Mechanistic Basis for Inhibition of the Extended Spectrum Class A β-Lactamase GES-1 by Tazobactam and Enmetazobactam

Expression of β-lactamases is the primary form of β-lactam antibiotic resistance in Gram-negative bacteria. Enmetazobactam is a penicillanic acid sulfone (PAS) that inhibits extended spectrum β-lactamases (ESBLs) by forming an acyl-enzyme complex that eventually breaks down to an irreversible lysinoalanine crosslink. In contrast, enmetazobactam inhibits the class A carbapenemase KPC-2 via an acyl-enzyme that does not lead to lysinoalanine crosslink formation. This difference correlates with greater inhibitory potency of enmetazobactam against class A ESBLs, compared to carbapenemases. The GES enzymes, unlike other class A β-lactamase families, show progression from carbapenem-inhibited to carbapenem-hydrolysing phenotypes through single point mutations. We present crystal structures of GES-1, a globally disseminated ESBL, as the enmetazobactam- and tazobactam-derived acyl-enzymes. The complexes differ in the identities of their respective covalent adducts, with the catalytic Ser70 acylated by a 214 Da enmetazobactam-derived fragment, whereas tazobactam has fragmented to a 70 Da aldehyde. The tautomeric form of the enmetazobactam-derived ligand is verified by high-level QM/MM calculations, revealing the trans -enamine as the most thermodynamically stable tautomer, that adopts an optimised conformation that best matches the experimentally observed electron density appended to the side chain oxygen of Ser70. In contrast to previous findings for the ESBL CTX-M-15, mass spectrometry provides no evidence for lysinoalanine crosslink formation on reaction of GES-1 with (enme)tazobactam, providing further evidence that PAS inhibitors inhibit different class A β-lactamases by different mechanisms. This work reveals new details of the basis for PAS inhibition of diverse β-lactamases, and will guide development of future β-lactamase inhibitors.