Substrate and stereochemical control of peptidoglycan crosslinking by transpeptidation by Escherichia coli PBP1B

Anita C Catherwood, Adrian J Lloyd, Julie A Tod, Smita Chauhan, Susan E Slade, Gregory Walkowiak, Nicola F Galley, Avinash Punekar, Katie Smart, Dean Rea, Neil D Evans, Michael J Chappell, David I Roper, Christopher G Dowson

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Abstract

Penicillin binding proteins (PBPs) catalyzing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of β-lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by Escherichia coli PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b) reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on β-lactam targeting of transpeptidation, and (d) demonstrate that both substrates have to be bound before transpeptidation occurs. The results allow characterization of high molecular weight PBPs as enzymes and not merely the targets of β-lactam acylation.

Original languageEnglish
Pages (from-to)5034-5048
Number of pages15
JournalJournal of the American Chemical Society
Volume142
Issue number11
Early online date12 Feb 2020
DOIs
Publication statusPublished - 18 Mar 2020

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    Catherwood, A. C., Lloyd, A. J., Tod, J. A., Chauhan, S., Slade, S. E., Walkowiak, G., Galley, N. F., Punekar, A., Smart, K., Rea, D., Evans, N. D., Chappell, M. J., Roper, D. I., & Dowson, C. G. (2020). Substrate and stereochemical control of peptidoglycan crosslinking by transpeptidation by Escherichia coli PBP1B. Journal of the American Chemical Society, 142(11), 5034-5048. https://doi.org/10.1021/jacs.9b08822