Human microsomal prostaglandin E synthase-1 -: Purification, functional characterization, and projection structure determination

Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH(2) to PGE(2) (V-max; 170 mumol min(-1) mg(-1)) and high k(cat)/K-m ( 310 mM(-1) s(-1)). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG(2) to 15-hydroperoxy-PGE(2) (V-max; 250 mumol min(-1) mg(-1)). The formation of 15-hydroperoxy-PGE(2) represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 mumol min(-1) mg(-1)), 5-hydroperoxyeicosatetraenoic acid (0.043 mumol min(-1) mg(-1)), and 15-hydroperoxy-PGE(2) (0.04 mumol min(-1) mg(-1)). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 mumol min(-1) mg(-1)). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm(3)/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1.