Peptidoglycan Cross-Linking Activity of l,d-Transpeptidases from Clostridium difficile and Inactivation of These Enzymes by β-Lactams.

In most bacteria, the essential targets of β-lactam antibiotics are the d,d-transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4→3 cross-links. The peptidoglycan of is unusual since it mainly contains 3→3 cross-links generated by l,d-transpeptidases. To gain insight into the characteristics of peptidoglycan cross-linking enzymes, we purified the three putative l,d-transpeptidase paralogues Ldt, Ldt, and Ldt, which were previously identified by sequence analysis. The catalytic activities of the three proteins were assayed with a disaccharide-tetrapeptide purified from the cell wall. Ldt and Ldt catalyzed the formation of 3→3 cross-links (l,d-transpeptidase activity), the hydrolysis of the C-terminal d-Ala residue of the disaccharide-tetrapeptide substrate (l,d-carboxypeptidase activity), and the exchange of the C-terminal d-Ala for d-Met. Ldt displayed only l,d-carboxypeptidase activity. Mass spectrometry analyses indicated that Ldt and Ldt were acylated by β-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of Ldt by these β-lactams was not detected. The acylation efficacy of Ldt and Ldt was higher for the carbapenems (480 to 6,600 M s) than for ampicillin and ceftriaxone (3.9 to 82 M s). In contrast, the efficacy of the hydrolysis of β-lactams by Ldt and Ldt was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that Ldt and Ldt are inactivated only by β-lactams of the carbapenem class due to a combination of rapid acylation and the stability of the resulting covalent adducts.