This proposal is an extension of our ongoing studies dealing with the structure, function, and regulation of NADPH-cytochrome P-450 oxidoreductase, cytochrome P-450, and epoxide hydrolase. The application is divided into three major parts, one for each of the enzymes listed above. The first deals with (1) the basal regulation of the oxidoreductase gene, (2) the mechanism of phenobarbital transcriptional activation, (3) the molecular basis for trans-stilbene oxide induction, and (4) the structure-function relationship of the oxidoreductase enzyme. The promoter region of the oxidoreductase gene is of particular interest since it lacks both TATA and CCAAT sequence motifs but instead contains multiple consensus sequences for Sp-1. In this respect, it differs from other genes encoding for members of the mixed-function oxidase system as well as the UDP-glucuronosyltransferase and the glutathione S-transferases. Our goal is to identify and characterize those factors and sequences important in the regulation of the oxidoreductase gene and also to elucidate the biochemical pathways for xenobiotic induction. Furthermore, in the absence of the crystallographic structure of the oxidoreductase protein, we will use computer modeling and site-directed mutagenesis to analyze the structure-function relationships of the enzyme. Our immediate goal is to identify amino acids that are functionally important for biological activity. Five domains are targeted for characterization and include the binding sites for the flavins (FAD and FMN), cytochrome P-450, NADPH, and the amino terminal anchor required for membrane integration. The second part of the proposal examines the basal regulation of the epoxide hydrolase gene along with the biochemical events responsible for the glucocorticoid receptor-dependent dexamethasone repression of the gene. Down-regulation appears to be linked to a proximal AP-1 site and may involve inhibition of AP-1 activity by the glucocorticoid receptor. The mechanisms by which various compounds (phenobarbital, trans-stilbene oxide, 2-acetylaminofluorene, and diethylnitrosamine) induce epoxide hydrolase also will be studied using promoter deletion mutants. It will be of particular interest to compare the mechanism of phenobarbital induction for genes lacking (oxidoreductase) and possessing (epoxide hydrolase and P-450 PCN-3) TATA and CCAAT regulatory sequences. In addition, the active site of epoxide hydrolase will be characterized using group specific chemical modifications in the presence and absence of an inhibitor followed by site-directed mutagenesis of the protected amino acid to establish biological relevancy. The third section of this application poses questions about the gene organization and regulation of the steroid-inducible P-450s (P-450III gene family). Emphasis will be placed on understanding the basis for the differential regulation of the P-450 PCN 2 and 3 genes.
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