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Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase.

Beassoni PR, Gallarato LA, Boetsch C, Garrido MN, Lisa AT - Enzyme Res (2015)

Bottom Line: In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa.The present study was aimed at performing the biochemical characterization of this enzyme.In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, FCEFQyN, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, Río Cuarto, 5800 Córdoba, Argentina.

ABSTRACT
Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn-1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) NH4 (+) is an activator of the enzyme and may function at concentrations lower than those of K(+); (iii) Zn(2+) is also an activator of paPpx and may substitute Mg(2+) in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg(2+) and capable of producing ATP regardless of the presence or absence of K(+) or NH4 (+) ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity.

No MeSH data available.


Related in: MedlinePlus

Kinetic characterization of paPpx(1–506) and N-paPpx(1–314). KM(app) (■) and catalytic efficiency (kcat/KM) (□) of paPpx (a) and N-paPpx(1–314) (b) for polyP of different chain length. Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with Mg2+ 5 mM and 80 mM K+. (c) Saturation curves of paPpx(1–506) (■) and N-paPpx(1–314) (□) with Mg2+. Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with polyP658 μM and 80 mM K+. (d) Saturation curves of paPpx with the monovalent ions K+ (○) and NH4+ (●). Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with Mg2+ 5 mM and polyP658 μM.
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fig1: Kinetic characterization of paPpx(1–506) and N-paPpx(1–314). KM(app) (■) and catalytic efficiency (kcat/KM) (□) of paPpx (a) and N-paPpx(1–314) (b) for polyP of different chain length. Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with Mg2+ 5 mM and 80 mM K+. (c) Saturation curves of paPpx(1–506) (■) and N-paPpx(1–314) (□) with Mg2+. Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with polyP658 μM and 80 mM K+. (d) Saturation curves of paPpx with the monovalent ions K+ (○) and NH4+ (●). Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with Mg2+ 5 mM and polyP658 μM.

Mentions: The kinetic behavior evaluated and apparent catalytic constants obtained for paPpx(1–506) and N-paPpx(1–314) are displayed in Figure 1 and summarized in Table 2. With saturating Mg2+ and K+ concentrations, paPpx(1–506) increased its affinity for polyP in the following order: polyP25 < polyP45 < polyP65 < polyP75. The decreasing KM(app) values were accompanied by a sharp rise in the catalytic efficiencies, with this behavior being more noticeable with the increment in the length of the substrate chain (Figure 1(a)). This trend did not occur with N-paPpx since both KM(app) and catalytic efficiency were similar independently of the length of the polyP chain (Figure 1(b), Table 2). Indeed, the analysis of the results obtained with polyP75 demonstrated that the full-length enzyme presented KM(app) that was approximately 23-fold lower than the one of N-paPpx(1–314) (1.30 ± 0.05 versus 30.67 ± 0.57 μM, respectively). This behavior was partially reversed by an experiment of complementation, where a mixture of N-paPpx(1–314)/C-paPpx(315–506), in a 1 : 10 ratio, produced an active enzyme with higher affinity for the substrate polyP75. The KM(app) value measured in the mix was 11 ± 2 μM, which represented an increase of ≈2.3-fold.


Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase.

Beassoni PR, Gallarato LA, Boetsch C, Garrido MN, Lisa AT - Enzyme Res (2015)

Kinetic characterization of paPpx(1–506) and N-paPpx(1–314). KM(app) (■) and catalytic efficiency (kcat/KM) (□) of paPpx (a) and N-paPpx(1–314) (b) for polyP of different chain length. Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with Mg2+ 5 mM and 80 mM K+. (c) Saturation curves of paPpx(1–506) (■) and N-paPpx(1–314) (□) with Mg2+. Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with polyP658 μM and 80 mM K+. (d) Saturation curves of paPpx with the monovalent ions K+ (○) and NH4+ (●). Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with Mg2+ 5 mM and polyP658 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4631893&req=5

fig1: Kinetic characterization of paPpx(1–506) and N-paPpx(1–314). KM(app) (■) and catalytic efficiency (kcat/KM) (□) of paPpx (a) and N-paPpx(1–314) (b) for polyP of different chain length. Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with Mg2+ 5 mM and 80 mM K+. (c) Saturation curves of paPpx(1–506) (■) and N-paPpx(1–314) (□) with Mg2+. Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with polyP658 μM and 80 mM K+. (d) Saturation curves of paPpx with the monovalent ions K+ (○) and NH4+ (●). Enzyme activity was measured in Tris-HCl buffer, pH 8.0, with Mg2+ 5 mM and polyP658 μM.
Mentions: The kinetic behavior evaluated and apparent catalytic constants obtained for paPpx(1–506) and N-paPpx(1–314) are displayed in Figure 1 and summarized in Table 2. With saturating Mg2+ and K+ concentrations, paPpx(1–506) increased its affinity for polyP in the following order: polyP25 < polyP45 < polyP65 < polyP75. The decreasing KM(app) values were accompanied by a sharp rise in the catalytic efficiencies, with this behavior being more noticeable with the increment in the length of the substrate chain (Figure 1(a)). This trend did not occur with N-paPpx since both KM(app) and catalytic efficiency were similar independently of the length of the polyP chain (Figure 1(b), Table 2). Indeed, the analysis of the results obtained with polyP75 demonstrated that the full-length enzyme presented KM(app) that was approximately 23-fold lower than the one of N-paPpx(1–314) (1.30 ± 0.05 versus 30.67 ± 0.57 μM, respectively). This behavior was partially reversed by an experiment of complementation, where a mixture of N-paPpx(1–314)/C-paPpx(315–506), in a 1 : 10 ratio, produced an active enzyme with higher affinity for the substrate polyP75. The KM(app) value measured in the mix was 11 ± 2 μM, which represented an increase of ≈2.3-fold.

Bottom Line: In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa.The present study was aimed at performing the biochemical characterization of this enzyme.In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, FCEFQyN, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, Río Cuarto, 5800 Córdoba, Argentina.

ABSTRACT
Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn-1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) NH4 (+) is an activator of the enzyme and may function at concentrations lower than those of K(+); (iii) Zn(2+) is also an activator of paPpx and may substitute Mg(2+) in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg(2+) and capable of producing ATP regardless of the presence or absence of K(+) or NH4 (+) ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity.

No MeSH data available.


Related in: MedlinePlus