title: A dynamic Asp-Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase.
abstract: Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden;
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Brock
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden;
authors.forename: Mats
authors.initials: M
authors.lastname: Hamberg
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden;
authors.forename: Navisraj
authors.initials: N
authors.lastname: Balagunaseelan
authors.affiliation: Department of Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-6304;
authors.forename: Michael
authors.initials: M
authors.lastname: Goodman
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Ralf
authors.initials: R
authors.lastname: Morgenstern
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden;
authors.forename: Emilia
authors.initials: E
authors.lastname: Strandback
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden; jesper.haeggstrom@ki.se bengt.samuelsson@ki.se.
authors.forename: Bengt
authors.initials: B
authors.lastname: Samuelsson
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden;
authors.forename: Agnes
authors.initials: A
authors.lastname: Rinaldo-Matthis
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden; jesper.haeggstrom@ki.se bengt.samuelsson@ki.se.
authors.forename: Jesper Z
authors.initials: JZ
authors.lastname: Haeggström
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004151:Dipeptides / Q000737:chemistry*; D005978:Glutathione / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008861:Microsomes / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D000071876:Prostaglandin-E Synthases; D011487:Protein Conformation
chemical_list: D004151:Dipeptides; D008024:Ligands; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases; D005978:Glutathione
keywords: MAPEG; inflammation; mPGES-1; mechanism; prostaglandin