The membrane anchor of penicillin‐binding protein PBP2a from <i>Streptococcus</i> <i>pneumoniae</i> influences peptidoglycan chain length

Helassa, Nordine; Vollmer, Waldemar; Breukink, Eefjan; Vernet, Thierry; Zapun, André

doi:10.1111/j.1742-4658.2012.08592.x

Cited by 27 publications

(36 citation statements)

References 51 publications

(86 reference statements)

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“…We observed a band at the top of the well (Figure 5b), indicating that the products were highly crosslinked and unable to enter the gel matrix. 44 In order to better analyze the product, we added lysostaphin, an endopeptidase that specifically cleaves S. aureus crosslinks, 45 to the reaction mixture before quenching. The lysostaphin-treated sample showed strong chemiluminescent signals across a wide range of molecular weights, corresponding to BDL-labeled linear peptidoglycan polymers of different lengths (Figure 5b).…”

Section: Resultsmentioning

confidence: 99%

Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams

et al. 2017

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Peptidoglycan is an essential crosslinked polymer that surrounds bacteria and protects them from osmotic lysis. Beta-lactam antibiotics target the final stages of peptidoglycan biosynthesis by inhibiting the transpeptidases that crosslink glycan strands to complete cell wall assembly. Characterization of transpeptidases and their inhibition by beta-lactams has been hampered by lack of access to substrate. We describe a general approach to accumulate Lipid II in bacteria and to obtain large quantities of this cell wall precursor. We demonstrate utility by isolating Staphylococcus aureus Lipid II and reconstituting the synthesis of crosslinked peptidoglycan by the essential penicillin-binding protein 2, PBP2, which catalyzes both glycan polymerization and transpeptidation. We also show that we can compare the potencies of different beta-lactams by directly monitoring transpeptidase inhibition. The methods reported here will enable a better understanding of cell wall biosynthesis and facilitate studies of next-generation transpeptidase inhibitors.

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Section: Resultsmentioning

confidence: 99%

Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams

et al. 2017

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“…Lys-containing lipid II and dansylated lipid II were prepared as described previously (41,42). Amidation of lipid II and subsequent purification was performed as described previously (21).…”

Section: Methodsmentioning

confidence: 99%

Substitutions in PBP2b from β-Lactam-resistant Streptococcus pneumoniae Have Different Effects on Enzymatic Activity and Drug Reactivity

Calvez

Breukink

Roper

et al. 2017

Journal of Biological Chemistry

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Edited by Gerald W. HartPneumococcus resists ␤-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of crosslinking the peptidoglycan, the major constituent of the bacterial cell wall. However, because ␤-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate. This question is addressed for the first time in this study, which compares the peptidoglycan crosslinking activity of PBP2b from susceptible and resistant strains with their inhibition by different ␤-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism.

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“…In vitro complete PG assembly resulting from both GT and TP activities has never been achieved with Gram-positive enzymes, but only with recombinant PBPs from Escherichia coli [11-13]. This is possibly due to the improper nature of the peptide stem available [2,14], or to missing interacting partners.…”

Section: Introductionmentioning

confidence: 99%

Reconstitution of Membrane Protein Complexes Involved in Pneumococcal Septal Cell Wall Assembly

et al. 2013

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The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.

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The membrane anchor of penicillin‐binding protein PBP2a from Streptococcus pneumoniae influences peptidoglycan chain length

Cited by 27 publications

References 51 publications

Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams

Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams

Substitutions in PBP2b from β-Lactam-resistant Streptococcus pneumoniae Have Different Effects on Enzymatic Activity and Drug Reactivity

Reconstitution of Membrane Protein Complexes Involved in Pneumococcal Septal Cell Wall Assembly

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