biostudies-literatureBeer MBLRD VACleveland Department of Veterans AffairsEuropean Research CouncilBiotechnology and Biological Sciences Research CouncilEngineering and Physical Sciences Research Council3284-3300https://www.ebi.ac.uk/biostudies/studies/S-EPMC12643063biostudies-literatureUnknown599(22)β-Lactamase-catalysed hydrolysis is the primary form of β-lactam antibiotic resistance in Gram-negative bacteria. The penicillanic acid sulfone (PAS) enmetazobactam is thought to inhibit extended-spectrum β-lactamases (ESBLs) by fragmentation of an initial acyl-enzyme to form an active-site lysinoalanine cross link. We investigate interactions of enmetazobactam and its congener tazobactam with GES-1, an ESBL with structural features of carbapenem-hydrolysing β-lactamases. Crystal structures show different breakdown products of the two inhibitors covalently bound to the catalytic Ser70, assigned using quantum mechanics/molecular mechanics (QM/MM) calculations. We find no evidence for lysinoalanine formation, with mass spectrometry indicating active enzyme regeneration, behaviour previously FEBS lettersMechanistic basis for inhibition of the extended-spectrum β-lactamase GES-1 by enmetazobactam and tazobactam.PMC12643063BB/M026280/1BB/T008741/11I01BX001974EP/W013738/1I01 BX001974101021207Papp-Wallace KMBeer MMulholland AJSpencer JBethel CRShapiro SHinchliffe PHanzevacki MTooke CLVan der Kamp MWBonomo RAfalseMechanistic basis for inhibition of the extended-spectrum β-lactamase GES-1 by enmetazobactam and tazobactam.β-Lactamase-catalysed hydrolysis is the primary form of β-lactam antibiotic resistance in Gram-negative bacteria. The penicillanic acid sulfone (PAS) enmetazobactam is thought to inhibit extended-spectrum β-lactamases (ESBLs) by fragmentation of an initial acyl-enzyme to form an active-site lysinoalanine cross link. We investigate interactions of enmetazobactam and its congener tazobactam with GES-1, an ESBL with structural features of carbapenem-hydrolysing β-lactamases. Crystal structures show different breakdown products of the two inhibitors covalently bound to the catalytic Ser70, assigned using quantum mechanics/molecular mechanics (QM/MM) calculations. We find no evidence for lysinoalanine formation, with mass spectrometry indicating active enzyme regeneration, behaviour previously 2025-01-01T00:00:00Z2025 Nov2026-06-05T16:51:50.471Z2026-05-19T03:11:56.287ZS-EPMC126430634094439210.1002/1873-3468.70155