The l,d-Transpeptidase Ldt from Is Poorly Inhibited by Carbapenems and Has a Unique Structural Architecture.

l,d-Transpeptidases (LDTs) are enzymes that catalyze reactions essential for biogenesis of the bacterial cell wall, including formation of 3-3 cross-linked peptidoglycan. Unlike the historically well-known bacterial transpeptidases, the penicillin-binding proteins (PBPs), LDTs are resistant to inhibition by the majority of β-lactam antibiotics, with the exception of carbapenems and penems, allowing bacteria to survive in the presence of these drugs. Here we report characterization of Ldt from the clinically important pathogen, . We show that survives inactivation of Ldt alone or in combination with PBP1b or PBP2, while simultaneous inactivation of Ldt and PBP1a is lethal. Minimal inhibitory concentrations (MICs) of all 13 β-lactam antibiotics tested decreased 2- to 8-fold for the Ldt deletion mutant, while further decreases were seen for both double mutants, with the largest, synergistic effect observed for the Ldt + PBP2 deletion mutant. Mass spectrometry experiments showed that Ldt forms complexes in vitro only with carbapenems. However, the acylation rate of these antibiotics is very slow, with the reaction taking longer than four hours to complete. Our X-ray crystallographic studies revealed that Ldt has a unique structural architecture and is the only known LDT to have two different peptidoglycan-binding domains.