Molecular and biochemical studies of a polyphosphate phosphatase activity from Pseudomonas aeruginosa PAO1

GALLARATO, LA; VICARIO, JC; BEASSONI, PR; GARRIDO, MN

Mendoza

Congreso; XXVI Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2008

Sociedad de Biologia de Cuyo

In P. aeruginosa PAO1 the PA5241 gene codifies for a polyphosphate phosphatase (PPX). Bioinformatics analysis of this PPX demonstrated a high homology with the E. coli enzyme. Comparative modelling indicated that P. aeruginosa PPX contains N- and C‑terminal domains. Biochemical studies indicated that the N‑terminal domain is responsible for the enzyme activity. The C‑terminal moiety might be related to the recognition of the length substrate. The overexpression (in E. coli  as N terminal fusion to 6xHis-tag, purified by affinity on Ni-agarose columns, and posterior thrombin 6xHis removal) of the full-length PPX (PPXfl), and the N(1-314) and C(314-506)  terminal domains led us to know some properties of the full enzyme and of the polypeptide N(1-314).  The kinetic studies demonstrated that the enzyme activity was dependent on Mg2+ (KA   5 mM), and was non-essential but cooperatively activated by K+  (n Hill  2; K0.5  15 mM). The KM values decreased, and the catalytic efficiency increased with the length of the polyphosphate chain (E.g., for polyphosphates 25, 45, 65 and 75 the KM values were approximately 11 µM, 7 µM, 4 µM and 1.5 µM, respectively). Site directed mutagenesis indicated that the E126, D149, G151, S154 and E156 residues were essential for the enzyme activity.