Mycobacterial cell walls are uniquely complex, comprised of mycolic acids, arabinogalactan and peptidoglycan (PG). The final step in PG biosynthesis involves the crosslinking of stem peptides through meso-diaminopimelic acid and D-alanine (4→3 linked) residues or two meso-DAP residues (3→3 linked) by D,D- and L,D-transpeptidases (Ldts), respectively. Mycobacterial PG is largely 3→3 crosslinked and both the Mycobacterium tuberculosis and Mycobacterium smegmatis genomes contain multiple genes encoding Ldts. In addition to catalyzing 3→3 crosslink formation, Ldts also incorporate non-canonical D-amino acids (NCDAAs) into PG and catalyze attachment of Braun lipoprotein in E. coli. The role these seemingly redundant M. smegmatis Ldts play in the proper maintenance of the cell wall is not well understood, and a previous study indicates mycobacterial Ldts can be grouped into distinct classes on the basis of their protein sequence. Homologs of M. smegmatis LdtA, LdtB, LdtC, LdtE and LdtF are found in M. tuberculosis; however no LdtG homolog is present in M. tuberculosis. Likewise, LdtD is found in M. tuberculosis but not in M. smegmatis, indicating these Ldts may serve unique functions in different mycobacterial species. Here, we present the cloning, overproduction and purification of the M. smegmatis Ldts and show they are acylated by (carba)penem antibiotics. Homology models reveal structural differences among M. smegmatis paralogs and M. tuberculosis homologs. Studies are ongoing to determine the roles these Ldts serve in proper maintenance of M. smegmatis PG.

Biochemical and structural analyses of mycobacterial L,D-transpeptidases

2019 Spring National Meeting

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