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

ABSTRACT: In most bacteria, the essential targets of ?-lactam antibiotics are the d,d-transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4?3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3?3 cross-links generated by l,d-transpeptidases. To gain insight into the characteristics of C. difficile peptidoglycan cross-linking enzymes, we purified the three putative C. difficile l,d-transpeptidase paralogues Ldt_Cd1, Ldt_Cd2, and Ldt_Cd3, which were previously identified by sequence analysis. The catalytic activities of the three proteins were assayed with a disaccharide-tetrapeptide purified from the C. difficile cell wall. Ldt_Cd2 and Ldt_Cd3 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_Cd1 displayed only l,d-carboxypeptidase activity. Mass spectrometry analyses indicated that Ldt_Cd1 and Ldt_Cd2 were acylated by ?-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of Ldt_Cd3 by these ?-lactams was not detected. The acylation efficacy of Ldt_Cd1 and Ldt_Cd2 was higher for the carbapenems (480 to 6,600 M^-1 s^-1) than for ampicillin and ceftriaxone (3.9 to 82 M^-1 s^-1). In contrast, the efficacy of the hydrolysis of ?-lactams by Ldt_Cd1 and Ldt_Cd2 was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that Ldt_Cd1 and Ldt_Cd2 are inactivated only by ?-lactams of the carbapenem class due to a combination of rapid acylation and the stability of the resulting covalent adducts.

SUBMITTER: Sutterlin L 

PROVIDER: S-EPMC5740366 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature