Kinetic features of L,D-transpeptidase inactivation critical for β-lactam antibacterial activity
- PMID: 23861815
- PMCID: PMC3701632
- DOI: 10.1371/journal.pone.0067831
Kinetic features of L,D-transpeptidase inactivation critical for β-lactam antibacterial activity
Abstract
Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldt(fm)) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldt(fm) does not confer resistance to β-lactams of the carbapenem class (imipenem MIC = 0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldt(fm) by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldt(fm) inactivation by ampicillin and ceftriaxone. For ampicillin, Ldt(fm) acylation was followed by rupture of the C(5)-C(6) bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldt(fm) blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.
Conflict of interest statement
Figures
![Figure 1](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3701632/bin/pone.0067831.g001.gif)
![Figure 2](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3701632/bin/pone.0067831.g002.gif)
![Figure 3](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3701632/bin/pone.0067831.g003.gif)
![Figure 4](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3701632/bin/pone.0067831.g004.gif)
![Figure 5](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3701632/bin/pone.0067831.g005.gif)
![Figure 6](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3701632/bin/pone.0067831.g006.gif)
References
-
- Zapun A, Contreras-Martel C, Vernet T (2008) Penicillin-binding proteins and beta-lactam resistance. FEMS Microbiol Rev 32: 361–385. - PubMed
-
- Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P (2008) The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev 32: 234–258. - PubMed
-
- Mainardi JL, Villet R, Bugg TD, Mayer C, Arthur M (2008) Evolution of peptidoglycan biosynthesis under the selective pressure of antibiotics in Gram-positive bacteria. FEMS Microbiol Rev 32: 386–408. - PubMed
-
- Mainardi JL, Legrand R, Arthur M, Schoot B, van Heijenoort J, et al. (2000) Novel mechanism of beta-lactam resistance due to bypass of DD-transpeptidation in Enterococcus faecium . J Biol Chem 275: 16490–16496. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources