Kinetic and Structural Characterization of Phosphorylcholine Phosphatase of Pseudomonas aeruginosa

BEASSONI, P. R.; RISSO, V. A.; OTERO, L.H.; FERREYRA, R. G.; LISA, A.T.; DOMENECH, C. E.; ERMÁCORA, M. R.

Montevideo

Congreso; VI International Conference of Biological Physics- ICBP2007, V Southern Cone Biophysics Congress, XXXVI Annual Meeting of the Argentinean Biophysical Society; 2007

International Union of Pure and Applied Physics, International Union for Pure and Applied Biophysics

Pseudomonas aeruginosa is an opportunistic pathogen responsible for acute and chronic nosocomial infections and represents an important problem for patients with severe burns, cystic fibrosis, and inmunocompromising diseases. We proposed a mechanism to explain the pulmonary infection through the coordinated and sequential action of hemolytic phospholipase C and phosphorylcholine phosphatase (PChP). Therefore, PChP may be a potential target enzyme to avoid the pathogenic action of this bacterium. The enzyme belongs to the haloacid dehalogenases (HAD) superfamily characterized by the presence of three motifs: I: 31DMDNT35; II, 166SAA168; and III, 261GDTPDSD The catalytic mechanism involves a nucleophylic attack on the O-P bond of the substrate. To catalyses the hydrolysis of phosphorylcholine, Mg2+ is one of the ions activators of PChP. Saturation curves performed with PCh allowed detect two sites with different affinity, and inhibition by high substrate concentration. Mathematical models to describe this kinetic behaviour have been developed in our laboratories and will be described during the cientific meeting. We are also interested in the structural characterization of this enzyme. For this reason, the recombinant enzyme was expressed in E. coli and purified from inclusion bodies by anion exchange chromatography under denaturing conditions. Pure urea denaturated protein was refolded by dialysis against physiological buffers in the presence or absence of Mg2+. In both conditions refolded PChP was active, but with very different tertiary structure. The enzyme refolded with Mg2+ showed a thermal stability 20 times higher than the refolded without Mg2+. However, its tertiary structure was modified by the addition of Mg2+. Kinetic experiments indicated that PChP contain two interaction sites for Mg2+ with different affinities. The high affinity site was necessary to avoid the inhibition caused by high substrate concentrations. As a consequence of these findings we were able to optimize the crystallization experiments.