Critical Impact of Peptidoglycan Precursor Amidation on the Activity of <scp>l,d</scp>‐Transpeptidases from <i>Enterococcus faecium</i> and <i>Mycobacterium tuberculosis</i>

Ngadjeua, Flora; Braud, Emmanuelle; Saidjalolov, Saidbakhrom; Iannazzo, Laura; Schnappinger, Dirk; Ehrt, Sabine; Hugonnet, Jean-Emmanuel; Mengin‐Lecreulx, Dominique; Patin, Delphine; Ethève-Quelquejeu, Mélanie; Fonvielle, Matthieu; Arthur, Michel

doi:10.1002/chem.201706082

Cited by 46 publications

(48 citation statements)

References 15 publications

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“…Fmoc-D-Glu-NHTrt and D-LacO t Bu were synthetized as previously described (see Supporting Information in Ngadjeua et al, 2018). Orthogonal Fmoc and 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl (ivDde) protecting groups were used for sequential assembly of the branched peptide stem (see Ngadjeua et al, 2018 for similar peptides containing a D-iAsn bridge). First, the main peptide stem (D-Lac-L-Ala-D-iGln-L-Lys-D-Ala 1or2 ) was elongated from the Wang-D-Ala resin by successive coupling reactions with Fmoc-protected amino-acids.…”

Section: Methodsmentioning

confidence: 99%

Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus

et al. 2019

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Peptidoglycan (PG) is an essential component of the cell envelope, maintaining bacterial cell shape and protecting it from bursting due to turgor pressure. The monoderm bacterium Staphylococcus aureus has a highly cross-linked PG, with ~90% of peptide stems participating in DD-cross-links and up to 15 peptide stems connected with each other. These cross-links are formed in transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs) of classes A and B. Most S. aureus strains have three housekeeping PBPs with this function (PBP1, PBP2, and PBP3) but MRSA strains have acquired a third class B PBP, PBP2a, which is encoded by the mecA gene and required for the expression of high-level resistance to β-lactams. Another housekeeping PBP of S. aureus is PBP4, which belongs to the class C PBPs, and hence would be expected to have PG hydrolase (DD-carboxypeptidase or DD-endopeptidase) activity. However, previous works showed that, unexpectedly, PBP4 has transpeptidase activity that significantly contributes to both the high level of cross-linking in the PG of S. aureus and to the low level of β-lactam resistance in the absence of PBP2a. To gain insights into this unusual activity of PBP4, we studied by NMR spectroscopy its interaction in vitro with different substrates, including intact peptidoglycan, synthetic peptide stems, muropeptides, and long glycan chains with uncross-linked peptide stems. PBP4 showed no affinity for the complex, intact peptidoglycan or the smallest isolated peptide stems. Transpeptidase activity of PBP4 was verified with the disaccharide peptide subunits (muropeptides) in vitro, producing cyclic dimer and multimer products; these assays also showed a designed PBP4(S75C) nucleophile mutant to be inactive. Using this inactive but structurally highly similar variant, liquid-state NMR identified two interaction surfaces in close proximity to the central nucleophile position that can accommodate the potential donor and acceptor stems for the transpeptidation reaction. A PBP4:muropeptide model structure was built from these experimental restraints, which provides new mechanistic insights into mecA independent resistance to β-lactams in S. aureus.

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Section: Methodsmentioning

confidence: 99%

Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus

et al. 2019

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“…However, the amidation of the mDAP residues in mycobacteria is essential for the formation of LD-cross-links (Ngadjeua et al . 2018). Additionally, the presence of zones of uncontrolled PG degradation in the ΔasnB mutant of B. subtilis suggests that mDAP amidation controls the activity of hydrolytic enzymes such as autolysins (Dajkovic et al .…”

Section: Introductionmentioning

confidence: 99%

Cell wall peptidoglycan inMycobacterium tuberculosis: An Achilles’ heel for the TB-causing pathogen

Maitra

Munshi

Healy³

et al. 2019

FEMS Microbiology Reviews

156

122

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Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis, remains one of the leading causes of mortality across the world. There is an urgent requirement to build a robust arsenal of effective antimicrobials, targeting novel molecular mechanisms to overcome the challenges posed by the increase of antibiotic resistance in TB. Mycobacterium tuberculosis has a unique cell envelope structure and composition, containing a peptidoglycan layer that is essential for maintaining cellular integrity and for virulence. The enzymes involved in the biosynthesis, degradation, remodelling and recycling of peptidoglycan have resurfaced as attractive targets for anti-infective drug discovery. Here, we review the importance of peptidoglycan, including the structure, function and regulation of key enzymes involved in its metabolism. We also discuss known inhibitors of ATP-dependent Mur ligases, and discuss the potential for the development of pan-enzyme inhibitors targeting multiple Mur ligases.

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“…54,55 Also, during the preparation of our manuscript it was shown, in vitro , that Ldts mediate cross-linking of synthetic Ldt substrates. 56 In contrast, there are currently no Ldt-specific probes to tag and visualize Ldt activity in live cells. We assembled a synthetic substrate of Ldt to specifically interrogate Ldt activity and better understand how the primary sequence of the stem peptide can modulate Ldt-mediated cross-linking.…”

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confidence: 99%

L,D-Transpeptidase Specific Probe Reveals Spatial Activity of Peptidoglycan Cross-Linking

et al. 2019

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Peptidoglycan (PG) is a cross-linked, meshlike scaffold endowed with the strength to withstand the internal pressure of bacteria. Bacteria are known to heavily remodel their peptidoglycan stem peptides, yet little is known about the physiological impact of these chemical variations on peptidoglycan cross-linking. Furthermore, there are limited tools to study these structural variations, which can also have important implications on cell wall integrity and host immunity. Cross-linking of peptide chains within PG is an essential process, and its disruption thereof underpins the potency of several classes of antibiotics. Two primary cross-linking modes have been identified that are carried out by D,D-transpeptidases and L,D-transpeptidases (Ldts). The nascent PG from each enzymatic class is structurally unique, which results in different cross-linking configurations. Recent advances in PG cellular probes have been powerful in advancing the understanding of D,D-transpeptidation by Penicillin Binding Proteins (PBPs). In contrast, no cellular probes have been previously described to directly interrogate Ldt function in live cells. Herein, we describe a new class of Ldt-specific probes composed of structural analogs of nascent PG, which are metabolically incorporated into the PG scaffold by Ldts. With a panel of tetrapeptide PG stem mimics, we demonstrated that subtle modifications such as amidation of iso-Glu can control PG cross-linking. Ldt probes were applied to quantify and track the localization of Ldt activity in Enterococcus faecium, Mycobacterium smegmatis, and Mycobacterium tuberculosis. These results confirm that our Ldt probes are specific and suggest that the primary sequence of the stem peptide can control Ldt cross-linking levels. We anticipate that unraveling the interplay between Ldts and other cross-linking modalities may reveal the organization of the PG structure in relation to the spatial localization of cross-linking machineries.

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Critical Impact of Peptidoglycan Precursor Amidation on the Activity of l,d‐Transpeptidases from Enterococcus faecium and Mycobacterium tuberculosis

Cited by 46 publications

References 15 publications

Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus

Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus

Cell wall peptidoglycan inMycobacterium tuberculosis: An Achilles’ heel for the TB-causing pathogen

L,D-Transpeptidase Specific Probe Reveals Spatial Activity of Peptidoglycan Cross-Linking

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