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*

Tove Hammarberg,Mats Hamberg,Anders Wetterholm,Henrik Hansson,Bengt Samuelsson,Jesper Z Haeggstro¨M

doi:10.1074/jbc.m808365200

Abstract

Microsomal prostaglandin E synthase type 1 (mPGES-1) converts prostaglandin endoperoxides, generated from arachidonic acid by cyclooxygenases, into prostaglandin E2. 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 C4 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 H2 into prostaglandin F2α. 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.

Highlights

Prostaglandin E2 (PGE2)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]
PGH2 is further isomerized into PGE2 by three distinct enzymes, cytosolic PGE synthase, microsomal PGE synthase type 1, and microsomal PGE synthase type 2 [3,4,5]
Cytosolic PGE synthase and microsomal PGE synthase type 2 seem to provide a basal synthesis of PGE2, microsomal PGE synthase type 1 (mPGES-1) appears to account for PGE2 synthesis under proinflammatory conditions

Summary

Introduction

Prostaglandin E2 (PGE2)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]. 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 A4, and an Arg residue, possibly involved in the activation of the GSH thiol. We used a homology model, based on the LTC4S structure, and site-directed mutagenesis to identify Arg-126 as a key catalytic residue in mPGES-1.

Results

Conclusion