Kinetic & physicochemical properties of Pseudomonas aeruginosa phosphorylcholine phosphatase variants

RAUL GABRIEL FERREYRA; BEASSONI, P. R.; VALERIA A RISSO; MARIO R. ERMACORA; LISA, T. A.; DOMENECH, C. E.

Rosario

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

P. aeruginosa phosphorylcholine phosphatase (PChP) is secreted to the periplasmic space after the cleavage of the signal peptide (sp). The aim of this work was to known in which manner could be able to alter kinetic and physicochemical properties of PChP. Enzymes with (PChP349) and without sp (PChP327) were expressed in E. coli as N-terminal fusion to intein. They were also expressed without intein and purified from inclusion bodies by anion exchange chromatography. The great difference in the catalytic efficiency between PChP327 and PChP349 was produced by an increased Km but not by a change of affinity for both substrates phosphorylcholine and p-NPP in the first binding site. The notably decreased the affinity of the enzyme for the second PCh binding site, increasing Km. Mass spectrometry showed that PChP349 is completely processed to a 327 protein and PChP327 had also the expected MW for the mature protein. N-terminal sequencing data confirmed these findings. CD spectra of PChP349 and PChP327 indicated typical alfa-helix predominance and a well-preserved tertiary structure, indicating that despite sp affects the catalytic properties of PChP, it does not affect its structure. These findings indicate that E. coli is capable to process PChP exactly as P. aeruginosa, and reveals the importance of the secretion pathway to produce an enzyme with high catalytic efficiency.