Double deletion mutation induced active site remodelling modulates substrate specificity in KPC-14 β-lactamase.

The emergence of KPC variants with altered substrate profiles poses a major challenge for β-lactam antibiotic therapy. KPC-14, which contains a double deletion of residues G242 and T243 (Y241_ΔGT_A244), exhibits enhanced resistance to ceftazidime and ceftazidime-avibactam while displaying reduced carbapenemase activity. However, the structural basis underlying this altered catalytic behaviour remains poorly understood. In this study, we built upon previous investigations and employed machine learning-driven enhanced sampling molecular dynamics simulations combined with unsupervised deep learning analysis to further investigate the conformational effects of the double deletion in KPC-14. As a result of the double deletion, the Ω-loop, 237-243 loop, and 266-275 loop exhibit increased flexibility in KPC-14 compared with KPC-2. A convolutional variational autoencoder identified distinct conformational landscapes for the two enzymes, with three dominant states observed for KPC-14 and two for KPC-2. Structural analysis revealed that deletion-induced repositioning of Y241 disrupts hydrophobic packing between active site loops and introduces new interaction patterns that reshape the catalytic environment. These rearrangements alter the orientation of S70, destabilise the E166 catalytic network, and modify the positioning of H274. Docking studies further showed that representative KPC-14 conformations more readily accommodate ceftazidime in catalytically competent binding geometries compared with KPC-2. Collectively, these structural changes impact the catalytic geometry required for efficient carbapenem hydrolysis while enlarging the active site cavity and facilitating accommodation of Oxyimino-cephalosporin side chains possessed by substrates such as ceftazidime. These findings provide a mechanistic explanation for the altered substrate specificity of KPC-14 and highlight how subtle structural perturbations can influence the catalytic landscape of class A β-lactamases.