The long-term objective of the research project is the elucidation of the biochemistry, molecular biology, and functions of the cytochrome P450 enzymes induced by barbiturates in the bacterium, Bacillus megaterium. Goals include the determination of the mechanism for barbiturate-mediated induction and structure-function relationships of these P450s in the bacterium and application of the findings to study analogous barbiturate-inducible systems in mammalian liver. This laboratory has identified and described 3 barbiturate-inducible P450s from B. megaterium. One, P450BM-3, contains both P450 and a NADPH:P450 reductase in the proteolytically separable domains of a single, soluble, 119 kDa polypeptide. It functions as a fatty acid monooxygenase independently of any other proteins and resembles liver microsomal systems in organization, sequence identity and mode of induction. Its gene has been cloned and sequenced (including the regulatory region), as has the complete gene for P450BM-1. P450BM-2 has not yet been cloned.
Specific aims for the next 4 years include (1) the cloning (using DNA-probe hybridization screening techniques), sequencing and expression of the P450BM-2 gene, (2) continuation of our efforts to identify and characterize all of the barbiturate-responsive transcription factors involved in the regulation of the B. megaterium P450s and to elucidate, at the gene level, the roles these transcription factors play in the normal and barbiturate-mediated regulation of expression of the three B. megaterium P450 genes, (3)comparative studies, utilizing recombinant DNA and protein-characterization techniques, of the barbiturate-mediated induction mechanisms of the bacterial and the analogous mammalian liver P450s including the cloning of the analogous (barbiturate-responsive) mammalian transcription factors, (4) functional characterization of rat regulatory proteins (currently Barbie box binding proteins) involved in transcription of barbiturate-inducible genes by co-transfection of primary rat hepatocytes with vectors expressing these proteins and reporter constructs containing the promoter sequences of the genes they regulate (currently rat CYP2B1) to drive expression of a luciferase gene, (5) the continued delineation of the structure-function relationships of the B. megaterium P450s including substrate-binding, specificity of oxygenation and electron transfer) utilizing enzymological techniques and sit-specific mutagenesis in conjunction with X-ray crystallography. The health-related implications of the proposed research include an increased understanding of the roles that liver cytochrome P450 enzymes and their inducers (including tumor promoters such as the barbiturates) play in carcinogenesis and in the development of tolerance to therapeutic drugs.
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