Tryptophan Fluorescence Quenching in β-Lactam-Interacting Proteins Is Modulated by the Structure of Intermediates and Final Products of the Acylation Reaction
Autor: | Mélanie Etheve-Quelquejeu, Fabrice Compain, Jean-Emmanuel Hugonnet, Michel Arthur, Adrian Dupuis, Laetitia Sutterlin, Clément Ourghanlian, Vincent Dubée, Sébastien Triboulet, Heiner Atze, Zainab Edoo |
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Rok vydání: | 2019 |
Předmět: |
0301 basic medicine
Stereochemistry Acylation Imine 030106 microbiology Reaction intermediate beta-Lactams Thioester beta-Lactamases Fluorescence spectroscopy Hydrophobic effect 03 medical and health sciences chemistry.chemical_compound Residue (chemistry) Catalytic Domain Serine polycyclic compounds 030304 developmental biology chemistry.chemical_classification 0303 health sciences Quenching (fluorescence) 030306 microbiology Chemistry Tryptophan Mycobacterium tuberculosis Mesomeric effect 3. Good health Spectrometry Fluorescence 030104 developmental biology Infectious Diseases Enzyme Covalent bond Peptidyl Transferases Lactam |
Zdroj: | ACS Infectious Diseases. 5:1169-1176 |
ISSN: | 2373-8227 |
Popis: | 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. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation. |
Databáze: | OpenAIRE |
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