Mutation of a Critical Arginine in Microsomal Prostaglandin E Synthase-1 Shifts the Isomerase Activity to a Reductase Activity That Converts Prostaglandin H2 into Prostaglandin F2α*

Abstract

Microsomal prostaglandin E synthase type 1 (mPGES-1) converts prostaglandin endoperoxides, generated from arachidonic acid by cyclooxygenases, into prostaglandin E 2 . This enzyme belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral membrane proteins, and because of its link to inflammatory conditions and preferential coupling to cyclooxygenase 2, it has received considerable attention as a drug target. Based on the high resolution crystal structure of human leukotriene C 4 synthase, a model of mPGES-1 has been constructed in which the tripeptide co-substrate glutathione is bound in a horseshoe-shaped conformation with its thiol group positioned in close proximity to Arg-126. Mutation of Arg-126 into an Ala or Gln strongly reduces the enzyme's prostaglandin E synthase activity (85-95%), whereas mutation of a neighboring Arg-122 does not have any significant effect. Interestingly, R126A and R126Q mPGES-1 exhibit a novel, glutathione-dependent, reductase activity, which allows conversion of prostaglandin H 2 into prostaglandin F 2␣ . Our data show that Arg-126 is a catalytic residue in mPGES-1 and suggest that MAPEG enzymes share significant structural components of their active sites. Prostaglandin E 2 (PGE 2 )3 is an abundant lipid mediator that signals via four receptors (EP1 to -4), resulting in an array of important biological actions in physiology as well as pathophysiology (1, 2). Biosynthesis of PGE 2 proceeds from arachidonic acid, which is converted to the unstable endoperoxide, prostaglandin H 2 , by cyclooxygenase type 1 and 2. PGH 2 is further isomerized into PGE 2 by three distinct enzymes, cytosolic PGE synthase, microsomal PGE synthase type 1 (mPGES-1), and microsomal PGE synthase type 2 (3-5). Although cytosolic PGE synthase and microsomal PGE synthase type 2 seem to provide a basal synthesis of PGE 2 , mPGES-1 appears to account for PGE 2 synthesis under proinflammatory conditions. Thus, mPGES-1 is up-regulated by mitogens and cytokines and is functionally coupled to cyclooxygenase type 2 (4, 6). Due to its key role in PGE 2 synthesis, mPGES-1 has attracted attention as a potential drug target in the areas of inflammation, pain, fever, and cancer (7).mPGES-1 is a member of the MAPEG superfamily of enzymes (8), which also includes two key proteins in the leukotriene cascade, viz. 5-lipoxygenase-activating protein and leukotriene C 4 synthase (LTC4S). Since all MAPEG members are integral membrane proteins, structural information on this family has been scarce. Recently, however, significant progress has been made in this area, and several high and low resolution structures have been solved by three-dimensional as well as two-dimensional crystallography (9 -12). The crystal structures of human LTC4S clearly provided the most detailed structural information, inter alia a unique, horse-shoe shaped binding conformation of GSH, a hydrophobic crevice presumably binding the lipid substrate leukotriene A 4 , and an Arg residue, possibly involved in...

A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E 2 synthase

“…Conversion of PGH 2 to PGE 2 by WT or mutated mPGES-1 were quantified by using GC-MS as described (12). For further details, please refer to SI Materials and Methods.…”

“…In particular, the crystal structures of human LTC4S provided detailed structural information, including an arginine residue that was later shown to activate the glutathione (GSH) thiolate (10,11). We have previously proposed that this conserved arginine residue is also essential for enzymatic activity in mPGES-1 as Arg-126 (12). The recent structural determination of mPGES-1, however, at an exceptionally high resolution of 1.16 Å, uncovered several unanticipated structural features (13).…”

Reduced prostaglandin F2α release from blood mononuclear leukocytes after oral supplementation of ω3 fatty acids: the OmegAD study

“…The generation of PGE and PGF proceeds from AA and the common precursor prostaglandin H. By means of an enzyme, 9KPGR, PGE can be converted to PGF ( 12 ). Various factors can alter the generation of either eikosanoid, such as mutations in generating enzymes, stimuli, and environment (30)(31)(32)(33). Thus, interactions of EPA, DHA, LPS, and PHA might be of signifi cance for generation of one or the other eikosanoid.…”

Convergence of the 5-LOX and COX-2 pathways: heme-catalyzed cleavage of the 5S-HETE-derived di-endoperoxide into aldehyde fragments

“…Cleavage of the carbon chain is accelerated by the presence of ferrous iron ( 4,5 ), heme ( 6 ), and it is also catalyzed by the enzyme thromboxane synthase ( 4,7 ). Here, we describe the nonenzymatic transformation of the 5 S -HETE derived di-endoperoxide in the presence of heme and ferrous iron (Fe 2+ ) in aqueous solution.…”

“…About 5 g of the isolated [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]labeled di-endoperoxide ( ≈ 15,000 cpm) was mixed immediately after collection off RP-HPLC with 1 ml of a freshly prepared solution of FeCl 2 in water (20 mM). After 1 min, the products were loaded onto a Waters Oasis HLB cartridge, washed with water, and eluted with methanol.…”