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Updated: Jun 3, 2026
08:06
The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions
Published on: February 1, 2018
Engineering an allosteric binding site for aminoglycosides into TEM1-β-Lactamase.
Alexander N Volkov1, Humberto Barrios, Pascale Mathonet
1Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium.
Chembiochem : a European Journal of Chemical Biology
|March 23, 2011
Summary
Scientists engineered a new enzyme, TEM1-β-lactamase, with a novel allosteric binding site for aminoglycoside antibiotics. This engineered enzyme demonstrates controlled activity upon kanamycin binding, enabling low-concentration detection.
Area of Science:
- Biochemistry
- Enzyme Engineering
- Synthetic Biology
Background:
- Allosteric enzyme regulation is crucial in biological systems.
- Current methods for engineering allosteric regulation involve creating hybrid proteins.
- Native β-lactamases, like TEM1-β-lactamase, are key in bacterial antibiotic resistance.
Purpose of the Study:
- To engineer a de novo allosteric aminoglycoside binding site into a monomeric enzyme, TEM1-β-lactamase.
- To investigate the mechanism of allosteric regulation by aminoglycosides in the engineered enzyme.
- To establish conditions for detecting aminoglycosides at low concentrations using the engineered enzyme.
Main Methods:
- Directed evolution was employed to create the allosteric binding site.
- Kinetic analyses were performed to study enzyme activity and regulation.
- Structural analyses were utilized to elucidate the binding and activation mechanism.
Main Results:
- An allosteric binding site for aminoglycosides (e.g., kanamycin) was successfully engineered into TEM1-β-lactamase.
- Aminoglycoside binding was shown to regulate the enzyme's activity.
- The activation mechanism involves the expulsion of an inhibitor from a fortuitous binding site.
- Conditions for sensitive detection of aminoglycosides were determined.
Conclusions:
- De novo engineering of allosteric regulation into enzymes is feasible using directed evolution.
- The engineered TEM1-β-lactamase provides a novel tool for studying allosteric mechanisms and detecting aminoglycosides.
- This work expands the possibilities for enzyme engineering and antibiotic resistance research.
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