Dataset Information

Reversible inactivation of a peptidoglycan transpeptidase by a β-lactam antibiotic mediated by β-lactam-ring recyclization in the enzyme active site.

ABSTRACT: β-lactam antibiotics act as suicide substrates of transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Nucleophilic attack of the β-lactam carbonyl by the catalytic residue (Ser or Cys) of transpeptidases results in the opening of the β-lactam ring and in the formation of a stable acyl-enzyme. The acylation reaction is considered as irreversible due to the strain of the β-lactam ring. In contradiction with this widely accepted but poorly demonstrated premise, we show here that the acylation of the L,D-transpeptidase Ldt_fm from Enterococcus faecium by the β-lactam nitrocefin is reversible, leading to limited antibacterial activity. Experimentally, two independent methods based on spectrophotometry and mass spectrometry provided evidence that recyclization of the β-lactam ring within the active site of Ldt_fm regenerates native nitrocefin. Ring strain is therefore not sufficient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics.

SUBMITTER: Edoo Z

PROVIDER: S-EPMC5567249 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Reversible inactivation of a peptidoglycan transpeptidase by a β-lactam antibiotic mediated by β-lactam-ring recyclization in the enzyme active site.

Edoo Zainab Z Arthur Michel M Hugonnet Jean-Emmanuel JE

Scientific reports 20170822 1

β-lactam antibiotics act as suicide substrates of transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Nucleophilic attack of the β-lactam carbonyl by the catalytic residue (Ser or Cys) of transpeptidases results in the opening of the β-lactam ring and in the formation of a stable acyl-enzyme. The acylation reaction is considered as irreversible due to the strain of the β-lactam ring. In contradiction with this widely accepted but poo ...[more]

PMID: 28831100

Similar Datasets

Project description:Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic-enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their beta-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its beta-lactam ring. In Tris-NaCl-MgCl(2) buffer at pH7.7 and 37 degrees C, the rate constants for the dissociation of the antibiotic-enzyme complexes were 2.8x10(-6), 1.5x10(-6) and 0.63x10(-6)s(-1) (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [(14)C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a beta-lactam-antibiotic-destroying enzyme but did not function as a beta-lactamase. Incubation at 37 degrees C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [(14)C]benzylpenicillin-enzyme complex. The rate constants were 1.6x10(-5)s(-1) and 0.8x10(-4)s(-1) respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.

Dataset Information

Reversible inactivation of a peptidoglycan transpeptidase by a β-lactam antibiotic mediated by β-lactam-ring recyclization in the enzyme active site.

Publications

Reversible inactivation of a peptidoglycan transpeptidase by a β-lactam antibiotic mediated by β-lactam-ring recyclization in the enzyme active site.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets