Substrate and Stereochemical Control of Peptidoglycan Cross-Linking by Transpeptidation by Escherichia coli PBP1B

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, beca...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2020-03, Vol.142 (11), p.5034-5048
Main Authors: Catherwood, Anita C, Lloyd, Adrian J, Tod, Julie A, Chauhan, Smita, Slade, Susan E, Walkowiak, Grzegorz P, Galley, Nicola F, Punekar, Avinash S, Smart, Katie, Rea, Dean, Evans, Neil D, Chappell, Michael J, Roper, David I, Dowson, Christopher G
Format: Article
Language:English
Subjects:
Bacterial Outer Membrane Proteins - chemistry
Biocatalysis
Enzyme Assays - methods
Escherichia coli - enzymology
Escherichia coli Proteins - chemistry
Kinetics
Penicillin-Binding Proteins - chemistry
Peptidoglycan - chemistry
Peptidoglycan Glycosyltransferase - chemistry
Polyisoprenyl Phosphate Monosaccharides - chemistry
Polyisoprenyl Phosphate Oligosaccharides - chemistry
Serine-Type D-Ala-D-Ala Carboxypeptidase - chemistry
Stereoisomerism
Substrate Specificity
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Summary: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.
ISSN:0002-7863
1520-5126
DOI:10.1021/jacs.9b08822