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

Abstract

CTX-M β-lactamases are prevalent antibiotic resistance enzymes and are notable for their ability to rapidly hydrolyze the extended-spectrum cephalosporin, cefotaxime. We hypothesized that the active site sequence requirements of CTX-M-mediated hydrolysis differ between classes of β-lactam antibiotics. Accordingly, we use codon randomization, antibiotic selection, and deep sequencing to determine the CTX-M active-site residues required for hydrolysis of cefotaxime and the penicillin, ampicillin. The study reveals positions required for hydrolysis of all β-lactams, as well as residues controlling substrate specificity. Further, CTX-M enzymes poorly hydrolyze the extended-spectrum cephalosporin, ceftazidime. We further show that the sequence requirements for ceftazidime hydrolysis follow those of cefotaxime, with the exception that key active-site omega loop residues are not required, and may be detrimental, for ceftazidime hydrolysis. These results provide insights into cephalosporin hydrolysis and demonstrate that changes to the active-site omega loop are likely required for the evolution of CTX-M-mediated ceftazidime resistance.

Synergistic effect of mannan extracted from Saccharomyces cerevisiae with antibiotics against multidrug resistant Escherichia coli

“…The results of AST are summarized in ). E. coli develops resistant to cephalosporins upon acquiring Expanded Spectrum Beta-Lactamases (ESBLs) such as TEM-1, TEM-2, and SHV-1 (Yasir et al, 2020), or it may be linked to the production of plasmidic class C βlactamases, such as the CMY enzyme (Judge et al, 2023). Often, trimethoprim and sulfamethoxazole are used together in various quantities depending on the target bacteria, providing synergistic bactericidal activity (Kawahara et al, 2023).…”

Revisiting the hydrolysis of ampicillin catalyzed by Temoneira‐1 β‐lactamase, and the effect of Ni(II), Cd(II) and Hg(II)

“…It is also noticeable that different DNA plasmids (i.e., potentially slightly different TEM‐1 β‐lactamase sequences), and partially different conditions were applied in these studies (see Table 1 ). Nevertheless, using the Michaelis–Menten formalism to treat the data, providing the k cat and K M or the k cat / K M values as kinetic parameters characterizing the enzymatic reaction is still a common procedure (Song and Tezcan, 2014 ; Bahr et al, 2021 ; Judge et al, 2023 ) most probably due to their comparability.…”

Positive epistasis drives clavulanic acid resistance in double mutant libraries of BlaC β-lactamase

“…β-Lactamases have been researched intensively with a focus on evolutionary studies, using both random and semi-rational approaches to screen for the variants with advantageous traits. Various positions in class A β-lactamases were mutated using saturation mutagenesis 38 – 41 , including 105, 130, 132 and 234 42 – 46 . Saturating multiple positions simultaneously was also attempted on three consecutive residues in TEM-1 β-lactamase 47 , 48 , and on two positions in SME-1 β-lactamase which are involved in disulfide bond formation 49 .…”