TY - JOUR
T1 - Substrate and Stereochemical Control of Peptidoglycan Cross-Linking by Transpeptidation by Escherichia coli PBP1B
AU - Catherwood, Anita C.
AU - Lloyd, Adrian J.
AU - Tod, Julie A.
AU - Chauhan, Smita
AU - Slade, Susan E.
AU - Walkowiak, Grzegorz P.
AU - Galley, Nicola F.
AU - Punekar, Avinash S.
AU - Smart, Katie
AU - Rea, Dean
AU - Evans, Neil D.
AU - Chappell, Michael J.
AU - Roper, David, I
AU - Dowson, Christopher G.
PY - 2020/3/18
Y1 - 2020/3/18
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85082099052&partnerID=8YFLogxK
U2 - 10.1021/jacs.9b08822
DO - 10.1021/jacs.9b08822
M3 - Article
C2 - 32048840
SN - 0002-7863
VL - 142
SP - 5034
EP - 5048
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 11
ER -