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

beta-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 beta-lactams, primarily carbapenems, leading to a stable covalent modification at the enzyme active site. A panel of 16 b-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, Ldt(Mt2). 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 beta-OH-butyryl adduct bound at the catalytic cysteine residue. The crystal structure of Ldt(Mt2) at 1.54 angstrom 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.