Engineering of a novel hybrid enzyme: an anti-inflammatory drug target with triple catalytic activities directly converting arachidonic acid into the inflammatory prostaglandin E2

Cyclooxygenase isoform-2 (COX-2) and microsomal prostaglandin E-2 synthase-1 (mPGES-1) are inducible enzymes that become up-regulated in inflammation and some cancers. It has been demonstrated that their coupling reaction of converting arachidonic acid (AA) into prostaglandin (PG) E-2 (PGE(2)) is responsible for inflammation and cancers. Understanding their coupling reactions at the molecular and cellular levels is a key step toward uncovering the pathological processes in inflammation. In this paper, we describe a structure-based enzyme engineering which produced a novel hybrid enzyme that mimics the coupling reactions of the inducible COX-2 and mPGES-1 in the native ER membrane. Based on the hypothesized membrane topologies and structures, the C-terminus of COX-2 was linked to the N-terminus of mPGES-1 through a transmembrane linker to form a hybrid enzyme, COX-2-10aa-mPGES-1. The engineered hybrid enzyme expressed in HEK293 cells exhibited strong triple-catalytic functions in the continuous conversion of AA into PGG(2) (catalytic-step 1), PGH(2) (catalytic-step 2) and PGE(2) (catalytic-step 3), a pro-inflammatory mediator. In addition, the hybrid enzyme was also able to directly convert dihomo-gamma-linolenic acid (DGLA) into PGG(1), PGH(1) and then PGE(1) (an anti-inflammatory mediator). The hybrid enzyme retained similar K-d and V-max values to that of the parent enzymes, suggesting that the configuration between COX-2 and mPGES-1 (through the transmembrane domain) could mimic the native conformation and membrane topologies of COX-2 and mPGES-1 in the cells. The results indicated that the quick coupling reaction between the native COX-2 and mPGES-1 (in converting AA into PGE(2)) occurred in a way so that both enzymes are localized near each other in a face-to-face orientation, where the COX-2 C-terminus faces the mPGES-1 N-terminus in the ER membrane. The COX-2-10aa-mPGES-1 hybrid enzyme engineering may be a novel approach in creating inflammation cell and animal models, which are particularly valuable targets for the next generation of NSAID screening.