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

Abstract

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.

Genetically modified proteins: functional improvement and chimeragenesis

“…3 Downloaded by [University of Exeter] at 18:01 28 July 2015 For the improvement of catalytic efficiency in the conditions of the membrane topologies and structures, the C-terminus of cyclooxygenase isoform-2 (COX-2) was linked to the N-terminus of microsomal prostaglandin E2 synthase-1 (mPGES-1) through a transmembrane linker to form a hybrid enzyme, COX-2-10aa-mPGES-1 (Table 1). 2 Under the necessity of the regulatory mechanism improvement through chimeragenesis, a bifunctional enzyme of hepatitis C (HCV) protein 3/4A (NS3/4A) comprising two separate domains with protease and helicase activities evolved for the more effective viral propagation (Table 1). 5 The functional analysis of the full-length protein and separately isolated catalytic domains suggested the existence of dynamic coupling through the interdomain communication.…”

Multiple roles of dihomo-γ-linolenic acid against proliferation diseases

“…Researches suggest that 15-lipoxygenase-derived hydroxy fatty acids inhibit the synthesis of AA-derived 5-lipoxygenase metabolites [11,12,28] (Figure 3). The observations are significant because elevated levels of AA-derived 5-lipoxygenase products, e.g., LTC 4 and LTB 4 , are associated with several pathologic inflammatory, hyperproliferative disorders [61,62].…”

“…DGLA can then be converted to arachidonic acid (AA), another 20-carbon ω-6 PUFA [11,12]. Both DGLA and AA are substrates of the lipid-peroxidizing enzyme COX.…”

Identification of Tumorigenesis from the Specific Coupling of Cyclooxygenase-2 with Microsomal Prostaglandin E2 Synthase-1 in vivo

“…To test the hypothesis, which says that the solo coupling of COX-2 with mPGES-I is pathogenic/tumorigenic, a system is required to mimic the solo coupling of COX-2 with mPGES-I and limit the involvement of cPGES and mPGES-2 for the pathogenic biosynthesis of PGE z in the cells. Recently, we successfully engineered a single enzyme, COX-2-10aa-mPGES-I by linking of the C-terminus of COX-2 to the N-terminus of the mPGES-I through a 10 amino acid linker (10aa) (17). Our enzymological study revealed that the COX-2-10aa-mPGES-l has a lower km than that of COX-2 coupled to cPGES and mPGES-2 with superior priority to convert the available AA directly to the pathogenic PGE z (17).…”

“…Thus, a solo inflammatory pathway for PGE z biosynthesis, mediated by the specific coupling reaction between COX-2 and mPGES-I in cells could be mimicked by expressing the COX-2-10aa-mPGES-l in the HEK293 cell line, even in the presence of the house keeping enzymes, cPGES and mPGES-2. 2 An HEK293 cell line stably expressing the (17).…”