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

Abstract

Penicillin binding proteins (PBPs) catalysing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of -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 -lactam targeting of transpeptidation and (d) demonstrate 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 -lactam acylation.

Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

“…An elegant recent report (Catherwood et al, 2020) described the use of a coupled D-Ala release assay to determine the kinetic parameters of the TPase activity of PBP1B Ec against different substrates and this study also confirmed the previously reported activation of the TPase of PBP1B by LpoB in the presence of detergents (Egan et al, 2014, Egan et al, 2018, Lupoli et al, 2014. The authors of the recent report (Catherwood et al, 2020) discussed that the LpoB-mediated TPase activation explains the essentiality of LpoB for PBP1B function in the cell. However, this view ignores previously published data demonstrating that the essentiality of LpoB can be readily explained by its primary effect, the >10-fold stimulation of PBP1B's GTase (Egan et al, 2014).…”

“…A limitation of the recent kinetic study is that authors used assay conditions (e.g. very low enzyme concentration) at which PBP1B Ec is virtually inactive without an activator (Catherwood et al, 2020) as demonstrated previously (Pazos et al, 2018, Muller et al, 2007, thus the study likely substantially overestimated the extent of TPase activation by LpoB. P. aeruginosa uses a structurally different lipoprotein activator, LpoP, to stimulate its PBP1B (Greene et al, 2018).…”

“…anchored lipoprotein activator LpoB (Typas et al, 2010, Egan et al, 2018, Lupoli et al, 2014, Catherwood et al, 2020.…”

Fundamental limits on the rate of bacterial growth and their influence on proteomic composition

“…The formation of these crosslinks occurs primarily during the polymerization of the murein subunits and is facilitated by a family of transpeptidase enzymes. The four primary transpeptidases of E. coli have only recently been quantitatively characterized in vivo, via liquid chromatography mass spectrometry, which revealed a notably slow kinetic turnover rate of˘2 crosslinking reactions formed per second per enzyme as noted above (Catherwood et al, 2020).…”

CryoEM structure of the antibacterial target PBP1b at 3.3 Å resolution

“…Furthermore, we hope that this cryoEM methodology will allow for the capture of structural information regarding the native acceptor and donor substrates, which have largely eluded crystallization in both class A and class B PBPs. Structural insight into these acceptor and donor substrates will allow for further appreciation of recent work to unravel these mechanisms through the use of continuous assays 21 and allow for the development of novel antibacterial compounds that target GTase activity. We believe that the work presented in this manuscript will revitalize the study of class A PBPs and that this and subsequent work will be of great assistance as we attempt to ward off a post-antibiotic era.…”