Modeling and Molecular Dynamics Simulations on the in situ Murine Cytochrome P450 4F System

Cytochrome P450s are major participants in the maintenance and well-being of cellular function and have important roles in the health and disease of living creatures. The ω-hydroxylation, catalyzed by CYP4 family members, has been observed to be an important metabolic pathway for the homeostasis of mammalian cells as it regulates inflammatory processes with the eicosanoid cascade of metabolites of the ω-6 polyunsaturated fatty acid, arachidonic acid. Many human CYP4F and murine Cyp4f subfamily members have recently gained interest for their usage as potential cancer biomarkers as the expression of these proteins are modified in tumor cells. 20-HETE, the ω-hydroxylated product of arachidonic acid, has gained attention for being the chief metabolic product of interest in vascular function, tumor progression and propagation. Whether or not individual Cyp4f isoforms are responsible for the production of this metabolite is of great interest to medicine as such insight could provide researchers with new avenues of study in the fight against cancer. One particular Cyp4f isozyme, Cyp4f13, has received relatively little study until only very recently and is the focus of the work presented in this thesis, as it has not fully had its role in eicosanoid metabolism understood. Using a combination of computational chemistry approaches, this study focuses on exploring the murine cytochrome P450 4f13 system and its active site using all-atomistic Molecular Dynamics Simulation of a homology model. With the embedded protein solvated and in situ environment replicated, the resting state of the substrate-free Cyp4f13 system was generated. Solvation of the active site was performed to explore the inner active cavity of the P450 system, with subsequent molecular docking and mutation of active site residues performed in order to gain insight into the interactions present in the protein-substrate complex. Protonation state changes were observed to have significant effects on both protein structure and arachidonate binding through electrostatic interactions. Leu137, Arg237, and Gly327 were modified and displayed drastic effects on predicted regiospecificity on the P450 substrate. With the insights obtained, we hope to further the understanding of murine Cyp4f13-catalyzed ω-hydroxylation of arachidonic acid.