New insights into the catalytic mechanism of Bombyx mori prostaglandin E synthase gained from structure-function analysis.

Prostaglandin E synthase (PGES) catalyzes the isomerization of PGH2 to PGE2. We previously reported the identification and structural characterization of Bombyx mori PGES (bmPGES), which belongs to Sigma-class glutathione transferase. Here, we extend these studies by determining the structure of bmPGES in complex with glutathione sulfonic acid (GTS) at a resolution of 1.37 Å using X-ray crystallography. GTS localized to the glutathione-binding site. We found that electron-sharing network of bmPGES includes Asn95, Asp96, and Arg98. Site-directed mutagenesis of these residues to create mutant forms of bmPGES mutants indicate that they contribute to catalytic activity. These results are, to our knowledge, the first to reveal the presence of an electron-sharing network in bmPGES.

Crystal structure of a Bombyx mori sigma-class glutathione transferase exhibiting prostaglandin E synthase activity.

BACKGROUND: Glutathione transferases (GSTs) are members of a major family of detoxification enzymes. Here, we report the crystal structure of a sigma-class GST of Bombyx mori, bmGSTS1, to gain insight into the mechanism catalysis. METHODS: The structure of bmGSTS1 and its complex with glutathione were determined at resolutions of 1.9Å and 1.7Å by synchrotron radiation and the molecular replacement method. RESULTS: The three-dimensional structure of bmGSTS1 shows that it exists as a dimer and is similar in structure to other GSTs with respect to its secondary and tertiary structures. Although striking similarities to the structure of prostaglandin D synthase were also detected, we were surprised to find that bmGSTS1 can convert prostaglandin H2 into its E2 form. Comparison of bmGSTS1 with its glutathione complex showed that bound glutathione was localized to the glutathione-binding site (G-site). Site-directed mutagenesis of bmGSTS1 mutants indicated that amino acid residues Tyr8, Leu14, Trp39, Lys43, Gln50, Met51, Gln63, and Ser64 in the G-site contribute to catalytic activity. CONCLUSION: We determined the tertiary structure of bmGSTS1 exhibiting prostaglandin E synthase activity. GENERAL SIGNIFICANCE: These results are, to our knowledge, the first report of a prostaglandin synthase activity in insects.

Human hematopoietic prostaglandin D synthase inhibitor complex structures.

In mast and Th2 cells, hematopoietic prostaglandin (PG) D synthase (H-PGDS) catalyses the isomerization of PGH(2) in the presence of glutathione (GSH) to produce the allergic and inflammatory mediator PGD(2). We determined the X-ray structures of human H-PGDS inhibitor complexes with 1-amino-4-{4-[4-chloro-6-(2-sulpho-phenylamino)-[1,3,5]triazin-2-ylmethyl]-3-sulpho-phenylamino}-9,10-dioxo-9,10-dihydro-anthracene-2-sulphonic acid (Cibacron Blue) and 1-amino-4-(4-aminosulphonyl) phenyl-anthraquinone-2-sulphonic acid (APAS) at 2.0 Å resolution. When complexed with H-PGDS, Cibacron Blue had an IC(50) value of 40 nM and APAS 2.1 µM. The Cibacron Blue molecule was stabilized by four hydrogen bonds and π-π stacking between the anthraquinone ring and Trp104, the ceiling of the active site H-PGDS pocket. Among the four hydrogen bonds, the Cibacron Blue terminal sulphonic group directly interacted with conserved residues Lys112 and Lys198, which recognize the PGH(2) substrate α-chain. In contrast, the APAS anthraquinone ring was inverted to interact with Trp104, while its benzenesulphonic group penetrated the GSH-bound region at the bottom of the active site. Due to the lack of extended aromatic rings, APAS could not directly hydrogen bond with the two conserved lysine residues, thus decreasing the total number of hydrogen bond from four to one. These factors may contribute to the 50-fold difference in the IC(50) values obtained for the two inhibitors.

The Caenorhabditis elegans DDX-23, a homolog of yeast splicing factor PRP28, is required for the sperm-oocyte switch and differentiation of various cell types.

DEAD/H-box proteins are involved in various aspects of RNA metabolism. Here we report the developmental function of a DEAD-box protein, DDX-23, in Caenorhabditis elegans, which has significant homology with the yeast splicing factor PRP28. We found by RNAi and mutant analyses that DDX-23 is essential for both embryonic and post-embryonic development, and required for differentiation of the majority of somatic tissues. When the germline function of ddx-23 was inhibited, hermaphrodite animals showed a reduced number of germ cells and failed to switch from spermatogenesis to oogenesis. These phenotypes were similar to those of the mutants of the three DEAH-box proteins (MOG-1, MOG-4, and MOG-5) whose yeast orthologs are involved in the pre-mRNA splicing pathway. We speculate that DDX-23 functions with the three MOG proteins in the same pathway to regulate tissue differentiation, robust germline proliferation, and the sperm/oocyte switch through modulations of ribonucleoprotein complexes.

