Distant Site Mutations in Clinical TEM Beta-Lactamase Variants Enhance Non-Covalent Binding to Ceftazidime: Insights from Spectroscopic and Biophysical Investigations

β-lactamases retain the central armamentarium against the β-lactams, resulting in surge of antibiotic resistance, primarily due to the hydrolysis of the amide bond of the four-membered ring. This study aimed to investigate the binding interactions of ceftazidime (CAZ) to TEM β- lactamase variants with distant site mutations isolated from clinical setting to explore the cause of their selection and dissemination due to indiscriminate use of β-lactams. Absorbance and fluorescence spectroscopy along with biophysical experimentation indicated facilitated binding of CAZ to the active site of the mutants than the wild type. The CAZ-TEM β-lactamase mutant interactions were predominantly hydrophobic compared to H-bonding and van der Waals forces in the CAZ-wild type complex. Additionally structural alteration to justify more rigid binding of CAZ to the mutants in contrast to the wild type enzyme was established in silico . Therefore, acquisition of distant site mutations with respect to the active site of the β-lactamase variants that rendered conformational flexibility to accommodate CAZ was evidenced. This study provided an insight to the bioactive interaction of CAZ with TEM β-lactamase variants that probably facilitated their selection from clinical settings in response to rampant usage of β-lactams.