DOI: 10.1101/472381Nov 19, 2018Paper

Tryptophan fluorescence quenching in β-lactam-interacting proteins is modulated by the structure of intermediates and final products of the acylation reaction

BioRxiv : the Preprint Server for Biology
Sébastien TribouletM Arthur

Abstract

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by L,D-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model L,D-transpeptidase (Ldtfm) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldtfm. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis . Fluoresc...Continue Reading

Related Concepts

Anions
Binding Sites, Antibody
Cysteine
Molecular Probe Techniques
Monobactams
Genus Mycobacterium
Mycobacterium tuberculosis
Penicillin G
Peptidoglycan
Peptidyltransferase

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