title: Engineering an allosteric binding site for aminoglycosides into TEM1-β-Lactamase.
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.
authors.affiliation: Laboratoire d'Ingénierie des Protéines et des Peptides, Institut des Sciences de la Vie, Université Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium.
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Volkov
authors.forename: Humberto
authors.initials: H
authors.lastname: Barrios
authors.forename: Pascale
authors.initials: P
authors.lastname: Mathonet
authors.forename: Christine
authors.initials: C
authors.lastname: Evrard
authors.forename: Marcellus
authors.initials: M
authors.lastname: Ubbink
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Declercq
authors.forename: Patrice
authors.initials: P
authors.lastname: Soumillion
authors.forename: Jacques
authors.initials: J
authors.lastname: Fastrez
mesh_terms: D000495:Allosteric Site; D000617:Aminoglycosides / Q000737:chemistry*; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D002151:Calorimetry; D019020:Directed Molecular Evolution; D007612:Kanamycin / Q000737:chemistry; D007700:Kinetics; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D007612:Kanamycin; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1