Role of Ambler Position 104 in Defining Substrate Specificity in the KPC Family of β-Lactamases

containing the P104K and P104R variants exhibited markedly increased resistance to ceftazidime, ceftazidime/avibactam, ceftazidime/relebactam, and cefiderocol, while carbapenem susceptibility remained largely unchanged. Steady-state kinetics confirmed enhanced hydrolysis of ceftazidime and cefiderocol by these variants. Molecular dynamics simulations and deep-learning analyses revealed that substitutions at position 104 alter W105 orientation, expand active-site volume, and increase hinge-loop flexibility, enabling accommodation of bulky substrates. These findings highlight the critical role of residue 104 in shaping substrate specificity and inhibitor susceptibility in KPC enzymes, with implications for antimicrobial therapy and resistance evolution.