Acyl-enzyme dynamics, tautomerisation and hydration regulate turnover of carbapenem antibiotics by the OXA-48 β-lactamase

Abstract OXA-48 is a globally disseminated class D serine β-lactamase that efficiently confers resistance to a range of β-lactam antibiotics, including carbapenems, the most potent such agents versus Enterobacterales (Escherichia coli and relatives). Here we characterise the interactions of OXA-48 with the acyl-enzyme complex intermediates formed on its reaction with the carbapenems meropenem and ertapenem using X-ray crystallography and molecular dynamics (MD) simulations. X-ray crystal structures identify acyl-enzymes in both the Δ1-imine and Δ2-enamine pyrroline tautomeric forms. MD simulations show the epimeric Δ2 tautomers of meropenem and ertapenem to more frequently adopt binding poses competent for hydrolysis, i.e. with an appropriate orientation of the carbapenem 6α-hydroxyethyl group and positioning of the water molecule required for deacylation; the results indicate that the Δ2 tautomers are preferred for deacylation over the Δ1-tautomer. MD simulations based on the crystal structures show that, compared to OXA-48, acyl-enzyme complexes of OXA-519 (a natural OXA-48 variant with a single Val120Leu substitution adjacent to the catalytic general base) more frequently sampled conformations favouring hydrolysis, or formation of the alternative β-lactone deacylation product. MD simulations of complexes derived from quantum mechanics/molecular mechanics (QM/MM) simulations show the meropenem-derived β-lactone product is better retained in the OXA-48 active site than hydrolysed meropenem, consistent with reversible β-lactone formation. Overall, our results demonstrate how acyl-enzyme tautomerisation, dynamics and hydration collectively modulate degradation of 1β-methyl carbapenems by class D β-lactamases of the OXA-48 group, and how subtle changes in active site structure potentiate such effects in the OXA-519 variant. Competing Interest Statement The authors have declared no competing interest. Abbreviations - MD - molecular dynamics - OXA - oxacillinase - RMSD - root mean-squared deviation - RMSF - root mean-squared fluctuation - SBL - serine β-lactamase