A secondary drug resistance mutation of TEM-1 β-lactamase that suppresses misfolding and aggregation

In Gram-negative bacteria, TEM-1 .beta -lactamase provides the major mechanism of plasmid-mediated .beta -lactam resistance. Natural variants of TEM-1 with increased antibiotic resistance have appeared in response to the use of extended-spectrum .beta -lactam antibiotics (e.g., ceftazidime) and beta -lactamase inhibitors (e.g., clavulanic acid). Some of the variant enzymes are more efficient at catalyzing beta -lactam hydrolysis, whereas others are more resistant to inhibitors. M182T is a substitution observed in both types of variant TEM-1 .beta -lactamases. This mutation is found only in combination with other amino acid substitutions, suggesting that it may correct defects introduced by other mutations that alter the specificity. An engineered core mutation, L76N, which diminishes the periplasmic beta -lactamase activity by 100-fold, was used as a model to understand the mechanism of suppression of the M182T mutation. Biochemical studies of the L76N enzyme alone and in combination with the M182T mutation indicate that the M182T substitution acts at the level of folding but does not affect the thermodynamic stability of TEM-1 beta -lactamase. Thus, the M182T substitution is an example of a naturally occurring mutation that has evolved to alter the folding pathway of a protein and confer a selective advantage during the evolution of drug resistance.