Biochemical Characterization and Physiological Properties ofEscherichia coliUDP-N-Acetylmuramate:l-Alanyl-γ-d-Glutamyl-meso- Diaminopimelate Ligase

Abstract

The UDP-N-acetylmuramate:L-alanyl-␥-D-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptide L-alanyl-␥-D-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme from Escherichia coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptide L-Ala-D-Glu and L-Ala very poorly. Replacement of meso-diaminopimelic acid by L-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of the E. coli mpl gene and/or the ldcA gene encoding the LD-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra-and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.The biosynthesis of bacterial cell wall peptidoglycan (murein) is a complex process involving many cytoplasmic and membrane steps (for a review, see reference 54). The main cytoplasmic precursors are a series of seven nucleotide compounds, ranging from UDP-N-acetylglucosaminewhose sequential formation is catalyzed by a set of highly specific enzymes designated the Mur synthetases MurA to MurF (54). Subsequent steps, which occur in the membrane, consist of the transfer of the MurNAc-pentapeptide and GlcNAc motifs to the undecaprenyl phosphate carrier lipid, generating lipid II, which is then translocated to the outer side of the membrane and used for polymerization reactions catalyzed by the penicillin-binding proteins.The cell wall should not be considered a static structure since permanent remodeling necessarily occurs during cell growth and division. This is believed to a result of balanced functioning of murein-hydrolyzing and murein-synthesizing activities, with the former degrading the old peptidoglycan structure to allow insertion of new material. In E. coli, as many as 18 murein hydrolases have been identified, and these enzymes belong to six different families and include lytic transglycosylases, amidases, and endopeptidases; there is a specific hydrolase for almost every covalent bond in the murein (24). The remodeling of the murein by these different "autolysins" results in dramatic turnover, estimated at 40 to 50% per generation time. Some cell wall peptides are rel...

Peptidoglycan Hydrolases of Escherichia coli

“…A similar phenotype was observed with dithiazoline-inhibited LdcA (10). In the absence of LdcA activity due to mutation or inhibition, the L-Ala-␥-D-Glu-(L)-meso-A 2 pm-(L)-D-Ala tetrapeptide accumulates and is used by Mpl to form UDPMurNAc-tetrapeptide (96,256). Tetrapeptide subunits are incorporated into PG instead of pentapeptide subunits, leading to a cross-linkage deficiency and lysis.…”

“…The incorporated fragments are further degraded by the cytoplasmic AmpD, NagZ, and LdcA hydrolases into the L-Ala-␥-D-Glu-(L)-meso-A 2 pm tripeptide, D-alanine, GlcNAc, and 1,6-anhydro-MurNAc (196). The tripeptide is ligated into UDP-MurNAc by the Mpl ligase, and the resulting tripeptide nucleotide precursor returns to the biosynthesis pathway (96,176). GlcNAc and 1,6-anhydro-MurNAc are both converted into GlcNAc-6-phosphate, which is available for reuse (119,196,204).…”

How Bacteria Consume Their Own Exoskeletons (Turnover and Recycling of Cell Wall Peptidoglycan)

“…Those authors' explanation for this phenotype is that in the absence of LdcA, tetrapeptide accumulates and is used by Mpl to form UDP-MurNAc-tetrapeptide (115). In fact, as noted above, the tetrapeptide is a good substrate for Mpl (46). The MurNAc-tetrapeptide is incorporated into the murein sacculus instead of MurNAc-pentapeptide.…”

“…Mpl was shown to utilize tripeptide, tetrapeptide, and pentapeptide with similar efficiencies (46). Mpl also accepts peptides containing lysine in place of Dap (5).…”

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…LdcA carboxypeptidase cleaves the terminal D-alanine from a tetrapeptide resulting in tripeptides [166]. In E. coli, murein peptide ligase (Mpl) recycles the tri-, tetra-and pentapeptides by ligating them to UDP-MurNAc [179,180]. Another enzyme, MurF can also ligate dipeptide D-Ala-D-Ala (a product of ATP-dependent ligase Ddl), to a UDP-MurNAc tripeptide to form a UDP-MurNAc pentapeptide [181][182][183].…”