Loss of a functionally and structurally distinct LD-transpeptidase, Ldt_Mt5, compromises cell wall integrity in mycobacterium tuberculosis

The final step of peptidoglycan (PG) biosynthesis in bacteria involves cross-linking of peptide side chains. This step in Mycobacterium tuberculosis is catalyzed by LD- and DD-transpeptidases that generate 3→3 and 4→3 transpeptide linkages, respectively. M. tuberculosis PG is predominantly 3→3 cross-linked, and Ldt_Mt2 is the dominant LD-transpeptidase. There are four additional sequence paralogs of Ldt_Mt2 encoded by the genome of this pathogen, and the reason for this apparent redundancy is unknown. Here, we studied one of the paralogs, Ldt_Mt5 , and found it to be structurally and functionally distinct. The structures of apo-LdtMt5 and its meropenem adduct presented here demonstrate that, despite overall architectural similarity to Ldt_Mt2 , the LdtMt5 active site has marked differences. The presence of a structurally divergent catalytic site and a proline-rich C-terminal subdomain suggest that this protein may have a distinct role in PG metabolism, perhaps involving other cell wall-anchored proteins. Furthermore, M. tuberculosis lacking a functional copy of LdtMt5 displayed aberrant growth and was more susceptible to killing by crystal violet, osmotic shock, and select carbapenem antibiotics. Therefore, we conclude that Ldt_Mt5 is not a functionally redundant LD-transpeptidase, but rather it serves a unique and important role in maintaining the integrity of the M. tuberculosis cell wall.