Our goals include the characterization of a soluble, 119 kD catalytically self-sufficient cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium and the elucidation of the induction mechanism.
The specific aims of the present proposal are: 1. Determination of the prosthetic groups of the monooxygenase. 2. Elucidation of the functional domains and determination of the amino acid sequence of the 119 kD polypeptide. 3. X-ray crystallography of the 119 kD polypeptide. 4. Characterization of two smaller cytochrome P-450 proteins also induced by barbiturates in B. megaterium including reconstitution and enzymological characterization of the associated monooxygenase systems. 5. Determination of the parameters and mechanism of barbiturate induction of the P-450-dependent monooxygenase systems of B. megaterium. This will include characterization of the biochemical events involved, elucidation of inducer structure-potency relationships, cloning of the 119 kD P-450 gene and functional expression of the cloned monooxygenase. The health-related implications of the proposed research include an increased understanding of how carcinogens may be produced from aromatic hydrocarbons and other xenobiotics by analogous P-450 enzymes in human tissues and how some of these same enzymes are regulated or induced by a variety of such xenobiotics including barbiturates.
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