Despite tremendous progress in recent years in understanding human drug metabolism, we do not know how individual cytochromes P450 bind and oxidize small molecules of great environmental interest with high affinity and/or specificity. Such compounds, including natural products and organic solvents, generally occur in mixtures, and it is often not clear how one compound can modulate the detoxification and bioactivation of other compounds. The long-term goal of our work is to understand the structural basis of mammalian P450 substrate specificity as the rational basis for prediction of drug-drug and drug-environment interactions, as well as individual differences in xenobiotic metabolism. The goal of the proposed research is to determine how human hepatic CYP2B6 can bind and oxidize small hydrocarbons such as monoterpenes (C10H16) efficiently and selectively in the absence of any functional groups in the compounds that can make specific contacts with active site residues or the heme iron. CYP2B6 is of particular importance in environmental health because it is induced by pesticides and polychlorinated biphenyls (PCBs) and metabolizes insecticides, herbicides, industrial chemicals, solvents, and PCBs. Furthermore, CYP2B6 activities can vary 25 to 80-fold among different individuals. Monoterpenes have been selected as the focus of the work because they represent a very important class of natural products that show great chemical diversity, are distributed widely in nature and used extensively in industrial and household settings, exhibit some very intriguing pharmacological and toxicological properties, and show interesting structure-activity relationships in their interactions with different P450s. The centra hypothesis is that efficient binding and oxidation of monoterpenes by CYP2B6 involve not only interactions in the heme pocket but also the allosteric action of these and other small organic molecules at a peripheral binding site, and that conformational changes enabled by enzyme plasticity facilitate optimal shape complementarity with the ligand. The rationale for the proposed work is that the findings will enable us to understand and predict the binding of the complete range of CYP2B6 ligands and how both endogenous and exogenous compounds may activate as well as inhibit CYP2B6-mediated catalysis. The hypothesis will be tested through pursuit of two specific aims: 1) To elucidate the kinetic and thermodynamic basis of monoterpene binding and catalysis by CYP2B6;2) To elucidate the structural basis of high affinity binding of monoterpenes to CYP2B6 and the functional role of a peripheral binding site. Key methods include X-ray crystallography, isothermal titration calorimetry, and absorbance and fluorescence spectroscopy. The innovation derives from the novel questions posed and the use of a fully integrated structural, biophysical, and biochemical approach to address a fundamental question about xenobiotic metabolism in relationship to environmental health. The significance of the work is that it will provide new and important information that is essential to understand and predict species and individual differences in the fate of environmental chemicals.
Cytochromes P450 are crucial enzymes found predominantly in the liver that are responsible for breaking down a wide variety of compounds to which humans are exposed, including drugs, environmental contaminants, and industrial chemicals. The proposed research will enable us to understand in detail how a human P450 binds and detoxifies a very important class of natural products that are found widely in nature and used extensively in households and in industry. The work is directly relevant to the mission of the National Institute of Environmental Health Sciences to reduce the burden of human illness and disability by understanding how the environment influences the development and progression of human disease.
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