Crystal Structure and Possible Catalytic Mechanism of Microsomal Prostaglandin E Synthase Type 2 (mPGES-2)

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Prostaglandin (PG) H2 (PGH2), formed from arachidonic acid, is an unstable intermediate and is converted efficiently into more stable arachidonate metabolites (PGD2, PGE2, and PGF2) by the action of three groups of enzymes. Prostaglandin E synthase catalyzes an isomerization reaction, PGH2 to PGE2. Microsomal prostaglandin E synthase type-2 (mPGES-2) has been crystallized with an anti-inflammatory drug indomethacin (IMN), and the complex structure has been determined at 2.6 Å resolution. mPGES-2 forms a dimer and is attached to lipid membrane by anchoring the N-terminal section. Two hydrophobic pockets connected to form a V shape are located in the bottom of a large cavity. IMN binds deeply in the cavity by placing the OMe-indole and chlorophenyl moieties into the V-shaped pockets, respectively, and the carboxyl group interacts with Sγ of C110 by forming a H-bond. A characteristic H-bond chain formation (N–H⋯Sγ–H⋯Sγ⋯H–N) is seen through Y107–C113–C110–F112, which apparently decreases the pKa of Sγ of C110. The geometry suggests that the Sγ of C110 is most likely the catalytic site of mPGES-2. A search of the RCSB Protein Data Bank suggests that IMN can fit into the PGH2 binding site in various proteins. On the basis of the crystal structure and mutation data, a PGH2-bound model structure was built. PGH2 fits well into the IMN binding site by placing the α and ω-chains in the V-shaped pockets, and the endoperoxide moiety interacts with Sγ of C110. A possible catalytic mechanism is proposed on the basis of the crystal and model structures, and an alternative catalytic mechanism is described. The fold of mPGES-2 is quite similar to those of GSH-dependent hematopoietic prostaglandin D synthase, except for the two large loop sections.

Introduction

Prostaglandins (PGs) have numerous and diverse biological effects on a variety of physiological and pathological events, such as contraction of smooth muscle, inflammation, and blood clotting. In humans, the most important PG precursor is arachidonic acid, a C20 polyunsaturated fatty acid with non-conjugated double bonds. PGs synthesized from arachidonic acid have the subscript 2 (the series 2 PGs), such as PGD2, PGE2, PGF2, and PGH2, which is an unstable intermediate formed from arachidonic acid by the action of PGH synthase in the arachidonate cascade. In mammalian systems, PGH2 is converted efficiently into more stable arachidonate metabolites, PGD2, PGE2, and PGF2, by the action of the respective synthases for these products.1 The crystal structures of PGH synthase (COX-1 and COX-2), PGD synthase (hPGDS), and PGF synthase (PGFS-1) have been determined.2, 3, 4 In particular, the structure and function of COX-1 and COX-2 have been studied intensively, and more than 30 different coordinate sets have been deposited in the RCSB Protein Data Bank (PDB).5

PGE2 was first discovered in sheep seminal vesicles. PGE2 is distributed widely in various organs, and exerts control over various biological activities, such as relaxation/contraction of smooth muscle,6 excretion of Na+,7 body temperature,8, 9 and the physiological sleep/wake cycle.10 The biosynthesis of PGE2 requires three sequential enzymatic steps: the release of arachidonic acid from membrane phospholipids by phospholipase A2 (PLA2),11, 12 conversion of arachidonic acid into PGH2 by COX-1 or COX-2,13 and transformation of PGH2 into PGE2 by PGES. Following inflammatory stimuli, PGE2 biosynthesis occurs in kinetically distinct phases: an immediate phase, occurring in seconds to minutes; and a delayed phase, occurring over hours.14 Stimuli that increase the concentration of cytoplasmic Ca2+ rapidly can elicit the immediate increase in PGE2 synthesis via functional coupling of pre-existing PG-biosynthetic enzymes. The second delayed increase in PGE2 biosynthesis is accompanied by the coordinated induction of COX-2 and several inducible PLA2 enzymes. It is generally thought that inflammation-associated PGE2 production is a result of this coupled induction of PLA2 and COX-2.11, 14, 15

PGES (EC 5.3.99.3) catalyzes the isomerization of PGH2 to PGE2. Over the last 30 years, several groups have purified this enzyme.16, 17, 18, 19 At least three different types of mammalian PGESs have been isolated. Ogorochi et al. and Meyer et al. independently purified the enzyme from the cytosol of human brain18 and Ascaridia galli,19 respectively. This cytosolic enzyme requires glutathione (GSH), belongs to the GSH-S-transferase (GSTase) family, and is named cPGES. The enzyme has been expressed in Escherichia coli.20 The membrane-associated PGES (mPGES-1) was partially purified from microsomal fractions of bovine and sheep vesicular glands, and was shown to require GSH.16, 17, 21 Jakobsson et al. expressed human GSH-specific, mPGES-1 in E. coli.22 cPGES and mPGES-1 in many organs are GSH-dependent enzymes.

