In VitroCross-Linking of Mycobacterium tuberculosis Peptidoglycan by l,d-Transpeptidases and Inactivation of These Enzymes by Carbapenems

Abstract

ABSTRACTTheMycobacterium tuberculosispeptidoglycan is cross-linked mainly byl,d-transpeptidases (LDTs), which are efficiently inactivated by a single β-lactam class, the carbapenems. Development of carbapenems for tuberculosis treatment has recently raised considerable interest since these drugs, in association with the β-lactamase inhibitor clavulanic acid, are uniformly active against extensively drug-resistantM. tuberculosisand kill both exponentially growing and dormant forms of the bacilli. We have purified the fivel,d-transpeptidase paralogues ofM. tuberculosis(Mt1 to -5) and compared their activities with those of peptidoglycan fragments and carbapenems. The five LDTs were functionalin vitrosince they were active in assays of peptidoglycan cross-linking (Mt5), β-lactam acylation (Mt3), or both (Mt1, Mt2, and Mt4). Mt3 was the only LDT that was inactive in the cross-linking assay, suggesting that this enzyme might be involved in other cellular functions such as the anchoring of proteins to peptidoglycan, as shown inEscherichia coli. Inactivation of LDTs by carbapenems is a two-step reaction comprising reversible formation of a tetrahedral intermediate, the oxyanion, followed by irreversible rupture of the β-lactam ring that leads to formation of a stable acyl enzyme. Determination of the rate constants for these two steps revealed important differences (up to 460-fold) between carbapenems, which affected the velocity of oxyanion and acyl enzyme formation. Imipenem inactivated LDTs more rapidly than ertapenem, and both drugs were more efficient than meropenem and doripenem, indicating that modification of the carbapenem side chain could be used to optimize their antimycobacterial activity.