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

Abstract

The Asp233‐Asp246 pair is highly conserved in Class A β‐lactamases, which hydrolyze β‐lactam antibiotics. Here, we characterize its function using CTX‐M‐14 β‐lactamase. The D233N mutant displayed decreased activity that is substrate‐dependent, with reductions in kcat/Km 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.

Toward physics‐based precision medicine: Exploiting protein dynamics to design new therapeutics and interpret variants

“…Pathogenic variants that diminish protein function will likely require different drug design strategies than variants that enhance protein function. Hence, new (Adamski et al, 2015;Kemp et al, 2021;Lu et al, 2023). Root mean square deviation of each mutant is given with respect to wildtype CTX-M-14.…”

“…The CTX‐M β‐lactamases mediate antibiotic resistance by hydrolyzing β‐lactam antibiotics (Brown et al, 2020 ). Because many CTX‐M clinical variants confer higher catalytic efficiency or protein stability, substantial effort was dedicated to solving their crystal structures (Adamski et al, 2015 ; Chen et al, 2005 ; Kemp et al, 2021 ; Lu et al, 2023 ). As is seen in Figure 1a , these CTX‐M variants have highly similar crystal structures with a backbone RMSD of <1 Å.…”

“…Bound cefotaxime substrate is shown in blue. (b) Comparison of catalytic rate ( k cat ) of ampicillin hydrolysis among variants depicted in (a) (Adamski et al, 2015 ; Kemp et al, 2021 ; Lu et al, 2023 ). Root mean square deviation of each mutant is given with respect to wildtype CTX‐M‐14.…”

Mapping the determinants of catalysis and substrate specificity of the antibiotic resistance enzyme CTX-M β-lactamase

“…The enzyme used in this study, CTX-M-14, is a member of the CTX-M-9 subgroup. CTX-M-14 has served as a model system for studies of CTX-M enzyme structure and mechanism [10][11][12] . CTX-M enzymes, like other class A enzymes, utilize a catalytic serine residue (Ser70) to attack the carbonyl carbon of the β-lactam ring, leading to the formation of a covalent, acyl-enzyme intermediate 13,14 .…”