Watanabe et al. reported that the GSH-non-specific PGES activity was distributed widely in the microsomal fractions of rat and sheep organs.23, 24 The enzyme activity in heart, spleen, and uterine microsomes did not specifically require GSH for its catalytic activity, although the catalytic rate is increased two- to fourfold in the presence of DTT, dihydrolipoic acid, GSH or other thiol compounds.25 This GSH-non-specific, membrane-associated PGES is named mPGES-2. A small amount of the N-terminal truncated enzyme was found in the microsomal fraction of bovine heart. The N-terminal sequence of the truncated enzyme starts from Glu88 of the protein derived from the human/monkey cDNA.24 The intact and the N-terminal truncated mPGES-2 show similar catalytic activity. The intact and the N-terminally truncated proteins expressed in E. coli have the same enzymatic properties as the enzyme purified from bovine heart microsomes.25 The amino acid sequence of mPGES-2 contains 110C-x-x-C113, which is seen in the active sites of glutaredoxin and thioredoxin.26 The C110S mutation abolishes the catalytic activity but the C113S mutated enzyme retains the catalytic activity, suggesting that C110 is involved in the catalytic reaction.26

Indomethacin (IMN) is a widely used non-steroidal anti-inflammatory drug and is generally known to exhibit its multiple biological functions by inhibiting COX.27 In spite of the therapeutic utility, however, the drug has significant adverse effects, a circumstance that limits its use. Of these, gastrointestinal and renal toxicities are of major concern.28, 29 Recent reports suggest that IMN and other non-steroidal anti-inflammatory drugs bind to prostaglandin F synthase (PGFS-1) and protect against the progression of gastrointestinal tumors.30, 31, 32 There is increasing evidence that these drugs may also protect against a variety of other cancers, including prostate carcinoma and, most recently, leukemia.33, 34, 35, 36, 37, 38, 39

Here, we present the crystal structure of N-terminal truncated mPGES-2 complexed with the non-steroidal anti-inflammatory drug IMN. The crystal structure indicates that IMN inhibits both PGH2 synthesis and PGE2 synthesis. On the basis of this structure and a PGH2-bound model structure, we propose a catalytic mechanism of the isomerization reaction from PGH2 to PGE2 by mPGES-2.

Section snippets

Overall structure

The enzyme used in this study is a recombinant protein whose N-terminal amino acid residues 1–87 were truncated and an extended His-tag (36 amino acid residues) was attached to residue 88. The crystal structure of N-terminally modified mPGES-2 complexed with the non-steroidal anti-inflammatory drug IMN is determined. The crystallographic refinement parameters (Table 1), final (2FoFc) and (FoFc) maps and conformational analysis by PROCHECK40 indicate that the structure of mPGES-2 has been

The fold of mPGES-2 is similar to those of hPGDS and GSTase

The crystal structure of the functionally similar hematopoietic PGD synthase (hPGDS) has been determined.3, 42, 43 hPGDS belongs to the GSTase family, and the fold of hPGDS is quite similar to those of known GSTases (α class,44 μ class,45 π class,46 and σ class47). Although mPGES-2 does not belong to the GSTase family, the fold of mPGES-2 is similar to those of hPGDS and GSTase. By using the program SARF2,48 the structure of mPGES-2 was compared with those of hPGDS (PDB code 1PD2) and human

Purification and crystallization

The N-terminal truncated (residues 1–87) monkey mPGES-2 gene was cloned into the pTrc-HisA vector and transformed in E. coli BL21,25 which were grown at 37 °C in 1 l of LB medium containing 50 mg of ampicillin and 100 mg of Fe(NO3)3. IPTG was added to a final concentration of 1 mM after 1.5 hours of culturing, and incubation was continued for an additional 15 hours. Cells were harvested by centrifugation and suspended in 60 ml of 50 mM Tris–HCl (pH 7.5), 0.5 mM EDTA. Cell lysis was carried out by

Acknowledgements

We express our thanks to the 19BM beamline staff at APS for assistance to Dr Hiroyuki Kojina for measurment of the enzyme activity and to Professor Richard H. Himes for a critical reading of the manuscript and very valuable comments. The work has been supported by grant GM37233 (to F.T.) from the National Institutes of Health. Use of the Argonne National Laboratory Structural Biology Center beamline at the Advanced Photon Source was supported by the US Department of Energy, Office of Energy

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