title: In vivo functional and molecular characterization of the Penicillin-Binding Protein 4 (DacB) of Pseudomonas aeruginosa.
abstract: Community and nosocomial infections by Pseudomonas aeruginosa still create a major therapeutic challenge. The resistance of this opportunist pathogen to β-lactam antibiotics is determined mainly by production of the inactivating enzyme AmpC, a class C cephalosporinase with a regulation system more complex than those found in members of the Enterobacteriaceae family. This regulatory system also participates directly in peptidoglycan turnover and recycling. One of the regulatory mechanisms for AmpC expression, recently identified in clinical isolates, is the inactivation of LMM-PBP4 (Low-Molecular-Mass Penicillin-Binding Protein 4), a protein whose catalytic activity on natural substrates has remained uncharacterized until now.
  
  
  
  We carried out in vivo activity trials for LMM-PBP4 of Pseudomonas aeruginosa on macromolecular peptidoglycan of Escherichia coli and Pseudomonas aeruginosa. The results showed a decrease in the relative quantity of dimeric, trimeric and anhydrous units, and a smaller reduction in monomer disaccharide pentapeptide (M5) levels, validating the occurrence of D,D-carboxypeptidase and D,D-endopeptidase activities. Under conditions of induction for this protein and cefoxitin treatment, the reduction in M5 is not fully efficient, implying that LMM-PBP4 of Pseudomonas aeruginosa presents better behaviour as a D,D-endopeptidase. Kinetic evaluation of the direct D,D-peptidase activity of this protein on natural muropeptides M5 and D45 confirmed this bifunctionality and the greater affinity of LMM-PBP4 for its dimeric substrate. A three-dimensional model for the monomeric unit of LMM-PBP4 provided structural information which supports its catalytic performance.
  
  
  
  LMM-PBP4 of Pseudomonas aeruginosa is a bifunctional enzyme presenting both D,D-carboxypeptidase and D,D-endopeptidase activities; the D,D-endopeptidase function is predominant. Our study provides unprecedented functional and structural information which supports the proposal of this protein as a potential hydrolase-autolysin associated with peptidoglycan maturation and recycling. The fact that mutant PBP4 induces AmpC, may indicate that a putative muropeptide-subunit product of the DD-EPase activity of PBP4 could be a negative regulator of the pathway. This data contributes to understanding of the regulatory aspects of resistance to β-lactam antibiotics in this bacterial model.
authors.affiliation: Departamento de Ciencias Preclínicas, Facultad de Medicina, Universidad de La Frontera, Temuco, Chile.|Laboratorio de División Celular Bacteriana y Resistencia a Antibióticos, Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid-CSIC, Madrid, Spain.
authors.forename: Cristian Gustavo
authors.initials: CG
authors.lastname: Aguilera Rossi
authors.affiliation: Grupo de Modelado Molecular, Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid-CSIC, Madrid, Spain.
authors.forename: Paulino
authors.initials: P
authors.lastname: Gómez-Puertas
authors.affiliation: Laboratorio de División Celular Bacteriana y Resistencia a Antibióticos, Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid-CSIC, Madrid, Spain. jayala@cbm.csic.es.
authors.forename: Juan Alfonso
authors.initials: JA
authors.lastname: Ayala Serrano
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002268:Carboxypeptidases / Q000378:metabolism; D002440:Cefoxitin / Q000494:pharmacology; D003428:Cross Infection; D004269:DNA, Bacterial / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D010457:Peptidoglycan / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D018440:beta-Lactam Resistance / Q000235:genetics; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D011994:Recombinant Proteins; D047090:beta-Lactams; D002440:Cefoxitin; D002268:Carboxypeptidases; D010450:Endopeptidases
keywords: Catalytic function; D,D-peptidase; LMM-PBP4; Macromolecular peptidoglycan; Pseudomonas aeruginosa; Purified muropeptides; Three-dimensional structure