Metabolic labeling of the bacterial peptidoglycan by functionalized glucosamine

Xu, Yang; Hernández-Rocamora, Víctor M.; Lorent, Joseph H.; Cox, Ruud C.; Wang, Xiaoqi; Xue, Bin; Stel, Marjon; Vos, Gaël M.; Bos, R.M. van den; Pieters, Roland J.; Gray, Joe; Vollmer, Waldemar; Breukink, Eefjan

doi:10.1016/j.isci.2022.104753

Cited by 9 publications

(16 citation statements)

References 79 publications

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“…32 As has been previously shown in OleD-expressing E. coli, UDP-GlcNAz is used by MurG to generate lipid II. 24 Figure 3. FACE gel of peptidoglycan sacculi.…”

Section: ■ Resultsmentioning

confidence: 99%

“…23 Using 2-chloro-4-nitrophenyl GlcNAz in E. coli expressing OleD, the authors were able to demonstrate the in situ production of UDP-GlcNAz and subsequent incorporation of GlcNAz into peptidoglycans. 24 In another approach, we have used the promiscuous Bifidobacterial enzyme NahK to generate a set of C6-modified UDP-GlcNAc derivatives in vivo as glycosyltransferase inhibitors. 25 NahK is a GlcNAc 1-kinase with a high tolerance for synthetic and modified substrates including GlcNAz 26,27 and has been used in chemoenzymatic syntheses of various 1-phosphosugars.…”

Section: ■ Introductionmentioning

confidence: 99%

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Metabolic Usage and Glycan Destinations of GlcNAz in E. coli

Eddenden,

Dooda,

Morrison

et al. 2023

ACS Chem. Biol.

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Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars, such as Nacetylglucosamine (GlcNAc), being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAccontaining glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc, N-azidoacetylglucosamine (GlcNAz), for metabolic labeling in Escherichia coli. The cytosolic production of UDP-GlcNAz was confirmed by using fluorescence-assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated was interrogated by analyzing potential sites including peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-β-1,6-N-acetylglucosamine (PNAG), lipopolysaccharide (LPS), and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) toward UDP-GlcNAz in vitro and the lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo confirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies.

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“…32 As has been previously shown in OleD-expressing E. coli, UDP-GlcNAz is used by MurG to generate lipid II. 24 Figure 3. FACE gel of peptidoglycan sacculi.…”

Section: ■ Resultsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Metabolic Usage and Glycan Destinations of GlcNAz in E. coli

Eddenden,

Dooda,

Morrison

et al. 2023

ACS Chem. Biol.

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“…[36,37,41] Furthermore, Lipid II analogues with an azido moiety on C6 of GlcNAc were well tolerated by PGTs in vitro. [40] Recently, C2 N-acetyl-modified MurNAc and GlcNAc probes have been used for metabolic labelling of peptidoglycan in live bacteria, [42,43] which implies that bacterial PGTs can tolerate these particular modifications for glycan polymerisation.…”

Section: Glycanmentioning

confidence: 99%

“…Correspondingly, several crystal and CryoEM structures of MoeA-bound PGTs (i. e., aPBPs and MGTs) revealed that MoeA resides in the glycosyl donor site and its carbohydrate and 3-PG moieties are extensively hydrogen bonded to a number of residues in the active site, locking the PGT into an inactive conformation. [49,50,[56][57][58][59][60] Given the strict substrate lipid requirement of the PGTs' donor sites, it remains unclear how the C 25 lipid of MoeA with unnatural double bond Lipid II probes can be created by modifying Lipid II on one or more of the following sites: N-acetyl groups on either sugar, [42,43] C6 on GlcNAc, [40] the terminus of the lipid tail, [38,39] and the third-fifth amino acids of the stem peptide. [32,38,39,44,45,48] geometry interacts with PGTs, as the molecular details are not resolved in current structural studies.…”

Section: Moenomycin a A Natural Product Inhibitor Of Pgtsmentioning

confidence: 99%

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Mechanistic Insights into the Activities of Major Families of Enzymes in Bacterial Peptidoglycan Assembly and Breakdown

Kwan

Qiao

2023

ChemBioChem

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Probing Peptidoglycan Synthesis in the Gut Commensal Akkermansia Muciniphila with Bioorthogonal Chemical Reporters

Sminia,

Aalvink,

de Jong

et al. 2024

ChemBioChem

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Our gut microbiota directly influences human physiology in health and disease. The myriad of surface glycoconjugates in both the bacterial cell envelope and our gut cells dominate the microbiota‐host interface and play a critical role in host response and microbiota homeostasis. Among these, peptidoglycan is the basic glycan polymer offering the cell rigidity and a basis on which many other glycoconjugates are anchored. To directly study peptidoglycan in gut commensals and obtain the molecular insight required to understand their functional activities we need effective techniques like chemical probes to label peptidoglycan in live bacteria. Here we report a chemically guided approach to study peptidoglycan in a key mucin‐degrading gut microbiota member of the Verrucomicrobia phylum, Akkermansia muciniphila. Two novel non‐toxic tetrazine click‐compatible peptidoglycan probes with either a cyclopropene or isonitrile handle allowed for the detection and imaging of peptidoglycan synthesis in this intestinal species.

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Metabolic labeling of the bacterial peptidoglycan by functionalized glucosamine

Cited by 9 publications

References 79 publications

Metabolic Usage and Glycan Destinations of GlcNAz in E. coli

Metabolic Usage and Glycan Destinations of GlcNAz in E. coli

Mechanistic Insights into the Activities of Major Families of Enzymes in Bacterial Peptidoglycan Assembly and Breakdown

Probing Peptidoglycan Synthesis in the Gut Commensal Akkermansia Muciniphila with Bioorthogonal Chemical Reporters

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