The long-term objective of the research project is the comprehensive elucidation of the biochemistry, molecular biology, and biological functions of the soluble cytochrome P-450 mono-oxygenases that are induced by barbiturates in the bacterium, Bacillus megaterium. To date, we have described three distinct P450 cytochromes (P450 BM-1/BM- 2/BM-3) from B. megaterium that are induced by barbiturates. One of these, P450BM-3 incorporates both a P450 and an NADPH:P450 reductase in proteolytically separable domains of a single, soluble, 119 kDa polypeptide and 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, more than it does the mitochondrial or other bacterial P450s. Its gene has been cloned and sequenced (including the complete 5' regulatory region). We have also cloned, sequenced and expressed the complete gene for P450 BM-1 and have purified but not yet cloned the third protein, P450 BM-2.
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; 2.) the elucidation, at the level of the gene, of the processes involved in the normal regulation of expression & the mechanism of barbiturate-mediated induction of the three B. megaterium P450s; 3.) the delineation of the structure-function relationships of P450 BM-3 (including substrate-binding, specificity of oxygenation and electron transfer) utilizing the site-specific mutagenesis in conjunction with X-ray crystallography; 4.) comparative studies, utilizing recombinant DNA and protein-characterization techniques, of the barbiturate-mediated induction mechanisms of the bacterial and the analogous mammalian liver P450s. 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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