Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.
Department of Molecular Biophysics and Integrated Bioimaging, Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Commun Biol. 2023 Jan 12;6(1):35. doi: 10.1038/s42003-023-04422-z.
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.
CTX-Mβ-内酰胺酶是普遍存在的抗生素耐药酶，其特点是能够迅速水解广谱头孢菌素头孢噻肟。我们假设 CTX-M 介导的水解的活性位点序列要求在β-内酰胺抗生素的类别之间存在差异。因此，我们使用密码子随机化、抗生素选择和深度测序来确定 CTX-M 活性位点残基水解头孢噻肟和青霉素、氨苄西林所必需的。该研究揭示了所有β-内酰胺水解所需的位置，以及控制底物特异性的残基。此外，CTX-M 酶对广谱头孢菌素头孢他啶的水解能力较差。我们进一步表明，头孢他啶水解的序列要求遵循头孢噻肟的要求，除了关键的活性位点ω环残基不需要，并且可能不利于头孢他啶水解。这些结果提供了对头孢菌素水解的深入了解，并表明活性位点ω环的变化可能是 CTX-M 介导的头孢他啶耐药进化所必需的。
