Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

An important mechanism of resistance to beta-lactam antibiotics is via their beta-lactamase-catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine beta-lactamases (SBLs) with carbapenems also produces beta-lactones. We report studies on the factors determining beta-lactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e. Trp(105), Val(120), and Leu(158), using OXA-48 numbering) impact on the relative levels of beta-lactones and hydrolysis products formed. Some variants, i.e. the OXA-48 V120L and OXA-23 V128L variants, catalyze increased beta-lactone formation compared with the WT enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show that some class D SBL variants catalyze formation of beta-lactones from all clinically relevant carbapenems regardless of the presence or absence of a 1 beta-methyl substituent. Analysis of reported crystal structures for carbapenem-derived acyl-enzyme complexes reveals preferred conformations for hydrolysis and beta-lactone formation. The observation of increased beta-lactone formation by class D SBL variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of beta-lactams to beta-lactones is important as a resistance mechanism.