Structure and Function of Pseudomonas aeruginosa Phosphorylcholine Phosphatase

BEASSONI, PR; LISA, AT; DOMENECH, CE

Villa General Belgrano, Córdoba

Workshop; I Argentine Workshop on Current Topics in Pseudomonas and Burkholderia Research; 2007

Universidad Nacional de Río Cuarto

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) catalyses the hydrolysis of phosphorylcholine (PCh), it contains two sites with different affinities for PCh, and is inhibited by high substrate concentration. The high affinity site is involved in the binding of the phosphate moiety; the low affinity site might recognize the alkylammonium moiety. Kinetic studies with PChP with and without the signal peptide indicated that it was the responsible for the decrease of catalytic efficiency and also increased the KM value of the low affinity site for PCh. The expression of PChP as untagged protein showed that it is processed by E. coli  as occurs in P. aeruginosa. Therefore, the processing mechanism and secretion pathway is shared by both bacteria and is important to produce an enzyme with higher catalytic efficiency. PChP belongs to the haloacid dehalogenase superfamily (HAD) and contains three characteristics motifs in positions 31DMDNT35, 166SAA168 and K242/261GDTPDSD267, respectively. Site-directed mutagenesis on some aminoacyl residues in motifs I, II, and III revealed that PChP shares a similar catalytic mechanism with all enzymes belonging to this superfamily. The aspartyl residue D31, acts as a nucleophile and it is phosphorylated during the phosphoryl transfer reaction. The seryl residue S166 orientates the substrate making the nucleophilic attack possible. The positive charged residue of lysine, K242, neutralises the charge of the phosphorylated intermediate of the enzyme. Finally, D262 and D267 plus D31 and D33 are involved in the coordination of Mg2+, which is required for enzyme activity. The recombinant mature PChP, is a stable globular monomer of 37,2 KDa with high a-helix content, and has a defined well preserved tertiary structure  that is modified by interaction with choline or Mg2+.