Characteristics of n-terminal domain of a PAO1 recombinant polyphosphatase Pseudomonas aeruginosa

BEASSONI, PR; GALLARATO, LA; GARRIDO, MN

Mar del Plata - Bs As

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

In Pseudomonas aeruginosa PAO1 the gene PA5241 encodes a polyphosphate phosphatase (PPX) scarcely studied. To characterize the enzyme it was obtained as a recombinant protein (rPPX) through an N-terminal fusion to intein (IMPACT-CN, NEB). The PM of rPPX was lower than predicted by its DNA sequence. Mass spectrometry and MALDI-TOF analysis demonstrated that rPPX had lost a 192 residues C-terminal fragment after overexpression and purification. The truncated rPPX had optimum activity on a pH range between 8.0-8.4. Mg was essential for its activity and the addition of 50mMKCl increased the activity 300%. Ca2+ ,Mn2+, Co2+ and Cu2+ were less effective than Mg2+ . rPPX hydrolyzed different sizes of polyphosphates (PP , PP , PP and PP ) exhibiting two affinity constants for each one. The catalytic efficiency data demonstrated that rPPX preferred PP over longer chain substrates. Multiple sequence analysis of different PPX exhibited sequence conservation in the N-terminal region. Comparative protein modeling of P. aeruginosa PPX with E. coli PPX (1U6Z) showed highly conserved residues which may be potential contributors to the active site or substrate binding.Our results suggest that the Cterminal domain is necessary to processive hydrolysis and a correct binding of polyphosphates higher than 50 Pi residues, whereas putativePPXactive site is located in N-terminal domain.