PMID:12876313
| Citation |
Goldberg, SD, Iannuccilli, W, Nguyen, T, Ju, J and Cornish, VW (2003) Identification of residues critical for catalysis in a class C beta-lactamase by combinatorial scanning mutagenesis. Protein Sci. 12:1633-45 |
|---|---|
| Abstract |
Despite their clinical importance, the mechanism of action of the class C beta-lactamases is poorly understood. In contrast to the class A and class D beta-lactamases, which contain a glutamate residue and a carbamylated lysine in their respective active sites that are thought to serve as general base catalysts for beta-lactam hydrolysis, the mechanism of activation of the serine and water nucleophiles in the class C enzymes is unclear. To probe for residues involved in catalysis, the class C beta-lactamase from Enterobacter cloacae P99 was studied by combinatorial scanning mutagenesis at 122 positions in and around the active site. Over 1000 P99 variants were screened for activity in a high-throughput in vivo antibiotic resistance assay and sequenced by 96-capillary electrophoresis to identify residues that are important for catalysis. P99 mutants showing reduced capability to convey antibiotic resistance were purified and characterized in vitro. The screen identified an active-site hydrogen-bonding network that is key to catalysis. A second cluster of residues was identified that likely plays a structural role in the enzyme. Otherwise, residues not directly contacting the substrate showed tolerance to substitution. The study lends support to the notion that the class C beta-lactamases do not have a single residue that acts as the catalytic general base. Rather, catalysis is affected by a hydrogen-bonding network in the active site, suggesting a possible charge relay system. |
| Links |
PubMed PMC2323950 Online version:10.1110/ps.0302903 |
| Keywords |
Base Sequence; Binding Sites; Catalysis; Drug Resistance, Bacterial/genetics; Escherichia coli/genetics; Hydrolysis; Kinetics; Models, Molecular; Mutagenesis, Site-Directed/genetics; Peptide Library; Protein Conformation; beta-Lactamases/chemistry; beta-Lactamases/classification; beta-Lactamases/genetics; beta-Lactamases/metabolism |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.