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Engineering an Allosteric Binding Site for Aminoglycosides into TEM1-β-Lactamase

Authors

  • Dr. Alexander N. Volkov,

    1. Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 4–5, Boîte 3, 1348 Louvain-la-Neuve (Belgium.), Fax: (+32) 10-472820
    2. Present address: Jean Jeener NMR Center, Structural Biology Brussels, Vrije Universiteit Brussel, Department of Molecular and Cellular Interactions, VUB-VIB, Pleinlaan 2, 1050 Brussels (Belgium)
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    • These authors contributed equally to this work.

  • Dr. Humberto Barrios,

    1. Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 4–5, Boîte 3, 1348 Louvain-la-Neuve (Belgium.), Fax: (+32) 10-472820
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    • These authors contributed equally to this work.

  • Dr. Pascale Mathonet,

    1. Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 4–5, Boîte 3, 1348 Louvain-la-Neuve (Belgium.), Fax: (+32) 10-472820
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    • These authors contributed equally to this work.

  • Dr. Christine Evrard,

    1. Unit of Structural Chemistry (CSTR), Université Catholique de Louvain, Place L. Pasteur 1, 1348 Louvain-la-Neuve (Belgium)
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  • Prof. Marcellus Ubbink,

    1. Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden (The Netherlands)
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  • Prof. Jean-Paul Declercq,

    1. Unit of Structural Chemistry (CSTR), Université Catholique de Louvain, Place L. Pasteur 1, 1348 Louvain-la-Neuve (Belgium)
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  • Prof. Patrice Soumillion,

    Corresponding author
    1. Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 4–5, Boîte 3, 1348 Louvain-la-Neuve (Belgium.), Fax: (+32) 10-472820
    • Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 4–5, Boîte 3, 1348 Louvain-la-Neuve (Belgium.), Fax: (+32) 10-472820
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  • Prof. Jacques Fastrez

    Corresponding author
    1. Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 4–5, Boîte 3, 1348 Louvain-la-Neuve (Belgium.), Fax: (+32) 10-472820
    • Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 4–5, Boîte 3, 1348 Louvain-la-Neuve (Belgium.), Fax: (+32) 10-472820
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Abstract

Allosteric regulation of enzyme activity is a remarkable property of many biological catalysts. Up till now, engineering an allosteric regulation into native, unregulated enzymes has been achieved by the creation of hybrid proteins in which a natural receptor, whose conformation is controlled by ligand binding, is inserted into an enzyme structure. Here, we describe a monomeric enzyme, TEM1-β-lactamase, that features an allosteric aminoglycoside binding site created de novo by directed-evolution methods. β-Lactamases are highly efficient enzymes involved in the resistance of bacteria against β-lactam antibiotics, such as penicillin. Aminoglycosides constitute another class of antibiotics that prevent bacterial protein synthesis, and are neither substrates nor ligands of the native β-lactamases. Here we show that the engineered enzyme is regulated by the binding of kanamycin and other aminoglycosides. Kinetic and structural analyses indicate that the activation mechanism involves expulsion of an inhibitor that binds to an additional, fortuitous site on the engineered protein. These analyses also led to the defining of conditions that allowed an aminoglycoside to be detected at low concentration.

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