Discovery and molecular basis of chloride as an allosteric activator and catalytic inhibitor for Class-D β-lactamases

Oxacillinase (OXA)-48-like carbapenemases are epidemic class D β-lactamases in Enterobacterales, resulting in high mortality. Though the chemical mechanism has been clearly established, for decades, the link between the biphasic kinetic behaviour of these enzymes, which significantly impacts antibiotic efficacy, and the state of carbamylated lysine has been elusive. Here, substituting N-carbamylated lysine73 with a chemically-stable N-acetyl lysine allows us to prove the origin of catalytic inhibition is not decarbamylation and enables us to capture an unprecedented inactive acyl-intermediate wedged in place by a chloride ion against the conserved residue arginine250. We here identify chloride as a “Janus effector” acting by allosteric activation of the burst phase and inhibition of the steady-state for a series of β-lactam substrates in kinetic assays. Chloride ions are necessarily present in both laboratory and clinical OXA activity assays and their inseparable role is now identified. Our finding suggests a new direction for the discovery of next-generation antibiotics specific for β-lactamases of Class D.