Transport and catabolism of murein tripeptide in Escherichia coli K-12

In bacteria, extracellular peptides are not only a source of nutrients but also play important roles in cell-cell communication. The primary mode of uptake of these peptides in bacteria is via ATP Binding Cassette (ABC) transporters. The substrate-binding protein (SBP) of these transporters captures extracellular peptides and delivers them to membrane-bound components for transport. Bacterial peptide-binding SBPs that function in ABC transporters have evolved predominantly to function as generalists, recognising peptides of particular lengths but with low or no sequence specificity. However, within the peptide-specific SBPs are examples that appear to have evolved subsequently to recognise specific peptides. Here we have provided the first biophysical evidence, using native electrospray mass spectrometry, for the binding of a specific peptide to an Escherichia coli SBP. This peptide is a cell wall component named murein tripeptide (Mtp, L-Ala--D-Glu-meso-Dap) and is transported by an ABC transporter containing the SBP MppA. We demonstrate that MppA recognises Mtp specifically and with high affinity (KD ~ 250 nM), as determined by protein fluorescence spectroscopy and isothermal titration calorimetry. The crystal structure of MppA in complex with Mtp has been solved in order to understand the structural basis of specific binding of Mtp to MppA. Comparison of the structure of MppA-Mtp with structures of general tripeptides bound to oligopeptide binding protein OppA, reveals close similarity in the protein chains which fold to form an enclosed interdomain pocket in which the respective peptides reside. The peptide ligands superimpose remarkably closely given the profound differences in their structure. Strikingly, the effect of the D-stereochemistry, which projects the side chain of residue 2 in Mtp in the direction of the main chain in a conventional tripeptide, is compensated by the side chain γ-linkage of the carboxylate of the D-Glu to the amino group of diaminopimelic acid. The resulting amide linkage mimicks the peptide bond between residues 2 and 3 of a conventional tripeptide. Specificity for Mtp is conferred by a series of ionic and polar residues of MppA which make charge-charge and dipole-charge interactions with the highly ionised peptide. Analysis of multiple bacterial genomes revealed that mppA is often situated near a gene called mpaA which encodes a putative amidase enzyme which is thought to hydrolyze murein tripeptide. This genetic organization is conserved among -proteobacteria and suggests that mppA and mpaA function together and are a part of peptide catabolic pathway. The MpaA enzyme from E. coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. MpaA was found to hydrolyze Mtp efficiently but it did not cleave murein tetrapeptide. Replacement of meso-Dap by L-Lys in the tripeptide resulted in much lower efficiency. Mass spectrometric analysis of purified MpaA suggested that it co-purifies with a zinc ion and exists as a dimer in solution. The first crystal structure of an MpaA protein from Vibrio harveyi was solved to 2.17A. The structural fold of V. harveyi MpaA was similar to eukaryotic carboxypeptidases. Analysis of the structure showed an obvious dimer interface between two monomers of MpaA and presence of a zinc ion in the active centre of each monomer consistent with the mass spectrometric data. The structure provides the basis for future modelling and mutational studies for extensive functional characterization of MpaA.