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Engineering an Allosteric Binding Site for Aminoglycosides into TEM1‐β‐Lactamase
Author(s) -
Volkov Alexander N.,
Barrios Humberto,
Mathonet Pascale,
Evrard Christine,
Ubbink Marcellus,
Declercq JeanPaul,
Soumillion Patrice,
Fastrez Jacques
Publication year - 2011
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201000568
Subject(s) - allosteric regulation , enzyme , allosteric enzyme , protein engineering , binding site , chemistry , biochemistry , active site , stereochemistry
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.