Activity Profiling of Mycobacterial L,D-Transpeptidases

) and non-tuberculous mycobacteria (NTM). l,d-Transpeptidases (Ldts) are attractive drug targets due to their essential role in peptidoglycan cell wall crosslinking, yet existing assays suffer from low throughput and limited sensitivity. We report a versatile, bead-based platform for high-throughput analysis of Ldt activity and inhibitor discovery. We incubated peptidoglycan stem peptides, either naturally harvested or synthetically immobilized on abiotic surfaces, with Ldts and a fluorescent acyl acceptor to quantitatively monitor crosslinking. After optimizing assay parameters, we profiled six Mycobacterium smegmatis Ldt paralogs, including the first characterization of a class 6 Ldt with chemically defined substrate sequences. Utilizing a series of acyl acceptors, we demonstrated modifications within the acyl acceptor that are tolerated by mycobacterial Ldts. Screening of β-lactam antibiotics revealed potent inhibition by (carba)penems, while cephalosporins, monobactams and penams showed negligible activity. The assay achieved excellent performance metrics and was successfully adapted to ELISA and 96-well formats, providing a powerful tool for discovering Ldt-targeted therapeutics against tuberculosis and related infections.