Binding and processing of β‐lactam antibiotics by the transpeptidase LdtMt2 from Mycobacterium tuberculosis

β‐lactam antibiotics represent a novel direction in the chemotherapy of tuberculosis that brings the peptidoglycan layer of the complex mycobacterial cell wall in focus as a therapeutic target. Peptidoglycan stability in Mycobacterium tuberculosis, especially during infection, relies on the nonconventional peptide cross‐links formed by l,d‐transpeptidases. These enzymes are known to be inhibited by β‐lactams, primarily carbapenems, leading to a stable covalent modification at the enzyme active site. A panel of 16 β‐lactam antibiotics was characterized by inhibition kinetics, mass spectrometry, and x‐ray crystallography to identify efficient compounds and study their action on the essential transpeptidase, LdtMt2. Members of the carbapenem class displayed fast binding kinetics, but faropenem, a penem type compound showed a three to four time higher rate in the adduct formation. In three cases, mass spectrometry indicated that carbapenems may undergo decarboxylation, while faropenem decomposition following the acylation step results in a small 87 Da β‐OH‐butyryl adduct bound at the catalytic cysteine residue. The crystal structure of LdtMt2 at 1.54 Å resolution with this fragment bound revealed that the protein adopts a closed conformation that shields the thioester bond from the solvent, which is in line with the high stability of this dead‐end complex observed also in biochemical assays. Database Structural data are available in Protein Data Bank under the accession numbers 5LB1 and 5LBG .