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

Abstract

CYP2J2 epoxygenase is a membrane bound cytochrome P450 that converts omega-3 and omega-6 fatty acids into physiologically active epoxides. In this work, we present a comprehensive comparison of the effects of N-terminal modifications on the properties of CYP2J2 with respect to the activity of the protein in model lipid bilayers using Nanodiscs. We demonstrate that the complete truncation of the N-terminus changes the association of this protein with the E.coli membrane but does not disrupt incorporation in the lipid bilayers of Nanodiscs. Notably, the introduction of silent mutations at the N-terminus was used 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. Taken together, it was determined that the presence of the N-terminus is not as critical as the presence of a membrane environment for efficient electron transfer from cytochrome P450 reductase to CYP2J2 for ebastine hydroxylation in Nanodiscs. This suggests that adopting 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.

Endocannabinoids Anandamide and 2-Arachidonoylglycerol Are Substrates for Human CYP2J2 Epoxygenase

“…Previous reports demonstrated the incorporation of CPR and P450 into NDs as verified by size-exclusion chromatography (Grinkova et al, 2010). Additionally, in a previous work, we prepared discs containing both CYP2J2 and CPR NDs (McDougle et al, 2013). Incubations for subsequent qualitative LC-MS analysis contained saturating concentrations of each substrate, whereas samples for quantitative kinetic analysis contained either 1, 5, 10, 20, 40, or 60 mM of each respective substrate.…”

“…The recombinant CYP2J2 (construct D34G) was expressed and purified, as published previously (McDougle et al, 2013;Zelasko et al, 2013). Briefly, DH5a cells containing the His-tagged and N-terminally truncated CYP2J2 plasmid and pTGro7 chaperonin plasmid were cultured in 30 ml Luria Bertani media with chloroamphenicol (20 mg/ml) and ampicillin (100 mg/ml) at 37°C and 250 rpm overnight.…”

“…Expression of the redox partner, cytochrome P450 reductase (CPR), from Rattus norvegicus was described previously (McDougle et al, 2013). Briefly, CPR starter cultures were used to inoculate 1 l Luria Bertani supplemented with ampicillin (100 mg/ml) and riboflavin (1 mg/l) and grown for 4 hours at 37°C and 220 rpm, before induction with isopropyl b-D-1-thiogalactopyranoside (1 ml 1 mM), and then grown for 18-20 hours at 33°C and 220 rpm.…”

“…Purified CYP2J2 was incorporated into the membrane bilayers of NDs as previously described, with the only modification being the addition of POPS (McDougle et al, 2013). POPS and POPC lipid stocks were combined in chloroform in a 20:80 molar ratio and dried down under a steady stream of N 2 .…”

Anti-inflammatory ω-3 endocannabinoid epoxides

“…Human CYP2J2 was heterologously expressed and incorporated into the lipid bilayers of nanodiscs as previously described (61). Incubation mixtures contained CYP2J2 nanodiscs (0.2 μM), CPR (0.6 μM), butylated hydroxytoluene (BHT) (0.1%), and incremental increases of either EPEA or DHEA (500 μL total volume) in 100 mM phosphate buffer (pH 7.4).…”