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 PGH2 to PGE2 (Vmax; 170 micromol min-1 mg-1) and high kcat/Km (310 mm-1 s-1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 micromol min-1 mg-1). The formation of 15-hydroperoxy-PGE2 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 micromol min-1 mg-1), 5-hydroperoxyeicosatetraenoic acid (0.043 micromol min-1 mg-1), and 15-hydroperoxy-PGE2 (0.04 micromol min-1 mg-1). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 micromol 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 cm3/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.
Thorén
Staffan
S
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden.
Weinander
Rolf
R
Saha
Sipra
S
Jegerschöld
Caroline
C
Pettersson
Pär L
PL
Samuelsson
Bengt
B
Hebert
Hans
H
Hamberg
Mats
M
Morgenstern
Ralf
R
Jakobsson
Per-Johan
PJ
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D015232:Dinoprostone / Q000096:biosynthesis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010544:Peroxidases / Q000378:metabolism; D000071876:Prostaglandin-E Synthases; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D010544:Peroxidases; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases; D005978:Glutathione; D015232:Dinoprostone