Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance

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Abstract

Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), e.g. cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than binding (KM). High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω-, 240- and 270-loops, that border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme, and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω-loop, adopts two distinct conformations in KPC-2, either pointing ‘in’ or ‘out’ of the active site; with only the ‘in’ form compatible with deacylation. The ‘out’ conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω-loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate.
Original languageEnglish
Article number016461
Number of pages14
JournalJournal of Biological Chemistry
Volume296
Early online date4 Jan 2021
DOIs
Publication statusE-pub ahead of print - 4 Jan 2021

Bibliographical note

Funding Information:
Funding and additional information—Research was supported by the Biotechnology and Biological Sciences Research Council (SWBioDTP [BB/J014400/1], studentship to C. L. T.) and the Medical Research Council (MR/T016035/1). C. J. S. thanks the Medical Research Council and the Wellcome Trust for funding. Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health to R. A. B. under award numbers R01AI100560, R01AI063517, and R01AI072219. This study was also supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs, award number 1I01BX001974 to R. A. B. from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development, and the Geriatric Research Education and Clinical Center VISN 10. 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. KPC diffraction data were collected at the BL13–XALOC beamline at ALBA Synchrotron with the collaboration of ALBA staff. We also thank Diamond Light Source for beamtime (proposals 172122 and 23269) and the staff of beamlines I24 and I04 for assistance.

Publisher Copyright:
© 2021 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.

Keywords

  • Serine β-lactamase
  • acyl-enzyme
  • enzyme catalysis
  • tructure-function
  • crystal structure
  • molecular dynamics
  • β-lactam
  • antibiotic resistance
  • ceftazidime

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