CONICET | Buscador de Institutos y Recursos Humanos

We study the structure/function relationship in proteins of Pseudomonas aeruginosa. Here, the advances with phosphorylcholine phosphatase (PchP) and exopolyphosphatase (PPX) are presented. PchP expression depends on the presence of choline or derivatives in culture media. In the same conditions, also hemolitic Phospholipase C (PlcH) is expressed and secreted to extracellular media. It was suggested that after sequential and coordinated action of PlcH and PchP on phosphatidilcholine (or sphingomielin) and phosphocholine, bacteria can obtain carbon, nitrogen and inorganic phosphate (Pi) to support colonization favoring pathogenesis. Thus, both enzymes can be considered as pharmaceutical targets. PchP belongs to HAD superfamily and contains three characteristics motifs in the active site: 31DMDNT35, 166S and K242/261GDTPDSD267. By site-directed mutagenesis the relevance of those residues was defined. PchP has no close homologues with solved structure. For this reason we have failed in obtain a high resolution model by comparative modeling. A combined approach between threading and comparative modeling techniques was used to obtain a tridimensional model based on atomic coordinates of M. janaschii phosphoserine phosphatase (PDB: 1f5s). Phyre server was used to obtain a sequence/structure alignment and MODELLER was used for comparative modeling. The obtained model was relaxed by molecular dynamics using NAMD software. A model of PchP with high similarity in the active site of HAD members was obtained. The site of interaction with Pi and the cofactor Mg2+ were located. Quaternay ammonium moiety rest to be located, but by comparison with human PHOSPHO1 and 2 we have some candidates with very promising results. Polyphosphates (PPn) have several functions, and besides of act as Pi reservoir, they were involved in pathogenesis and stress response. PPn are sinthetized by polyphosphate kinase (PPK) and hydrolyzed by PPX. PPX is a processive enzyme, Mg2+ and K+ dependent. An homology model of PPX was obtained by comparative modeling using ICM-Pro software based on E. coli PPX (ecPPX) structure (PDB: 1u6z). The final model (paPPX) was relaxed by molecular dynamics in presence of Mg2+ in the active site. N-terminal domain is the catalytic one and C-terminal domain would be the responsible of PPn chain recognition. By site directed mutagenesis and comparison with ecPPX, the active site was proposed. According to sulfate ions binding and electrostatic potential calculations performed in ecPPX it was proposed that PPn might be bound to a positive cleft in the dimeric interfase of this enzyme. The comparison of the electrostatic potential in paPPX, calculated using APBS, indicated that in this latter enzyme the positive cleft is absent. Only the half of the ecPPX potential is shared with paPPX. This is a very interesting point which may suggests that the positive cleft may have another function or paPPX may be an evolved protein loosing part of that function, or on the contrary be an ancestor of other PPX.

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