Molecular Recognition Mechanism of Hematopoietic Prostaglandin D Synthase with its Cofactor and Substrate

Abstract

Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is a Sigma class member of the glutathione S-transferase (GST) superfamily, and requires glutathione (GSH) as a cofactor. H-PGDS catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2), which acts as an allergic and inflammatory mediator. The overproduction of PGD2 by H-PGDS causes allergic and inflammatory reactions in the necrotic muscle fibers of Duchenne muscular dystrophy (DMD) patients. Therefore, H-PGDS inhibitors are thought to have potential for use in drug therapy for DMD.The catalytic mechanism of H-PGDS remains unclear, since essential information, such as the binding affinity and stoichiometry of GSH and PGH2 to H-PGDS, have not been precisely determined yet. Therefore, to clarify the issues related to the cofactor and substrate recognition of H-PGDS, we investigated the interaction between H-PGDS and PGH2 in the presence and absence of GSH.For this purpose, isothermal titration calorimetry (ITC) was performed using a substrate mimetics U-44069 in the place of PGH2 since PGH2 is quite unstable in most solutions. Recombinant H-PGDS was prepared using an E. coli expression system and used for the ITC measurements. The results showed that U-44069 binds to H-PGDS in the presence of the cofactor, GSH, but the binding was not significant in the absence of GSH. Therefore, these results suggest that the GSH binding promotes the interaction between H-PGDS and PGH2, and that GSH plays an important role, not only for the catalytic reaction but also for substrate binding.