Phosphorylcholine phosphatase from Pseudomonas aeruginosa. Some key residues in involved in substrate interaction

BOETSCH, C; VERGARA-JAQUE, A; OTERO, LH; GONZALEZ-NILO; BEASSONI, PR

Termas de Chillan, Chile

Congreso; The first international conference on Bioinformatics SOIBIO 2010; 2010

Sociedad Iberoamericana de Bioinformatica

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pch) to produce choline and inorganic phosphate and presents a distinct inhibition by high substrate concentration. Acting jointly with haemolitic phospholipase C, it is involved in colonization and pathogenesis. PchP belongs to the HAD superfamily and posses the 3 characteristic motifs forming the active site [1].D31 is the nucleophilic residue acting on the O-P bond in the substrate, S166 orients the substrate, K242 stabilizes the phosphorylated intermediate of the enzyme and D33, D262, D267 together with D31 forms the Mg2+ pocket [2]. Nothing is known about the interaction with the non-phosphate moiety of the substrate.PchP was modeled combining protein fold recognition and comparative modeling by satisfaction of spatial restraints. Based on the comparison with human enzymes PHOSPHO1, a phsohocholine/phosphoetanolamine phosphatase also belonging to the HAD superfamily, PHOSPHO2 and Choline Binding Proteins of Gram (+) bacteria we selected the residues E42, E43 and the aromatic triplet 82YYY84 for study.Using a global approach including site-directed mutagenesis, kinetic and docking studies we describe an extended active site including E42, E43 and Y84. Alanine substitutions in those residues abolish the inhibition caused by high substrate concentration. We present a model including two molecules of substrate docked in the active site of wt PchP. In silico mutations affect the size and shape of the pocket which would prevent the entrance of a second molecule of substrate explaining the resistance to substrate inhibition in mutants.