| Autor: |
Goldberg, Shalom D., Iannuccilli, William, Nguyen, Tuan, Ju, Jingyue, Cornish, Virginia W. |
| Zdroj: |
Protein Science: A Publication of the Protein Society; 2003, Vol. 12 Issue 8, p1633-1645, 13p |
| Abstrakt: |
Despite their clinical importance, the mechanism of action of the class C β-lactamases is poorly understood. In contrast to the class A and class D β-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 β-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 β-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 β-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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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