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Functional Characterization of Penicillin-Binding Protein 1b from Streptococcus pneumoniae
- Publication Year :
- 2003
- Publisher :
- American Society for Microbiology, 2003.
-
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 β-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.
- Subjects :
- Electrophoresis
Cefotaxime
medicine.drug_class
Proteolysis
Antibiotics
Molecular Sequence Data
Oligosaccharides
Biology
Muramoylpentapeptide Carboxypeptidase
medicine.disease_cause
Microbiology
Benzylpenicillin
Fluorescence
Bacterial Proteins
Multienzyme Complexes
Glycosyltransferase
Streptococcus pneumoniae
medicine
Penicillin-Binding Proteins
Fluorometry
Trypsin
Amino Acid Sequence
Molecular Biology
Lipid II
medicine.diagnostic_test
Base Sequence
Pathogenic bacteria
Penicillin G
Molecular biology
Enzymes and Proteins
Uridine Diphosphate N-Acetylmuramic Acid
Anti-Bacterial Agents
Protein Structure, Tertiary
Kinetics
Biochemistry
Hexosyltransferases
Peptidyl Transferases
biology.protein
Carrier Proteins
medicine.drug
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....a7113d2b842b713158b06d0613615dc7