Critical active-site residues in P. aeruginosa phosphorylcholine phosphatase

BEASSONI, PR; OTERO, LH; MASSIMELLI, MJ; LISA, AT; DOMENECH, CE

Rosario, Argentina

Congreso; XLII Reunión Anual, Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is synthesized when the bacteria are grown with choline or metabolic derivatives. With Mg2+, PChP catalyses the hydrolysis of both phosphorylcholine (PCh) and p‑nitrophenylphosphate (p-NPP). PChP contained three conserved motifs characteristic of the haloacid dehalogenases (HAD) superfamily. In PChP, motifs I, II and III correspond to the residues 31DMDNT35, 166SAA168 and K242/261GDTPDSD267, respectively. The catalytic importance of the conserved residues in these motifs on the enzyme activity was analyzed by site-directed mutagenesis. The substitution of D31 and D33 by glutamate caused a complete loss of activity whereas substitution of D262 and D265 by glutamates caused depletion in Kcat of two-three orders of magnitude, but conserves the catalytic sites involved in the hydrolysis of PCh or p‑NPP. S166 is also important to catalyze the hydrolysis of both substrates. The substitution of lysine (K242) by arginine or glutamine revealed the importance of the positive charged group, either from the amino or guanidinium groups. Therefore, D31 may be the nucleophile that gets phosphorylated during phosphoryl transfer reaction. The phosphorylated intermediate may be stabilized by D33. In motif III, D265 and D267 may be involved in coordination of cofactor Mg2+ together with D31 and D33 of motif I forming the Mg2+ binding pocket.