CYP2J2 epoxygenase is a membrane bound cytochrome P450, which converts omega-3 and omega-6 fatty acids into physiologically active epoxides. In this work, we functionally characterize CYP2J2 in model lipid bilayer Nanodiscs. We present a comprehensive comparison of the effects of N-terminal modifications on the properties of CYP2J2 and incorporate novel findings with respect to the activity of the protein in lipid bilayers of Nanodiscs. We demonstrate that complete truncation of the N-terminus changes the association of this protein with the E.coli membrane but does not disrupt incorporation of this protein into the lipid bilayers of Nanodiscs. Additionally, we introduce silent mutations at the N-terminus to express full length CYP2J2 in E. coli while maintaining wild-type functionality. We further show that lipid bilayers are essential for the productive use of NADPH for ebastine hydroxylation by CYP2J2. We show that the presence of N-terminus is not as critical as the presence of membrane environment for efficient electron transfer from cytochrome P450 reductase to CYP2J2 for ebastine hydroxylation in Nanodiscs. This shows that adapting the native-like conformation of CYP2J2 and cytochrome P450 reductase in lipid bilayers is essential for effective use of reducing equivalents from NADPH for ebastine hydroxylation.

Functional studies of N-terminally modified CYP2J2 epoxygenase in model lipid bilayers

246th National Meeting (2013)

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