Characterization of the global stabilizing substitution A77V and its role in the evolution of CTX-M β-lactamases.
The widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of β-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active site-associated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 β-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extended-spectrum β-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of β-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levels in vivo and protein stability in vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in β-lactamase evolution and a common mechanism in protein evolution.
Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA.
Meha P
MP
Patel
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.
Bartlomiej G
BG
Fryszczyn
Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA Department of Pharmacology, Baylor College of Medicine, Houston, Texas, USA timothyp@bcm.edu.
Timothy
T
Palzkill
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002442:Ceftazidime / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D019143:Evolution, Molecular; D009154:Mutation; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D002442:Ceftazidime; D001618:beta-Lactamases