A distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha and Betaproteobacteria

Most bacteria are surrounded by an essential protective mesh-like structure called peptidoglycan, made of glycan chains crosslinked through short peptides by enzymes known as transpeptidases. Of these, penicillin-binding DD-transpeptidases connect adjacent peptide stems between their 4 th and 3 rd amino acids (4,3-type), D-alanine and a meso-diaminopimelic acid (mDAP) in Gram negatives, whereas LD-transpeptidases make the 3,3-type between mDAP 3 residues. While these two processes explain the formation of crosslinks in most bacteria, recent investigations involving non-model species have brought to light novel crosslinking mechanisms that point to the existence of less-explored groups of peptidoglycan crosslinking enzymes. Here, we present the identification and characterization of a novel LD-transpeptidase found in the acetic acid bacterial Gluconobacter oxydans , named LDT Go , which performs 1,3-type crosslinks between L-Ala 1 and mDAP 3 . LDT Go -like proteins are conserved among Alpha and Betaproteobacteria species that do not encode LD3,3-transpeptidases. Using a highly active ortholog, we demonstrated in vitro that this enzyme can work with non-terminal peptide bonds in the crosslinking process. This property is different from the strict specificity of typical LD- and DD-transpeptidases, which only deal with terminal peptide bonds. The high-resolution crystal structure of LDT Go revealed significant distinctions when compared to 3,3-type LD-transpeptidases. These include a proline-rich region near the N-terminus that restricts substrate access to the active site, and an unprecedented cavity designed to accommodate both the glycan chain and the peptide stem from donor muropeptides, a feature that exhibits broad conservation among LD1,3-transpeptidases. Finally, we demonstrated the involvement of DD-crosslinking turnover in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between structurally distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans .