Phosphorylcholine Phosphatase of Pseudomonas aeruginosa: structural Insights into Inhibition Site

BOETSCH, C; BUSTOS GUAJARDO, D; ALZATE MORALES, J; VERGARA-JAQUE, A; LISA, AT; BEASSONI, PR

Rosario

Congreso; 50th. Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Phosphorylcholine phosphatase (PchP) has been involved in the pathogenesis of Pseudomonas aeruginosa, hydrolyzing the substrate phosphorylcholine (Pcho) producing choline and phosphate. PchP exhibits distinctive inhibition behavior caused by high substrate concentrations that has never been reported for another Pcho hydrolyzing enzyme. We proposed the existence of two sites for Pcho: an active one and other for inhibition, based on the recently published crystallographic structure of PchP plus kinetic data. Docking and molecular dynamics simulations were employed to analyze the binding of Pcho to the protein and characterize the different binding sites. Free Energy Perturbation method (FEP) was used to evaluate the binding relative free energy (Gbind) of Pcho by native PchP and E42A mutant. The results confirmed the existence of a second binding pocket in PchP, which is adjacent to the catalytic site and acting as an inhibitory site. The simulations revealed the interactions which determine the binding of Pcho molecules within both sites. Finally, the energetic and structural analysis performed on the E42A mutant showed that 42E residue is key in the affinity of Pcho for the inhibition site, and provides relevant information to explain the access of this substrate to the enzyme.