title: Site-directed mutations and kinetic studies show key residues involved in alkylammonium interactions and reveal two sites for phosphorylcholine in Pseudomonas aeruginosa phosphorylcholine phosphatase.
abstract: Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pcho) to produce choline and inorganic phosphate. PchP belongs to the haloacid dehalogenase superfamily (HAD) and possesses the three characteristic motifs of this family: motif I ((31)D and (33)D), motif II ((166)S), and motif III ((242)K, (261)G, (262)D and (267)D), which fold to form the catalytic site that binds the metal ion and the phosphate moiety of Pcho. Based on comparisons to the PHOSPHO1 and PHOSPHO2 human enzymes and the choline-binding proteins of Gram-(+) bacteria, we selected residues (42)E and (43)E and the aromatic triplet (82)YYY(84) for site-directed mutagenesis to study the interactions with Pcho and p-nitrophenylphosphate as substrates of PchP. Because mutations in (42)E, (43)E and the three tyrosine residues affect both the substrate affinity and the inhibitory effect produced by high Pcho concentrations, we postulate that two sites, one catalytic and one inhibitory, are present in PchP and that they are adjacent and share residues.
authors.affiliation: Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, X5800BYA Río Cuarto, Córdoba, Argentina. pbeassoni@exa.unrc.edu.ar
authors.forename: Paola R
authors.initials: PR
authors.lastname: Beassoni
authors.forename: Lisandro H
authors.initials: LH
authors.lastname: Otero
authors.forename: Cristhian
authors.initials: C
authors.lastname: Boetsch
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Domenech
authors.forename: Fernado D
authors.initials: FD
authors.lastname: González-Nilo
authors.forename: Angela T
authors.initials: AT
authors.lastname: Lisa
mesh_terms: D000473:Alkanes / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010767:Phosphorylcholine / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D000473:Alkanes; D001426:Bacterial Proteins; D000644:Quaternary Ammonium Compounds; D010767:Phosphorylcholine; D010744:Phosphoric Monoester Hydrolases