Interactions of hydrolyzed β-lactams with the L1 metallo-β-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products

DOI: 10.1016/j.jbc.2023.104606

Authors: Philip Hinchliffe (University of Bristol) , Karina Calvopina (University of Oxford) , Patrick Rabe (University of Oxford) , Maria F. Mojica (Western Reserve University; CWRU-Cleveland VA Medical Center for Antimicrobial Resistance and Epidemiology (Case VA CARES); Louis Stokes Cleveland Department of Veterans Affairs Medical Center; Universidad El Bosque) , Christopher J. Schofield (University of Oxford) , Gary I. Dmitrienko (University of Waterloo) , Robert A. Bonomo (CWRU-Cleveland VA Medical Center for Antimicrobial Resistance and Epidemiology (Case VA CARES); Louis Stokes Cleveland Department of Veterans Affairs Medical Center; Case Western Reserve University School of Medicine) , Alejandro J. Vila (CWRU-Cleveland VA Medical Center for Antimicrobial Resistance and Epidemiology (Case VA CARES); nstituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR); Universidad Nacional de Rosario) , James Spencer (University of Bristol)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 399

State: Published (Approved)
Published: March 2023
Diamond Proposal Number(s): 17212 , 23269

Abstract: L1 is a dizinc subclass B3 metallo-β-lactamase (MBL) that hydrolyzes most β-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed β-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole) and carbapenem (tebipenem, doripenem and panipenem) classes. Despite differences in their structures, all the β-lactam-derived products hydrogen bond to Tyr33, Ser221 and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modelled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1β-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1β-hydrogen-containing carbapenems), or is displaced by the C-6 carboxylate (1β-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur non-enzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3’ leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (β) faces, and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and β-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

Journal Keywords: metallo β-lactamase; antibiotic resistance; β-lactam; carbapenem; hydrolysis; X-ray crystallography; L1 carbapenemase

Diamond Keywords: Enzymes; Bacteria

Subject Areas: Biology and Bio-materials, Medicine, Chemistry


Instruments: I03-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Added On: 19/03/2023 16:55

Discipline Tags:

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

Technical Tags:

Diffraction Macromolecular Crystallography (MX)