Crystallization of prostaglandin-H synthase for X-ray structure analysis

Prostaglandin-H (PGH) synthase from ram seminal vesicles is a dimeric integral membrane protein of molecular weight 140 kDa. PGH synthase is a key enzyme in the biosynthesis of prostaglandins, has cyclooxygenase and peroxidase activities, and contains heme as a coenzyme. In the peroxidation step of its reaction. PGH synthase can use xenobiotics as co-substrates and can catalyze the metabolic activation of carcinogens such as diethylstilbestrol. To gain a detailed understanding of the inner workings of PGH synthase, we are investigating its three-dimensional structure by X-ray crystallography. A purification procedure was established that yields stable homogeneous PGH synthase that is at least 80% holoenzyme. The crucial aspect is the proper choice of type and concentration of detergent in all steps of the procedure. Single crystals can be obtained from concentrated solutions of PGH synthase in the presence of polyethylene glycol 4000 as a precipitant. Crystallization occurs during gas phase equilibration with a concentrated salt solution. The enzyme solution becomes turbid and forms a second liquid phase in which PGH synthase crystals grow up to 0.2 mm in length in the course of days. Manipulation of these crystals is very difficult due to the small volume of the growth phase. The crystals dissolved rapidly in all aqueous media into which they were transferred for mounting in X-ray capillaries. Therefore, we have not yet been able to demonstrate their true X-ray scattering power. A crystal provisionally dry mounted diffracted to about 8 A resolution.