Catalytic properties of Pseudomonas aeruginosa phosphorylcholine phosphatase overexpressed in Escherichia coli

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

Villa Carlos Paz, Córdoba

Congreso; XXXVIII Reunión Anual, Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2002

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

P. aeruginosa produces phosphorylcholine phosphatase when grown on choline, betaine or dimethylglycine as carbon or nitrogen source. By a transposon mutagenesis the gene responsible for the production of this enzyme was located in its genome. The pcP gene was cloned and overexpressed in E. coli using the IMPACTTM system (BioLabs). The PcP protein was obtained after self-cleaving of the overexpressed intein-PcP recombinant protein bound to a chitin affinity column. Measured at pH 5, the Km for p-nitrophenylphosphate (p-NPP) and the KA values for Mg2+ were 2 mM and 1.2 mM, respectively. The inhibition by Al3+ ion was cooperative with a Hill coefficient (napp) of 3.2 and a Kapp of 0.24 mM. By measuring the enzyme activity with p-NPP and utilizing phosphorylcholine (PC) as inhibitor, two interaction sites (napp= 2) for PC were detected. It was confirmed with PC as substrate. The cationic compounds choline and betaine produced hyperbolic mixed and linear mixed inhibition, respectively. These data were consistent with previous studies observed with the purified enzyme from P. aeruginosa. Nevertheless, some catalytic properties of the overexpressed phosphatase measured with PC were different. Therefore, these data indicated that the expression-cloning strategies may fail in achieving the expression of an entirely functional and equivalent enzyme in a heterologous host.