Mutagenesis and structural analysis reveal the CTX-M β-lactamase active site is optimized for cephalosporin catalysis and drug resistance

CTX-M beta-lactamases are a widespread source of resistance to beta-lactam antibiotics in Gram-negative bacteria. These en-zymes readily hydrolyze penicillins and cephalosporins, including oxyimino-cephalosporins such as cefotaxime. To investigate the preference of CTX-M enzymes for cephalo-sporins, we examined eleven active-site residues in the CTX-M-14 beta-lactamase model system by alanine mutagenesis to assess the contribution of the residues to catalysis and specificity for the hydrolysis of the penicillin, ampicillin, and the cephalo-sporins cephalothin and cefotaxime. Key active site residues for class A beta-lactamases, including Lys73, Ser130, Asn132, Lys234, Thr216, and Thr235, contribute significantly to substrate binding and catalysis of penicillin and cephalosporin substrates in that alanine substitutions decrease both kcat and kcat/KM. A second group of residues, including Asn104, Tyr105, Asn106, Thr215, and Thr216, contribute only to substrate binding, with the substitutions decreasing only kcat/KM. Importantly, calcu-lating the average effect of a substitution across the 11 active-site residues shows that the most significant impact is on cefotaxime hydrolysis while ampicillin hydrolysis is least affected, suggesting the active site is highly optimized for cefotaxime catalysis. Furthermore, we determined X-ray crystal structures for the apo-enzymes of the mutants N106A, S130A, N132A, N170A, T215A, and T235A. Surprisingly, in the structures of some mutants, particularly N106A and T235A, the changes in structure propagate from the site of substitution to other regions of the active site, suggesting that the impact of substitutions is due to more widespread changes in structure and illustrating the interconnected nature of the active site.

Automated summary

CTX-M beta-lactamases are widespread in Gram-negative bacteria and play a significant role in resistance to beta-lactam antibiotics. This study investigates the preference of CTX-M enzymes for cephalosporins through alanine mutagenesis, revealing key active site residues that contribute to catalysis and specificity. The impact of substitutions on the structure of the active site is also examined.