CONICET | Buscador de Institutos y Recursos Humanos

Pseudomonas aeruginosa is a ubiquitous and opportunistic microorganism with a great ability to adapt itself to different environments. In humans, this bacterium is responsible for urinary, respiratory, corneal and surgical infections [1]. We have proposed a mechanism that would explain the pulmonary infection through the coordinated action of hemolytic phospholipase C on phosphatidylcholine or sphingomyelyn, and on phosphorylcholine. Therefore, P. aeruginosa phosphorylcholine phosphatase (PChP) is a potential pharmaceutical target. PChP catalyzes the hydrolysis of phosphorylcholine in choline and phosphate. This enzyme consists of three conserved motifs that are characteristic of the bacterial haloacid dehalogenases (HAD) hydrolase superfamily [2,3]. The aim of this work was to characterize biophysically and biochemically the properties of P. aeruginosa PChP. 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. Analysis by SEC-FPLC showed a single species with a retention time corresponding to a slightly expanded globular protein with the expect size. CD and fluorescence analysis showed the well preserved secondary and tertiary structure. Protein stability was studied by thermal denaturation between 0 and 95 ºC, monitoring elipticity at 220 nm. This experiment showed that the unfolding process was highly cooperative, with a transition temperature of 52ºC. Crystallization trials were carried out and tetragonal crystals were obtained in both the presence and absence of metals. The results obtained permits to undertake the structural characterization of this protein at atomic resolution and to envisage rational design of inhibitors for PChP.

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