Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH 2 , a common precursor of various prostanoids, to produce PGD 2 , an endogenous somnogen and nociceptive modulator, in the brain. L-PGDS is a member of the lipocalin superfamily and binds lipophilic substances, such as retinoids and bile pigments, suggesting that L-PGDS is a dual functional protein acting as a PGD 2 -synthesizing enzyme and a transporter for lipophilic ligands. In this study we determined by NMR the three-dimensional structure of recombinant mouse L-PGDS with the catalytic residue Cys-65. The structure of L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel -barrel and a long ␣-helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins, and the cavity contained two pockets. Molecular docking studies, based on the result of NMR titration experiments with retinoic acid and PGH 2 analog, revealed that PGH 2 almost fully occupied the hydrophilic pocket 1, in which Cys-65 was located and all-trans-retinoic acid occupied the hydrophobic pocket 2, in which amino acid residues important for retinoid binding in other lipocalins were well conserved. Mutational and kinetic studies provide the direct evidence for the PGH 2 binding mode. These results indicated that the two binding sites for PGH 2 and retinoic acid in the large cavity of L-PGDS were responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by retinoic acid.
Lipocalin-type prostaglandin (PG)2 D synthase (L-PGDS, prostaglandin H 2 D-isomerase, EC 5.3.99.2) (1-3) catalyzes the isomerization of the 9,11-endoperoxide group of PGH 2 , a common precursor of various prostanoids, to produce PGD 2 with 9-hydroxy and 11-keto groups, an endogenous somnogen (4) and a modulator of pain responses (5), in the presence of sulfhydryl compounds. L-PGDS is the only enzyme among members of the lipocalin gene family (6) that is composed of a group of lipid-transporter proteins, such as retinol-binding protein, -lactoglobulin, major urinary protein, aphorodisin (6 -8), epididymal retinoic acid-binding protein (9), and tear lipocalin (10). L-PGDS has three Cys residues, Cys-65, Cys-89, and Cys-186, conserved among all species. Two of these Cys residues, Cys-89 and Cys-186, form a disulfide bridge, which is highly conserved among most, but not all lipocalins (6). This disulfide bridge can be removed without a significant loss of the enzymatic activity. On the other hand, Cys-65 is unique to L-PGDS, as it has never been found in other lipocalins. Moreover, the replacement of Cys-65 with Ser/Ala by site-directed mutagenesis led to complete loss of the catalytic activity of the recombinant rat (11), human, mouse, chicken (12), and bull and frog (13) enzymes, indicating that the Cys-65 residue is the key residue for the reaction catalyzed by L-P...