Kinetic, molecular and In silico approaches shows two interaction sites for phosphorylcholine in Pseudomonas aeruginosa phosphorylcholine phosphatase.

BEASSONI, PR; BOETSCH, C; OTERO, LH; LISA, AT; DOMENECH, CE

Cordoba

Congreso; Segundo Congreso Argentino de Bioinformática; 2011

A2B2C Asociación Argentina de Bioinformática y Biología Computacional

Background: Phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pch) to produce choline and inorganic phosphate. This protein acts in a coordinated and sequential manner with hemolytic phospholipase C, and both proteins are involved in the colonization and pathogenesis of these bacteria. PchP belongs to the haloacid dehalogenase superfamily (HAD) superfamily and possesses the three characteristic motifs of this family: motif I, 31D and 33D; motif II, 166S; and motif III, 242K, 261G, 262D and 267D, which fold to form the catalytic site which binds the metal ion and the phosphate moiety of phosphorylcholine. Based on comparisons to the human enzymes PHOSPHO1, a phosphocholine/phosphoetanolamine phosphatase also belonging to the HAD superfamily, PHOSPHO2 and choline binding proteins of Gram (+) bacteria, we selected residues E42, E43 and the aromatic triplet 82YYY84 for an interaction study with phosphorylcholine and p-nitrophenylphosphate, which are both substrates of PchP. Results: Using a comprehensive approach including homology modeling, site-directed mutagenesis, kinetic and docking studies, we show the possible conformations of binding for both substrates (Figure 1) and we show that the residues mentioned above are involved in the non-phosphate moiety of these substrates. Furthermore, we demonstrate that hPHOSPHO1 and PchP show highly significant differences, despite the functional similarity between them, and that both belong to the HAD superfamily. Conclusions: We propose a model of the PchP active site with two molecules of Pch docked, providing an explanation for the inhibition caused by this substrate in high concentrations (Figure 1B) In silico mutagenesis plus molecular dynamics resulted in a rearrangement of the active site that altered the size and shape of the binding pocket, and a second substrate molecule would be unable to bind, which explains the lack of inhibition by high concentrations of substrate in vitro mutants.