known 7a-hydroxysteroid dehydrogenase elaborating bacterium such as Bacteroides fmgilis (12, 13) or Escherichia coli (14, 15). Clostridium absonum 7a-hydroxysteroid dehydrogenases (NADPand NAD-linked) and 70-hydroxysteroid dehydrogenase (NADP-linked) are inducible by several bile acid substrates and a non-substrate, deoxycholic acid, and are repressed by the respective urso end product (10, 11). Up to this time little has been reported on the enzyme system in the lecithinase-lipase-negative clostridia isolated by Edenharder et al. (3, 4). The 7P-hydroxysteroid dehydrogenases in I? pmductus and E. aerofaciens are NADP linked, constitutively synthesized, of low total activity, and are unstable (6, 8). The primary significance of 7a-hydroxyl group epimerization appears to be the lower toxicity of ursodeoxyAbbreviations: TLC, thin-layer chromatography; HSDH, hydroxysteroid dehydrogenase; ATCC, American Type Culture Collection; VPI, Virginia Polytechnic Institute; NADP, nicotinamide adenine dinudeotide phosphate; NAD, nicotinamide adenine dinucleotide; CA, cholic acid (3~~,7a,12a-trihydroxy-5fl-cholan-24-oic acid); UC, ursocholic acid (3a,7fl,12~t-trihydmxy-58-cholan-24-oic a id); 7-KDC, 7-ketodeoxycholic acid (3a,12a-dihydroxy-7-oxo-5fl-cholan-24-oic a id); CDC, chenodeoxycholic acid (301,7a-dihydroxy-5fl-cholan-24-oic acid); UDC, ursodeoxycholic acid (3a,7fl-dihydroxy-5fl-cholan-24-oic a id); 7-KLC, 7-ketolithocholic acid (3a-hydroxy-7-oxo-5fl-cholan-24-oic acid); DC, deoxycholic acid (3a,12a-dihydroxy-5fl-cholan-24-oic acid); GCDC, glycochenodeoxycholic acid (3a,7u-dihydroxy-5fl-cholanoyl glycine); G u m , glycoursodeoxycholic acid (3~,7fl-dihydroxy-5fl-cholanoyl glycine); E D C , taurochenodeoxycholic acid (3a,7a-dihydroxy-5fl-cholanoyltaurine); TUDC, taurounodeoxycholic acid (3~,7fl-dihydroxy-5flcholanoyl taurine). 344 Journal of Lipid Research Volume 26, 1985 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 0, 2013 w w w .j.org D ow nladed fom cholic acid than chenodeoxycholic acid due to a lesser hydrophobicity of the former (16). With C. absonum, polyacrylamide gel electrophoresis has shown that no less than five new polypeptide bands, other than those associated with enzyme activity, are present in cultures induced by either chenodeoxycholic acid or deoxycholic acid (17). Thus induction of 7a17fl-hydroxysteroid dehydrogenases may represent an entire shift in the synthesis of proteins to facilitate survival of an organism in a "hostile" bile salt environment. Recent interest in ursodeoxycholic acid and bacterial organisms capable of synthesizing this bile acid has arisen from its oral administration for the dissolution of human cholesterol gallstones (18-24). Commercial microbiological synthesis of ursodeoxycholic acid remains a possibility, but C. absonum does not appear to be the best candidate due to its inability to grow in high concentrations of chenodeoxycholic acid (1). However, C. absonum 70hydroxysteroid dehydrogenase purified by Procion Red affinity chromatography (25) is now being used to spectrophotometrically quantify ursodeoxycholic acid in ursodeoxycholic acid-rich biles (26). Recently we have demonstrated that a soil isolate of Clostridium limosum can convert primary bile acids to 7phydroxy and 7-keto products (27). It is the purpose of this communication to study the inducibility of both 7cr17phydroxysteroid dehydrogenases in C. limosum and to preliminarily characterize these enzymes. MATERIALS AND METHODS