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

Philip Hinchliffe, Karina Calvopiña, Patrick Rabe, Maria F. Mojica, Christopher J. Schofield, Gary I Dmitrienko, Robert A. Bonomo, Alejandro J Vila, James Spencer*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

2 Citations (Scopus)

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.
Original languageEnglish
Article number104606
JournalJournal of Biological Chemistry
Volume299
Issue number5
Early online date15 Mar 2023
DOIs
Publication statusPublished - 1 May 2023

Bibliographical note

Funding Information:
This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH) to R. A. B., A. J. V. and J. S. under Award Number R01AI100560 and to G. I. D., C. J. S., and J. S. under award number R21-AI123835 . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs. G. I. D. acknowledges support from the Canadian Institutes of Health Research , under award number PJT-427834 . We thank BrisSynBio, a BBSRC/EPSRC-funded Synthetic Biology Research Centre (L01386X), for funding the Tecan Infinite 200 pro microplate reader.

Funding Information:
We thank Diamond Light Source for beamtime (proposals MX17212 and MX23269) and the staff of beamlines I03 and I24 for assistance with crystal testing and data collection. We thank Prof. Dr Christina Redfield for her support and the Department of Biochemistry (Oxford) for the use of the 750 MHz NMR spectrometer. P. H. K. C. and P. R. investigation; P. H. K. C. P. R. M. F. M. C. J. S. G. I. D. R. A. B. A. J. V. and J. S. methodology, formal analysis, and writing – original draft. This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH) to R. A. B. A. J. V. and J. S. under Award Number R01AI100560 and to G. I. D. C. J. S. and J. S. under award number R21-AI123835. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs. G. I. D. acknowledges support from the Canadian Institutes of Health Research, under award number PJT-427834. We thank BrisSynBio, a BBSRC/EPSRC-funded Synthetic Biology Research Centre (L01386X), for funding the Tecan Infinite 200 pro microplate reader.

Publisher Copyright:
© 2023 The Authors

Fingerprint

Dive into the research topics of 'Interactions of hydrolyzed β-lactams with the L1 metallo-β-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products'. Together they form a unique fingerprint.

Cite this