The long-term objective of this proposal is to described, at the molecular level, the mechanism of bile acid 7-alpha-7-beta- dehydroxylation in the intestinal Eubacterium sp. VPI 12708. This biotransformation has important medical implications in regard to treatment of cholesterol gallstones with chenodeoxycholic acid or ursodeoxycholic acid and colon carcinogenesis. The specific objectives are as follows: 1) Purify and characterize the cholic acid inducible polypeptides (77,000; 45,000; 27,000 and 23,500) and prepare specific antibody to each; 2) Clone each polypeptide into E. coli; 3) Determine the nucleotide sequence of the structural and regulatory regions of each gene; 4) Express each polypeptide in E. coli alone and in combination in order to better define its role in this biotransformation; 5) Measure enzyme activities (7- alpha-7-beta-dehydroxylase, Delta-6-reductase, conjugase and cholic acid binding) in cell extracts of E. coli synthesizing each polypeptide; 6) Elucidate the structure and mechanism of synthesis of a novel bile acid nucleotide who's formation appears to be catalyzed by the inducible 27,000 dalton polypeptide; 7) Define the role of NAD+ and ATP in the formation of this bile acid nucleotide; 8) Elucidate the pathway of metabolism of this bile acid nucleotide in cell extracts of Eubacterium sp. VPI 12708. The final objective will be to determine the distribution of each polypeptide and its gene in other intestinal bacteria.
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