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 these findings to elucidate analogous barbiturate-inducible systems in mammalian liver. We've described 3 distinct P450s from B. megaterium that are barbiturate-induced. One, p450 BM-3m, incorporates both a 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 protein. P450 BM-3 resembles the 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 P450 BM-1, P450BM-2 has been purified but not yet cloned.
Our specific aims for the next five years include (1) the cloning (using either immunochemical or DNA-probe hybridization screening techniques), sequencing and expression of the gene, including the regulatory portion, encoding P450 BM-2, 92) the identification and characterization of the barbiturate- responsive transcription factors involved in the regulation of the B. megaterium P450s and the elucidation, at the level of the gene, of the mechanisms of 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 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 to drive expression of a chloramphenicol acetyltransferase gene. We will also determine the tissue distribution of Barbie box-binding protein mRNA, following barbiturate administration to rats, by Northern analysis. (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 site-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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