Mutation of the conserved Asp-Asp pair impairs the structure, function, and inhibition of CTX-M Class A β-lactamase

The Asp233-Asp246 pair is highly conserved in Class A beta-lactamases, which hydrolyze beta-lactam antibiotics. Here, we characterize its function using CTX-M-14 beta-lactamase. The D233N mutant displayed decreased activity that is substrate-dependent, with reductions in k(cat)/K-m ranging from 20% for nitrocefin to 6-fold for cefotaxime. In comparison, the mutation reduced the binding of a known reversible inhibitor by 10-fold. The mutant structures showed movement of the 213-219 loop and the loss of the Thr216-Thr235 hydrogen bond, which was restored by inhibitor binding. Mutagenesis of Thr216 further highlighted its contribution to CTX-M activity. These results demonstrate the importance of the aspartate pair to CTX-M hydrolysis of substrates with bulky side chains, while suggesting increased protein flexibility as a means to evolve drug resistance.

Automated summary

The study characterized the function of the Asp233-Asp246 pair in Class A beta-lactamases using CTX-M-14 beta-lactamase. The D233N mutation led to decreased activity depending on the substrate, while also reducing the binding of a reversible inhibitor. Structural analysis revealed changes in loop movement and hydrogen bond loss, highlighting the importance of the aspartate pair in substrate hydrolysis and suggesting increased protein flexibility as a means to evolve drug resistance.