Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.
Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-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 beta-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 sight 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.
Petrosino
J F
JF
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
Palzkill
T
T
D000595:Amino Acid Sequence; D000667:Ampicillin / Q000494:pharmacology; D000668:Ampicillin Resistance / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002442:Ceftazidime / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010406:Penicillins / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
D002511:Cephalosporins; D004269:DNA, Bacterial; D010406:Penicillins; D000667:Ampicillin; D002442:Ceftazidime; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1