Geneviève Auger

Crystal structure of UDP-N-acetylmuramoyl-L- alanine:D-glutamate ligase from Escherichia coli

UDP‐N‐acetylmuramoyl‐L‐alanine:D‐glutamate ligase (MurD) is a cytoplasmic enzyme involved in the biosynthesis of peptidoglycan which catalyzes the addition of D‐glutamate to the nucleotide precursor UDP‐N‐acetylmuramoyl‐L‐alanine (UMA). The crystal structure of MurD in the presence of its substrate UMA has been solved to 1.9 Å resolution. Phase information was obtained from multiple anomalous dispersion using the K‐shell edge of selenium in combination with multiple isomorphous replacement. The structure comprises three domains of topology each reminiscent of nucleotide‐binding folds: the N‐ and C‐terminal domains are consistent with the dinucleotide‐binding fold called the Rossmann fold, and the central domain with the mononucleotide‐binding fold also observed in the GTPase family. The structure reveals the binding site of the substrate UMA, and comparison with known NTP complexes allows the identification of residues interacting with ATP. The study describes the first structure of the UDP‐N‐acetylmuramoyl‐peptide ligase family.

Colicin M Exerts Its Bacteriolytic Effect via Enzymatic Degradation of Undecaprenyl Phosphate-linked Peptidoglycan Precursors

Colicin M was earlier demonstrated to provoke Escherichia coli cell lysis via inhibition of cell wall peptidoglycan (murein) biosynthesis. As the formation of the O-antigen moiety of lipopolysaccharides was concomitantly blocked, it was hypothesized that the metabolism of undecaprenyl phosphate, an essential carrier lipid shared by these two pathways, should be the target of this colicin. However, the exact target and mechanism of action of colicin M was unknown. Colicin M was now purified to near homogeneity, and its effects on cell wall peptidoglycan metabolism reinvestigated. It is demonstrated that colicin M exhibits both in vitro and in vivo enzymatic properties of degradation of lipid I and lipid II peptidoglycan intermediates. Free undecaprenol and either 1-pyrophospho-MurNAc-pentapeptide or 1-pyrophospho-MurNAc-(pentapeptide)-Glc-NAc were identified as the lipid I and lipid II degradation products, respectively, showing that the cleavage occurred between the lipid moiety and the pyrophosphoryl group. This is the first time such an activity is described. Neither undecaprenyl pyrophosphate nor the peptidoglycan nucleotide precursors were substrates of colicin M, indicating that both undecaprenyl and sugar moieties were essential for activity. The bacteriolytic effect of colicin M therefore appears to be the consequence of an arrest of peptidoglycan polymerization steps provoked by enzymatic degradation of the undecaprenyl phosphate-linked peptidoglycan precursors.

Colicin M hydrolyses branched lipids II from Gram-positive bacteria.

Lipids II found in some Gram-positive bacteria were prepared in radioactive form from l-lysine-containing UDP-MurNAc-pentapeptide. The specific lateral chains of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus (di-L-alanine, D-isoasparagine, and pentaglycine, respectively) were introduced by chemical peptide synthesis using the Fmoc chemistry. The branched nucleotides obtained were converted into the corresponding lipids II by enzymatic synthesis using the MraY and MurG enzymes. All of the lipids were hydrolysed by Escherichia coli colicin M at approximately the same rate as the meso-diaminopimelate-containing lipid II found in Gram-negative bacteria, thereby opening the way to the use of this enzyme as a broad spectrum antibacterial agent.