title: Critical active-site residues identified by site-directed mutagenesis in Pseudomonas aeruginosa phosphorylcholine phosphatase, a new member of the haloacid dehalogenases hydrolase superfamily.
abstract: Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP), the product of the PA5292 gene, is synthesized when the bacteria are grown with choline, betaine, dimethylglycine, or carnitine. In the presence of Mg(2+), PChP catalyzes the hydrolysis of both phosphorylcholine (PCh) and p-nitrophenylphosphate (p-NPP). PCh saturation curve analysis of the enzyme with or without the signal peptide indicated that the peptide was the fundamental factor responsible for decreasing the affinity of the second site of PChP for PCh, either at pH 5.0 or pH 7.4. PChP contained three conserved motifs characteristic of the haloacid dehalogenases superfamily. In the PChP without the signal peptide, motifs I, II, and III correspond to the residues (31)DMDNT(35), (166)SAA(168), and K(242)/(261)GDTPDSD(267), respectively. To determine the catalytic importance of the D31, D33, T35, S166, K242, D262, D265, and D267 on the enzyme activity, site-directed mutagenesis was performed. D31, D33, D262, and D267 were identified as the more important residues for catalysis. D265 and D267 may be involved in the stabilization of motif III, or might contribute to substrate specificity. The substitution of T35 by S35 resulted in an enzyme with a low PChP activity, but conserves the catalytic sites involved in the hydrolysis of PCh (K(m1) 0.03 mM: , K(m2) 0.5 mM: ) or p-NPP (K(m) 2.1 mM: ). Mutating either S166 or K242 revealed that these residues are also important to catalyze the hydrolysis of both substrates. The substitution of lysine by arginine or by glutamine revealed the importance of the positive charged group, either from the amino or guanidinium groups, because K242Q was inactive, whereas K242R was a functional enzyme.
authors.affiliation: Biologia Molecular, Universidad Nacional de Rio Cuarto, km 601, Ruta 36, Rio Cuarto, 5800, Argentina.
authors.forename: Paola R
authors.initials: PR
authors.lastname: Beassoni
authors.forename: Lisandro H
authors.initials: LH
authors.lastname: Otero
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Massimelli
authors.forename: Angela T
authors.initials: AT
authors.lastname: Lisa
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Domenech
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics*; D021382:Protein Sorting Signals; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics
chemical_list: D021382:Protein Sorting Signals; D010744:Phosphoric Monoester Hydrolases