The bacterial 7 alpha-dehydroxylation of cholic acid and chenodeoxycholic acid generates the secondary bile acids, deoxycholic acid and lithocholic acid, respectively. Secondary bile acids normally represent about 20% to 25% of the total biliary bile acid pool in man. However, deoxycholic acid increases to about 40% of the biliary bile acid pool following removal of the gallbladder (cholecystectomy). Secondary bile acids are much more toxic to eucaryotic cells than their corresponding primary bile acids and have been implicated in playing an important role in certain cholestatic liver diseases and colon carcinogenesis. Treatment of certain cholestatic liver diseases or cholesterol gallstone dissolution by hydrophilic bile acids is complicated by bacterial 7-alpha-dehydroxylation. Our long range goal is to try and understand the basic enzymology and genetics of intestinal bile acid 7-alpha-dehydroxylation and develop a specific inhibitor of this bacterial biotransformation.
The specific aims of this application are: l) Complete the cloning, sequencing and analysis of a large bile acid inducible operon which is involved in 7-alpha- dehydroxylation. Clone, sequence and analyze a second putative bile acid inducible operon in this bacterium; 2) Determine the function each polypeptide encoded by these operons plays in bile acid 7-alpha- dehydroxylation using both biochemical and genetic approaches. These include subcloning and expression of each gene, reconstitution of the bile acid 7-alpha-dehydroxylation pathway in vitro, and purification of various bile acid inducible activities. Specifically, we propose to: l) Subclone, express, purify and characterize the baiA1 and baiA3 gene products which we hypothesize are 3-alpha-hydroxysteroid dehydrogenase isoenzymes; 2) Subclone and express the baiE, and baiF genes, and determine if these genes encode a bile acid 7-alpha and 7-beta dehydratases; 3) Purification, cloning and sequencing of the bile acid delta 4 and delta 6-reductases; Subclone and express the baiG gene, which has high amino acid sequence identity to the tetracycline resistance gene, and determine if this gene encodes a bile acid transporter/exporter.
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