A Two Amino Acid Duplication, L167E168, in the Ω-Loop Drastically Decreases Carbapenemase Activity of KPC-53, a Natural Class A β-Lactamase

KPC-53 enzyme is a natural KPC variant which showed a duplication of L167E168 residues in the Omega-loop structure. The bla(K)(PC-53) gene was cloned both into pBC-SK and pET-24a vectors, and the recombinant plasmids were transferred by transformation in Escherichia coli competent cells to evaluate the antimicrobial susceptibility and to produce the enzyme. Compared to KPC-3, the KPC-53 was less stable and showed a dramatic reduction of k(cat) and K-cat/K-m versus several beta-lactams, in particular carbapenems. Indeed, a-2000-fold reduction was observed in the k(cat) values of KPC-53 for imipenem and meropenem. Concerning inhibitors, KPC-53 was susceptible to tazobactam and clavulanic acid but maintained resistance to avibactam. The molecular modeling indicates that the L167E168 duplication in KPC-53 modifies the interactions between residues involved in the catalytic pocket, changing the flexibility of the S2-loop, which is directly coupled with the catalytic properties of the KPC enzymes.

Automated summary

KPC-53 enzyme is a natural variant of KPC with a duplication of L167E168 residues, which results in decreased stability and significant reductions in activity against beta-lactam antibiotics, particularly carbapenems. The enzyme is susceptible to tazobactam and clavulanic acid but remains resistant to avibactam. Molecular modeling shows that the duplication in KPC-53 alters the interactions between residues involved in catalysis, impacting enzyme flexibility and activity.