Identification of residues critical for catalysis in a class C beta-lactamase by combinatorial scanning mutagenesis.
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.
Goldberg
Shalom D
SD
Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA.
Iannuccilli
William
W
Nguyen
Tuan
T
Ju
Jingyue
J
Cornish
Virginia W
VW
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D019151:Peptide Library; D011487:Protein Conformation; D001618:beta-Lactamases / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
D019151:Peptide Library; D001618:beta-Lactamases