Site-directed mutagenesis of Pseudomonas aeruginosa phosphorylcholine phosphatase gene expressed in E. coli

BEASSONI, PR; MASSIMELLI, MJ; FORRELLAD, MA; BARRA, JL; GARRIDO, MN; LISA, AT; DOMENECH, CE

Iguazú, Misiones

Congreso; XL Reunión Anual, Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2004

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

P. aeruginosa phosphorylcholine phosphatase, PChP, is synthesized when the bacteria are grown with choline or its derivatives. It catalyzes the hydrolysis of phosphorylcholine in choline and Pi. The gene PA5292 (pchP), responsible for the synthesis of PChP, was expressed in E. coli ER2566 and site-directed mutagenesis was carried out using a QuickChange XL Kit (Stratagene). DNA sequences of mutant plasmid were determined to confirm the mutation points. The expressed self-cleavage PChP‑Intein tag proteins were purified by a chitin-binding column (IMPACT-CN, New England BioLabs). Bioinformatic data revealed that PChP contained three conserved motifs characteristic of the bacterial HAD hydrolase superfamily. A totally conserved motif I, and two other less well conserved motifs, II and III are found in the residues 53-57, 188-190 and 283‑288, respectively. Motif I has affinity for divalent ions as Mg2+. Conservative mutants D53E, D55E and T57S and nonconservative D53A, D55A and T57A in motif I led to a complete loss of PChP activity. The gram positive bacterial choline binding domain, GW(V/L)(K/Q)D(N/K)(G/D)TWYYL (N/D)S(S/D)G (A/S)MAT, was not found in PChP, but probably the 104YYY106 or 149TYY151residues from PChP may be involved in the binding of choline.