Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase

Bury, Daniel; Dahmane, Ismahene; Derouaux, Adeline; Dumbre, Shrinivas G.; Herdewijn, Piet; Matagne, André; Breukink, Eefjan; Mueller-Seitz, Erika; Petz, Michael; Terrak, Mohammed

doi:10.1016/j.bcp.2014.11.003

Cited by 8 publications

(15 citation statements)

References 30 publications

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“…3E). This result can be explained on the basis of our previous finding showing a positive cooperativity between the donor and acceptor sites of the GTase domain 22 . Indeed, using surface plasmon resonance we showed that the binding of lipid II or analogs to the acceptor site of Staphylococcus aureus MtgA facilitates the binding of moenomycin to the donor site by allosteric effect.…”

Section: Competition Of Antibiotics With Nbd-lipid Ii:proteins Using supporting

confidence: 58%

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ

Boes

Olatunji

Mohammadi

et al. 2020

Sci Rep

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Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled lipid II, four lipid ii-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. in addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.

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Section: Competition Of Antibiotics With Nbd-lipid Ii:proteins Using supporting

confidence: 58%

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ

Boes

Olatunji

Mohammadi

et al. 2020

Sci Rep

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“…In the free inner membrane-embedded enzyme, to start the catalytic cycle, lipid II binds to the donor site, and a second lipid II molecule binds the extended ␣4 helix loop at the acceptor site, which is observed unperturbed by crystal packing for the first time in our E. coli PBP1b-aztreonam crystal structure (Fig. 6, A-D) (6,26,29). Upon formation of the precatalytic complex with both donor-and acceptor-bound, the general base Glu 233 , which we now observe directly in the electron density of the PBP1b GTase active site (Figs.…”

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confidence: 67%

Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B

King

Wasney

Nosella

et al. 2017

Journal of Biological Chemistry

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Edited by F. Peter GuengerichIn Escherichia coli, the peptidoglycan cell wall is synthesized by bifunctional penicillin-binding proteins such as PBP1b that have both transpeptidase and transglycosylase activities. The PBP1b transpeptidase domain is a major target of ␤-lactams, and therefore it is important to attain a detailed understanding of its inhibition. The peptidoglycan glycosyltransferase domain of PBP1b is also considered an excellent antibiotic target yet is not exploited by any clinically approved antibacterials. Herein, we adapt a pyrophosphate sensor assay to monitor PBP1b-catalyzed glycosyltransfer and present an improved crystallographic model for inhibition of the PBP1b glycosyltransferase domain by the potent substrate analog moenomycin. We elucidate the structure of a previously disordered region in the glycosyltransferase active site and discuss its implications with regards to peptidoglycan polymerization. Furthermore, we solve the crystal structures of E. coli PBP1b bound to multiple different ␤-lactams in the transpeptidase active site and complement these data with gel-based competition assays to provide a detailed structural understanding of its inhibition. Taken together, these biochemical and structural data allow us to propose new insights into inhibition of both enzymatic domains in PBP1b.

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“…Tetrasaccharide- and hexasaccharide-containing precursors (lipid IV or lipid VI) are poor or no substrates on their own, but can be incorporated into higher oligomers when lipid II is polymerized [ 80 , 85 ]. It has been recently shown for the monofunctional S. aureus GTase MtgA that a low concentration of lipid II (binding to the acceptor site) increases the binding affinity of moenomycin to the donor site, indicating an allosteric activation of the donor site and positive cooperativity between both sites [ 86 ].…”

Section: Processivity and Substrate Specificity Of Gtasesmentioning

confidence: 99%

“… precursor/substrate S. pneumoniae PBP1a, PBP2a, PBP2b, and PBP2x — amidation of lipid II [ 75 ] — TPase activity requires the presence of an amidated iso-Gln residue at position 2 of the stem peptide. S. aureus MtgA — lipid II [ 86 ] — Binding of lipid II enhances the affinity of moenomycin to the glycan acceptor site. environmental conditions C. crescentus PBP2 and PBP1A — osmolarity of growth medium [ 129 ] — Upshift in the osmolarity of growth medium enhances localization to mid-cell relocating the PG growth site.…”

Section: Regulation Of Penicillin-binding Protein Activitymentioning

confidence: 99%

Activities and regulation of peptidoglycan synthases

Egan

Biboy

Veer

et al. 2015

Phil. Trans. R. Soc. B

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Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.

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Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase

Cited by 8 publications

References 30 publications

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ

Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B

Activities and regulation of peptidoglycan synthases

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