The cytochrome P450 enzymes (CYPs) are essential for the biosynthesis of numerous natural products, steroid hormones, and eicosanoids, as well as the clearance of most drugs. Due to their central role in xenobiotic disposition, CYPs mediate many adverse drug interactions of therapeutic significance. The mechanisms of CYP catalyzed O2 activation and substrate oxidation have been challenging to unravel, in large part because of the reactivity of intermediates. The CYPs that cleave C-C bonds are among the most mechanistically flexible of such enzymes;however, it is not usually realized that the pathways and reactive intermediates of this group of CYPs have not yet been investigated extensively. Most studies on CYP have been primarily focused on the hydroxylating CYPs. Thus relatively little attention has been paid to the CYP enzymes which use multiple oxidants and catalyse the more complicated transformations. Thus the presently available experimental data cannot be generally extrapolated to the C-C bond cleaving CYPs. Mycobacterium tuberculosis CYP51 constitutes a valuable and prototypical example for the study of O2 activation and C-C bond cleavage mechanisms. Moreover, many mycobacterial, trypanosomal, and fungal pathogens utilize bond cleaving CYPs in their own biosynthetic pathways, each of which is a drug target. Given that these pathogens are responsible for millions of deaths annually, there is a profound need for a clearer mechanistic understanding of these particular enzymes in support of the development of therapeutics of broad public health importance. The long-term goal of this project is to understand the catalytic mechanisms of C-C bond cleaving CYPs, and to answer questions surrounding how these enzymes tune the reactivity of their putative oxygen intermediates. Applying molecular dynamics simulation and hybrid quantum mechanics/molecular mechanics techniques (QM/MM), the objective of the first Specific Aim is to explore the several possible reaction mechanisms utilized by M. tuberculosis CYP51 to activate O2 and perform substrate oxidation. The objective of the second Specific Aim is to validate the computationally-derived structure-function relationships governing the lifetimes of reactive oxygen intermediates. To meet these objectives, organic chemical syntheses of catalytic intermediates, site-directed mutagenesis, stopped-flow UV-vis, and resonance Raman techniques will be utilized. Guided by computational results, the objective of the third Specific Aim is to characterize relevant CYP intermediates using cryoradiolysis and resonance Raman spectroscopy to shed light on the C-C bond cleavage mechanism. Taken together, the interplay between these three Specific Aims will provide a feedback loop between theory and experiment, allowing incremental refinement of mechanistic hypotheses to provide a more complete understanding of reactive oxygen intermediate chemistry in CYP enzymes with important implications for human health.

Public Health Relevance

The cytochromes P450 are among the most ubiquitous enzymes, and in humans, catalyze several reactions in hormone biosynthesis and have a dominant role in the metabolism of foreign substances. Furthermore, bond cleaving biosynthetic cytochromes in pathogenic bacteria are emerging as drug targets for the treatment of infectious diseases. Understanding the structure-function relationships and the mechanisms of these biosynthetic enzymes will provide important insight towards the development of new therapeutics and the understanding of mechanisms of the entire cytochrome P450 enzyme superfamily.

National Institute of Health (NIH)
Research Project (R01)
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Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
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Okita, Richard T
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Virginia Commonwealth University
Schools of Pharmacy
United States
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