Zebrafish and chicken lipocalin-type prostaglandin D synthase homologues: Conservation of mammalian gene structure and binding ability for lipophilic molecules, and difference in expression profile and enzyme activity.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a bifunctional protein possessing both the ability to synthesize PGD(2) and to serve as a carrier protein for lipophilic molecules. L-PGDS has been extensively studied in mammalian species, whereas little is known about non-mammalian forms. Here, we identified and characterized the L-PGDS homologues from non-mammals such as zebrafish and chicken. Phylogenetic analysis revealed that L-PGDSs of mammalian and non-mammalian organisms form a "L-PGDS sub-family" that has been evolutionally separated from other lipocalin gene family proteins. The genes for zebrafish and chicken L-PGDS homologues consisted of 6 exons, and all of the exon/intron boundaries were completely identical to those of mammalian L-PGDS genes. Zebrafish and chicken L-PGDS genes were clustered with several lipocalin genes in the chromosome, as in the case of mouse and human genes. Gene expression profiles were different among chicken, mouse, human, except for conservation of abundant expression in the brain and heart. The chicken L-PGDS homologue carried weak PGDS activity, whereas the zebrafish protein did not show any of the activity. However, when the amino-terminal region of the zebrafish L-PGDS homologue was exchanged for that of mouse L-PGDS carrying the Cys residue essential for PGDS activity, this chimeric protein showed weak PGDS activity. Both zebrafish and chicken L-PGDS homologues bound thyroxine and all-trans retinoic acid, like mammalian L-PGDSs and other lipocalin gene family proteins. These results indicate that non-mammalian and mammalian L-PGDS genes evolved from the same ancestral gene and that the non-mammalian L-PGDS homologue was the primordial form of L-PGDS but whose major function was and is to serve as a carrier protein for lipophilic molecules. During molecular evolution, the mammalian L-PGDS protein might have acquired effective PGDS activity through substitution of several amino acid residues, especially in the amino-terminal region including the Cys residue, which is essential for PGDS activity.

First determination of the inhibitor complex structure of human hematopoietic prostaglandin D synthase.

Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is responsible for the production of PGD(2) as an allergy or inflammation mediator in mast and Th2 cells. We determined the X-ray structure of human H-PGDS complexed with an inhibitor, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) at 1.9 A resolution in the presence of Mg(2+). The styryl group of the inhibitor penetrated to the bottom of the active site cleft, and the tetrazole ring was stabilized by the stacking interaction with Trp104, inducing large movement around the alpha5-helix, which caused the space group of the complex crystal to change from P2(1) to P1 upon binding of BSPT. The phthalhydrazidyl group of BSPT exhibited steric hindrance due to the cofactor, glutathione (GSH), increasing the IC(50) value of BSPT for human H-PGDS from 36.2 micro M to 98.1 micro M upon binding of Mg(2+), because the K(m) value of GSH for human H-PGDS was decreased from 0.60 micro M in the presence of EDTA to 0.14 micro M in the presence of Mg(2+). We have to avoid steric hindrance of the GSH molecule that was stabilized by intracellular Mg(2+) in the mM range in the cytosol for further development of structure-based anti-allergic drugs.

Mechanism of metal activation of human hematopoietic prostaglandin D synthase.

Here we report the crystal structures of human hematopoietic prostaglandin (PG) D synthase bound to glutathione (GSH) and Ca2+ or Mg2+. Using GSH as a cofactor, prostaglandin D synthase catalyzes the isomerization of PGH2 to PGD2, a mediator for allergy response. The enzyme is a homodimer, and Ca2+ or Mg2+ increases its activity to approximately 150% of the basal level, with half maximum effective concentrations of 400 microM for Ca2+ and 50 microM for Mg2+. In the Mg2+-bound form, the ion is octahedrally coordinated by six water molecules at the dimer interface. The water molecules are surrounded by pairs of Asp93, Asp96 and Asp97 from each subunit. Ca(2+) is coordinated by five water molecules and an Asp96 from one subunit. The Asp96 residue in the Ca2+-bound form makes hydrogen bonds with two guanidium nitrogen atoms of Arg14 in the GSH-binding pocket. Mg2+ alters the coordinating water structure and reduces one hydrogen bond between Asp96 and Arg14, thereby changing the interaction between Arg14 and GSH. This effect explains a four-fold reduction in the K(m) of the enzyme for GSH. The structure provides insights into how Ca2+ or Mg2+ binding activates human hematopoietic PGD synthase.

Pronounced eosinophilic lung inflammation and Th2 cytokine release in human lipocalin-type prostaglandin D synthase transgenic mice.

PGD(2) is a major lipid mediator released from mast cells, but little is known about its role in the development of allergic reactions. We used transgenic (TG) mice overexpressing human lipocalin-type PGD synthase to examine the effect of overproduction of PGD(2) in an OVA-induced murine asthma model. The sensitization of wild-type (WT) and TG mice was similar as judged by the content of OVA-specific IgE. After OVA challenge, PGD(2), but not PGE(2), substantially increased in the lungs of WT and TG mice with greater PGD(2) increment in TG mice compared with WT mice. The numbers of eosinophils and lymphocytes in the bronchoalveolar lavage (BAL) fluid were significantly greater in TG mice than in WT mice on days 1 and 3 post-OVA challenge, whereas the numbers of macrophages and neutrophils were the same in both WT and TG mice. The levels of IL-4, IL-5, and eotaxin in BAL fluid were also significantly higher in TG mice than in WT mice, although the level of IFN-gamma in the BAL fluid of TG mice was decreased compared with that in WT mice. Furthermore, lymphocytes isolated from the lungs of TG mice secreted less IFN-gamma than those from WT mice, whereas IL-4 production was unchanged between WT and TG mice. Thus, overproduction of PGD(2) caused an increase in the levels of Th2 cytokines and a chemokine, accompanied by the enhanced accumulation of eosinophils and lymphocytes in the lung. These results indicate that PGD(2) plays an important role in late phase allergic reactions in the pathophysiology of bronchial asthma.