Protein-protein interactions and mechanistic insights for CYP2J2 and CYP5A1

Eicosanoids are signaling molecules formed from the oxidation of -3 and -6 fatty acids. CYP2J2 and CYP5A1 are two key enzymes involved in the formation of eicosanoids. CYP2J2 is a classical P450 which requires transfer of electrons from its obligate redox partner, cytochrome P450 reductase (CPR). Here the triphasic kinetics of the potentially rate-limiting first electron transfer from CPR to CYP2J2 are reported. These rates are similar to previously reported rates of other CYPs and show differences with CYP2J2 in the presence of the substrates arachidonic acid (AA) and ebastine. When CYP2J2 and CPR were not allowed to pre-incubate, use of a truncated CYP2J2 without the hydrophobic N-terminus caused a 200-fold decrease in the kinetics of electron transfer. In a step towards being able to gain structural data for CYP2J2, the protein was mutated at its surface to increase solubility. Individual mutations of I236D and F239H altered the metabolism of AA and/or ebastine, however there was complementation in the double mutant such that substrate metabolism was near that of wild type levels. Further investigation into the reaction cycle of CYP2J2 involved mutation of the highly conserved I-helix residue T315 near the heme. This mutation not only altered the rate of AA metabolism, but also altered the ratio of hydroxylation to epoxidation products. A similar study was carried out with the non-classical CYP5A1, which isomerizes prostaglandin H2 (PGH2) to thromboxane A2 (TXA2) rather than being reduced prior to hydroxylation or epoxidation of a substrate. This CYP has I346 in the position normally occupied by highly conserved I-helix threonine, so in this study the mutants I346T and I346S were created. Each of these hydroxyl-containing mutants showed increased isomerization of PGH2. In addition, I346T appeared to have a more constrained active site than I346S based on the ability to bind substrate analogs. A previously unknown protein-protein interaction between CYP5A1 and CPR was also shown via a direct titration and by monitoring the ability of CPR to produce H2O2 in the presence of CYP5A1. These studies of protein-protein interactions and of a key active site residue in both CYP2J2 and CYP5A1 complement each other