Bacterial peptidoglycan is constructed by cross-linking sugar chains carrying pentapeptide building blocks with two o-alanine residues at the C-terminus. Incorporation into the polymer and subsequent breakdown of peptidoglycan releases a tetrapeptide with a single n-alanine residue. Removal of this residue is necessary for the tripeptide to receive a new D-Ala-D-Ala dipeptide in the synthetic pathway, but proteases are generally unable to work with substrates having residues of unusual chirality close to the scissile bond. Processing of the tetrapeptide is carried out by a dedicated to-carboxypeptidase, which is of interest as a novel drug target. We describe the high resolution crystal structure of the enzyme from E. coli, and demonstrate the dimeric structure is highly conserved. (C) 2018 Elsevier Inc. All rights reserved.