Structure and Function of the First Full-Length Murein Peptide Ligase (Mpl) Cell Wall Recycling Protein

Abstract

Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships.

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4

“…The region downstream to parI, encoding the gene with the locus tag Psyc_0981 is similar to the MU phage [18]. In addition to explaining the orphan status of ParI, horizontal gene transfer of parI into P. arcticus by a phage may explain why ParI does not exhibit typical cold-adapted features previously documented for other Psychrobacter enzymes such as the branched-chain 2-keto acid decarboxylase and murein peptide ligase which have optimal temperature of activity at 30°C [52,53].…”

Lasso Peptides and Murein Peptide Ligase Inhibitors as Novel Anti-Mycobacterial Agents

“…The choice of potential inhibitors of Mpl was based on structural analogy with the putative natural peptide substrates identified in E.coli [4,5]. These small peptides have been synthesised via solid-phase synthesis and have been tested using M. aurum through the SpotI whole-cell high-throughput screening assay [6].…”

“…Recently, among the class of lasso peptides, interesting stable cyclic peptides of bacterial origin were reported, including the anti-mycobacterial lariatins A and B, followed by the newly-discovered anti-TB pseudo-lasso peptide lassomycin [1][2][3]. Furthermore the identification in M. tuberculosis of an orthologue of an enzyme found in E. coli [4,5] involved in peptidoglycan recycling encouraged us to try to identify and evaluate its activity and find potential inhibitors. …”

Bacterial cell‐wall recycling

“…However, Mpl is known to accept the tri‐, tetra‐, and pentapeptides 7a , 7b , and 7c equally well and the ligation with 7c produces 2 directly 130 . A crystal structure of Mpl was solved recently 131 . A minor pathway for recycling of the tripeptide 7c involves its breakdown into the individual amino acids by the sequential action of MpaA, YcjG, and PepD.…”

“…130 A crystal structure of Mpl was solved recently. 131 A minor pathway for recycling of the tripeptide 7c involves its breakdown into the individual amino acids by the sequential action of MpaA, YcjG, and PepD. MpaA amidase cleaves the ␥ -d-Glu−m-DAP bond of 7c to generate l-Ala-␥d-Glu and m-DAP.…”