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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

DOI: 10.1128/mbio.01793-21 DOI Help

Authors: Philip Hinchliffe (University of Bristol) , Catherine L. Tooke (University of Bristol) , Christopher R. Bethel (Louis Stokes Cleveland Department of Veterans Affairs) , Benlian Wang (Case Western Reserve University School of Medicine) , Christopher Arthur (University of Bristol) , Kate J. Heesom (University of Bristol) , Stuart Shapiro (Allecra Therapeutics SAS) , Daniela M. Schlatzer (Case Western Reserve University School of Medicine) , Krisztina M. Papp-Wallace (Louis Stokes Cleveland Department of Veterans Affairs; Case Western Reserve University School of Medicine) , Robert A. Bonomo (Louis Stokes Cleveland Department of Veterans Affairs; Case Western Reserve University School of Medicine; CWRU-Cleveland VAMC Center for Antimicrobial Resistance and Epidemiology (Case VA CARES)) , James Spencer (University of Bristol)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Mbio

State: Published (Approved)
Published: May 2022
Diamond Proposal Number(s): 23269

Open Access Open Access

Abstract: 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 Enterobacterales. 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.

Diamond Keywords: Bacteria

Subject Areas: Biology and Bio-materials, Medicine

Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Other Facilities: BL13-XALOC at ALBA

Added On: 01/06/2022 08:50


Discipline Tags:

Pathogens Antibiotic Resistance Infectious Diseases Health & Wellbeing Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)