Evolutionary study of metallo-β-lactamase IMP-1 reveals a specific allosteric network mediated by distal mutations

Metallo-β-lactamases (MβLs) are a large contributor to antibiotic resistance. To elucidate how MβLs have evolved with the introduction and use of antibiotics, the mutations and evolution of IMP-1 from Pseudomonas aeruginosa were investigated in media with continuous and discontinuous cephalothin (CEF) concentration gradients. The results revealed 6-point mutations, K160R, G165V, A179V, K206T, K214R, and K214E. Compared with IMP-1, the minimum inhibitory concentrations (MICs) of A179V, K214R, K214E, and K160R increased at least 64-fold, G165V increased 16-fold, and K206T increased 4-fold. The k_cat/K_m of CEF by K160R and A179V increased by 841% and 495%, respectively. Furthermore, the K_a of K160R and A179V increased by 68.8% and 64.1%, respectively, suggesting that resistance was associated with enhanced substrate binding and catalytic efficiency. Molecular dynamics (MD) simulations revealed that the active site exhibited a structural change from an ordered to a disordered conformation. Simultaneously, the flexibility of loop1 and loop4 was significantly increased. These variations led to substantial alterations in the geometry and dynamic properties of the binding pocket. Consequently, these conformational changes enhanced the accessibility and catalytic competence of the Zn²⁺ center, optimizing substrate positioning and promoting efficient turnover. In K160R, structural changes at Arg-160 are spatially transmitted to the active region via a network of interactions between the Arg-160 side chain and its adjacent active pocket near amino acid side chains. These findings reveal a sophisticated evolutionary strategy employed by MβLs and delineate a novel allosteric network that could be exploited for the rational design of next-generation enzyme inhibitors.