A New Mechanism for β‐Lactamases: Class D Enzymes Degrade 1β‐Methyl Carbapenems through Lactone Formation

Abstract

Abstractβ‐Lactamases threaten the clinical use of carbapenems, which are considered antibiotics of last resort. The classical mechanism of serine carbapenemase catalysis proceeds through hydrolysis of an acyl‐enzyme intermediate. We show that class D β‐lactamases also degrade clinically used 1β‐methyl‐substituted carbapenems through the unprecedented formation of a carbapenem‐derived β‐lactone. β‐Lactone formation results from nucleophilic attack of the carbapenem hydroxyethyl side chain on the ester carbonyl of the acyl‐enzyme intermediate. The carbapenem‐derived lactone products inhibit both serine β‐lactamases (particularly class D) and metallo‐β‐lactamases. These results define a new mechanism for the class D carbapenemases, in which a hydrolytic water molecule is not required.

19F NMR Monitoring of Reversible Protein Post‐Translational Modifications: Class D β‐Lactamase Carbamylation and Inhibition

“…However, the K m values for meropenem and nitrocefin were greater for OXA‐48 T213C* compared to wild‐type OXA‐48. The product profile of OXA‐48 T213C* with meropenem resembled what was observed for wild‐type OXA‐48, with both enzymes producing a similar ratio of carbapenem‐derived hydrolysis and lactone products (Figure S5) …”

“…Based on kinetics tudies (Table S1), the k cat values determined for OXA-48 T213C* with meropenema nd nitrocefin weres imilar (but not identical) to wild-type OXA-48, while the activity of OXA-48 L158C*w as relatively poor.H owever,t he K m values for meropenem and nitrocefin were greater for OXA-48 T213C* compared to wild-type OXA-48.T he product profile of OXA-48 T213C*w ith meropenem resembled what was observed for wild-type OXA-48, with both enzymes producing as imilarr atio of carbapenem-derived hydrolysisa nd lactone products ( Figure S5). [9] Circulard ichroism analyses indicate that the secondary structureo fb oth variants resembles that of wild-type enzyme ( Figure S6). Substitution of Leu158 appears to have ad eleterious effect on enzymea ctivity and carbamylation, as manifested clearly in 19 FNMR spectra acquired at lower pH (FigureS7).…”

Non‐Hydrolytic β‐Lactam Antibiotic Fragmentation by l,d‐Transpeptidases and Serine β‐Lactamase Cysteine Variants

“…The acyl‐enzyme complexes of SBLs with some other β‐lactams favor alternative (i.e., not C5–C6) fragmentation reactions, as reported for the SBL inhibitors clavulanic acid, sulbactam, and tazobactam . Alternative enzyme‐catalyzed mechanisms not involving C−C fragmentation have also been observed, including the degradation of carbapenems by class D SBLs via lactone formation …”

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

“…The β-lactone products are proposed to be formed by initial nucleophilic attack of the C-6 hydroxyethyl hydroxyl group onto the ester carbonyl of the AEC. In support of this mechanism and arguing against their formation via reaction of a non-covalently bound complex, at least in some cases, the lactones can react reversibly with class D SBLs to give a covalently modified complex (14). The carbapenem derived β-lactones are less potent SBL inhibitors than the parent carbapenems, but with optimization to enable them to form stable acylenzyme complexes, β-lactones may form the basis of a new type of SBL inhibitor (14).…”