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Nat Chem Biol. 2017 Jul;13(7):793-798. doi: 10.1038/nchembio.2388. Epub 2017 May 29.

Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams.

Author information

1
Department of Microbiology and Immunology, Harvard Medical School, Boston, Massachusetts, USA.
2
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
3
Department of Microbiology, Ohio State University, Columbus, Ohio, USA.

Abstract

Peptidoglycan is an essential crosslinked polymer that surrounds bacteria and protects them from osmotic lysis. β-lactam antibiotics target the final stages of peptidoglycan biosynthesis by inhibiting the transpeptidases that crosslink glycan strands to complete cell wall assembly. Characterization of transpeptidases and their inhibition by β-lactams have been hampered by lack of access to a suitable substrate. We describe a general approach to accumulate Lipid II in bacteria and to obtain large quantities of this cell wall precursor. We demonstrate the utility of this strategy by isolating Staphylococcus aureus Lipid II and reconstituting the synthesis of crosslinked peptidoglycan by the essential penicillin-binding protein 2 (PBP2), which catalyzes both glycan polymerization and transpeptidation. We also show that we can compare the potencies of different β-lactams by directly monitoring transpeptidase inhibition. The methods reported here will enable a better understanding of cell wall biosynthesis and facilitate studies of next-generation transpeptidase inhibitors.

PMID:
28553948
PMCID:
PMC5478438
DOI:
10.1038/nchembio.2388
[Indexed for MEDLINE]
Free PMC Article

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