title: Functional characterization of penicillin-binding protein 1b from Streptococcus pneumoniae.
abstract: The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by beta-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.
authors.affiliation: Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, UJF, UMR5075, 38027 Grenoble cedex 1, France.
authors.forename: Anne Marie
authors.initials: AM
authors.lastname: Di Guilmi
authors.forename: Andréa
authors.initials: A
authors.lastname: Dessen
authors.forename: Otto
authors.initials: O
authors.lastname: Dideberg
authors.forename: Thierry
authors.initials: T
authors.lastname: Vernet
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins; D002439:Cefotaxime / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D006602:Hexosyltransferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002267:Muramoylpentapeptide Carboxypeptidase; D009844:Oligosaccharides / Q000378:metabolism / Q000494:pharmacology; D010400:Penicillin G / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013296:Streptococcus pneumoniae / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D009844:Oligosaccharides; D046915:Penicillin-Binding Proteins; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D014357:Trypsin; D002439:Cefotaxime; C419802:moenomycin; D010400:Penicillin G