Using a molecular model and kinetic experiments in the presence of divalent cations to study the active site and catalysis of Pseudomonas aeruginosa phosphorylcholine phosphatase.
Phosphorylcholine phosphatase (PchP) of Pseudomonas aeruginosa, a product of the PA5292 gene, catalyzes the hydrolysis of phosphocholine to choline and inorganic phosphate (Pi). Phosphocholine is produced after hemolytic phospholipase C (PlcH) acts upon phosphatidylcholine or sphingomyelin. Therefore, PlcH and PchP are involved in the pathogenesis of P. aeruginosa. PchP belongs to the HAD superfamily as it contains three conserved sequences motifs. In mature PchP, the motifs I, II, and III are (31)DMDNT(35), (166)S, and (261)GDTPDSD(267), respectively. Kinetic characterization of wild-type and mutated proteins, obtained by site-directed mutagenesis, in addition to a molecular model of PchP helped us to understand the contribution of key residues in the conserved motifs I, II and III that are involved in the catalysis of p-nitrophenylphosphate processing after the addition of Mg(2+), Zn(2+) or Cu(2+) (these are activators of PchP activity). Our results are explained by invoking the concept of chemical hardness and softness introduced by Pearson in 1963 and its extension that "hard acids prefer to coordinate to hard bases and soft acids to soft bases" [Parr and Pearson, J. Am. Chem. Soc., 105, 7512-7516 (1983)].
Beassoni
Paola R
PR
Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, 5800 Río Cuarto, Córdoba, Argentina.
Otero
Lisandro H
LH
Lisa
Angela T
AT
Domenech
Carlos E
CE
D020816:Amino Acid Motifs / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D006868:Hydrolysis; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010767:Phosphorylcholine / Q000737:chemistry / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D008670:Metals; D010767:Phosphorylcholine; D017855:Transferases (Other Substituted Phosphate Groups); C477519:sphingomyelin synthase, Pseudomonas aeruginosa; D010744:Phosphoric Monoester Hydrolases