Using a molecular model and kinetic experiments in the presence of divalent cations to study the active site and catalysis of Pseudomonas aeruginosa phosphorylcholine phosphatase (BBAPRO-08-237R1)

BEASSONI, PAOLA RITA; OTERO, LISANDRO HORACIO; LISA, ANGELA TERESA; DOMENECH, CARLOS EDUARDO

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS

ELSEVIER SCIENCE BV

Lugar: New York. ; Año: 2008 vol. 1784 p. 2038 - 2044

1570-9639

Phosphorylcholine phosphatase (PchP) of Pseudomonas aeruginosa, a product of the PA5292 gene, catalyzes the hydrolysis of phosphocholine to choline and inorganic phosphate (Pi). Phosphocholine is produced after hemolytic phospholipase C (PlcH) acts upon phosphatidylcholine or sphingomyelin. Therefore, PlcH and PchP are involved in the pathogenesis of P. aeruginosa. PchP belongs to the HAD superfamily as it contains three conserved sequences motifs. In mature PchP, the motifs I, II, and III are (31)DMDNT(35), (166)S, and (261)GDTPDSD(267), respectively. Kinetic characterization of wild-type and mutated proteins, obtained by site-directed mutagenesis, in addition to a molecular model of PchP helped us to understand the contribution of key residues in the conserved motifs I, II and III that are involved in the catalysis of p-nitrophenylphosphate processing after the addition of Mg(2+), Zn(2+) or Cu(2+) (these are activators of PchP activity). Our results are explained by invoking the concept of chemical hardness and softness introduced by Pearson in 1963 and its extension that "hard acids prefer to coordinate to hard bases and soft acids to soft bases" [Parr and Pearson, J. Am. Chem. Soc., 105, 7512-7516 (1983)]