Penicillanic Acid Sulfones Inactivate the Extended-Spectrum β-Lactamase CTX-M-15 through Formation of a Serine-Lysine Cross-Link: an Alternative Mechanism of β-Lactamase Inhibition

• Published in: mBio 2022-06, Vol.13 (3), p.e0179321-e0179321
• Main Authors: Hinchliffe, Philip, Tooke, Catherine L, Bethel, Christopher R, Wang, Benlian, Arthur, Christopher, Heesom, Kate J, Shapiro, Stuart, Schlatzer, Daniela M, Papp-Wallace, Krisztina M, Bonomo, Robert A, Spencer, James
• Format: Article
• Language: English
• Subjects: AAI101; antibiotic resistance; CTX-M-15; enmetazobactam; inhibitor; Research Article; Structural Biology; tazobactam
• ISSN: 2150-7511; 2150-7511
• DOI: 10.1128/mbio.01793-21

β-Lactamases hydrolyze β-lactam antibiotics and are major determinants of antibiotic resistance in Gram-negative pathogens. Enmetazobactam (formerly AAI101) and tazobactam are penicillanic acid sulfone (PAS) β-lactamase inhibitors that differ by an additional methyl group on the triazole ring of enmetazobactam, rendering it zwitterionic. In this study, ultrahigh-resolution X-ray crystal structures and mass spectrometry revealed the mechanism of PAS inhibition of CTX-M-15, an extended-spectrum β-lactamase (ESBL) globally disseminated among . CTX-M-15 crystals grown in the presence of enmetazobactam or tazobactam revealed loss of the Ser70 hydroxyl group and formation of a lysinoalanine cross-link between Lys73 and Ser70, two residues critical for catalysis. Moreover, the residue at position 70 undergoes epimerization, resulting in formation of a d-amino acid. Cocrystallization of enmetazobactam or tazobactam with CTX-M-15 with a Glu166Gln mutant revealed the same cross-link, indicating that this modification is not dependent on Glu166-catalyzed deacylation of the PAS-acylenzyme. A cocrystal structure of enmetazobactam with CTX-M-15 with a Lys73Ala mutation indicates that epimerization can occur without cross-link formation and positions the Ser70 Cβ closer to Lys73, likely facilitating formation of the Ser70-Lys73 cross-link. A crystal structure of a tazobactam-derived imine intermediate covalently linked to Ser70, obtained after 30 min of exposure of CTX-M-15 crystals to tazobactam, supports formation of an initial acylenzyme by PAS inhibitors on reaction with CTX-M-15. These data rationalize earlier results showing CTX-M-15 deactivation by PAS inhibitors to involve loss of protein mass, and they identify a distinct mechanism of β-lactamase inhibition by these agents. β-Lactams are the most prescribed antibiotic class for treating bacterial diseases, but their continued efficacy is threatened by bacterial strains producing β-lactamase enzymes that catalyze their inactivation. The CTX-M family of ESBLs are major contributors to β-lactam resistance in , preventing effective treatment with most penicillins and cephalosporins. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcome such resistance. Here, we describe how exposure to enmetazobactam and tazobactam, BLIs based on a penicillanic acid sulfone (PAS) scaffold, leads to a protein modification in CTX-M-15, resulting in irremediable inactivation of this most commonly encou