Hydrogen-bonding changes cause differences in imipenem breakdown activity in OXA-48 variants

Abstract

The {beta}-lactamase OXA-48 efficiently hydrolyses carbapenem antibiotics, especially imipenem. Carbapenem resistance is a rising clinical concern, and is frequently associated with OXA-48 and its variants. OXA-48 variants carrying different mutations in the {beta}5-{beta}6 loop differ in hydrolytic activity towards imipenem. OXA-517 has a higher KM, but similar kcat for imipenem hydrolysis, compared to OXA-48, whereas OXA-163 and -405, which have similar mutations in the {beta}5-{beta}6 loop, are less active. Multiscale simulations (using quantum mechanics/molecular mechanics, QM/MM) of deacylation of the respective imipenem acylenzymes show this to be most efficient when the deacylating water (DW) acts as a hydrogen bond (H-bond) donor to imipenem, and the carboxylated Lys73 base is less hydrated. Calculated barriers for deacylation correlate very well with experimental data, but for OXA-163 and -405 only when DW acts as a H-bond acceptor. Dynamics simulations of imipenem acylenzyme complexes show that mutations in the {beta}5-{beta}6 loop change the active site H-bond network. In OXA-48, the DW H-bonding pattern linked to high activity is more frequently sampled, and in OXA-517 it is stabilised through H-bonding to Thr213; explaining the higher kcat values compared to OXA-163 and -405, where this is not the case. Furthermore, simulations of non-covalent imipenem complexes indicate that increased KM for OXA-517 is linked to lower binding affinity, caused by repositioning of bound imipenem. Our work identifies the molecular basis for differences in imipenem hydrolytic activity between OXA-48 variants, offering detailed insights into how active site interactions alter the dynamics and reaction efficiencies related to antibiotic resistance.