Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase

Abstract

␤-Lactamase is a bacterial protein that provides resistance against ␤-lactam antibiotics. TEM-1 ␤-lactamase is the most prevalent plasmid-mediated ␤-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant ␤-lactamases have evolved with activity toward a variety of ␤-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant ␤-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 ␤-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 ␤-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active site study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants.

Contribution of Natural Amino Acid Substitutions in SHV Extended-Spectrum β-Lactamases to Resistance against Various β-Lactams

“…D179 and R164 form a salt bridge and are the anchor points of the omega loop that contains the catalytically important E166 (20). Disruption of this salt bridge leads to extension of the substrate profile (2,28). For unknown reasons, many natural TEM ESBLs are altered in position 164 (22), but none are altered in position 179, while in SHV ESBLs the opposite is true.…”

Extended-Spectrum β-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance Threat

“…It is interesting that laboratory mutants of TEM-1 that contain mutations at positions other than the ones described in nature have been constructed (18,130,180,182). It has been suggested that the naturally occurring TEM-type ESBLs are the result of fluctuating selective pressure from several ␤-lactams within a given institution rather than selection with a single agent (18).…”

Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis

“…The agar plates containing 1 mg/ml ampicillin were used to select clones from the library that provide wild-type levels of ampicillin resistance because this is the highest concentration of ampicillin on which an E. coli cell containing the TEM-1 plasmid can grow (33,34). The plates were then incubated at 37°C overnight.…”