The long range goal of these studies is to increase our knowledge of bile salt metabolism in the body. I propose to use specific intestinal bacteria as model systems to help us address several basic questions in the arena of Bile Acid Molecular Biology. For example, how does a bile acid molecule activate or repress a gene? What constitutes a bile acid binding site or domain on a protein? The specific aims of this proposal are: 1) Elucidate the biochemical mechanism of bile acid 7-dehydroxylation in the intestinal Eubacterium sp. VPI 12708; 2) clone, characterize and determine the nucleotide sequence of a cholic acid inducible operon that appears to code for the polypeptides that are involved in bile acid 7-dehydroxylation; 3) purify and characterize the cholic acid inducible NADH:flavin oxidoreductase and cholic acid binding activities; 4) clone and determine the nucleotide sequence of the bile salt hydrolase gene from Clostridium perfringens. The mechanism of 7-dehydroxylation will be elucidated by demonstrating the proposed intermediates in this pathway and by using custom synthesized radiolabeled bile acids as substrates for 7- dehydroxylation will be elucidated by demonstrating the proposed intermediates in this pathway and by using custom synthesized radiolabeled bile acid metabolism will be cloned into lambda gt-11 or lambda-DASH bacteriophage vectors, detected using synthetic DNA probes and sequenced by the Sanger dideoxy technique. NADH:flavin oxidoreductase and cholic acid binding activities will be purified using DEAE-cellulose, gel filtration, cibacron blue and DEAE-HPLC techniques. In regard to health relatedness, the gut flora generates 20 to 25% of the biliary bile acid pool (secondary bile acids). We have shown in the rat that the hydrophobic secondary bile acids may be important regulators of hepatic cholesterol and bile acid metabolism. Dissolution of cholesterol gallstones by chenodeoxycholic acid and ursodeoxycholic acid is complicated by intestinal bile acid 7-dehydroxylation. If our proposed new mechanism of 7-dehydroxylation is correct, it should allow for a more rational synthesis of bile acid analogs that are resistant to 7-dehydroxylation.
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