tailieunhanh - Báo cáo khoa học: Broad antibiotic resistance profile of the subclass B3 metallo-b-lactamase GOB-1, a di-zinc enzyme

The metallo-b-lactamase (MBL) GOB-1 was expressed via a T7 expression system in Escherichia coliBL21(DE3). The MBL was purified to homoge-neity and shown to exhibit a broad substrate profile, hydrolyzing all the tested b-lactam compounds efficiently. | 1FEBS Journal Broad antibiotic resistance profile of the subclass B3 metallo-p-lactamase GOB-1 a di-zinc enzyme Louise E. Horsfall1 Youssef Izougarhane1 Patricia Lassaux1 Nathalie Selevsek2 Benoit M. R. Lienard3 Laurent Poirel4 Michael B. Kupper5 Kurt M. Hoffmann5 Jean-Marie Frere1 Moreno Galleni1 and Carine Bebrone1 1 Centre d Ingenierie des Proteines Universite de Liege Belgium 2 Department of BiochemicalEngineering Saarland University Saarbrucken Germany 3 Chemistry Research Laboratory University of Oxford UK 4 Service de Bacteriologie-Virologie INSERM U914 Emerging Resistance to Antibiotics Hopitalde Bicetre Assistance Publique Hopitaux de Paris Faculte de Medecine Paris Sud France 5 Institute of Molecular Biotechnology RWTH-Aachen University Germany Keywords antibiotic resistance GOB metallo-b-lactamase zinc-binding site b-lactamase Correspondence C. Bebrone Centre d lngenierie des Proteines Universite de Liege Allee de 6 Aout B6 Sart-Tilman Liege Belgium Fax 32 43 663 364 Tel 32 43 663 348 E-mail Website http Received 16 September 2010 revised 5 January 2011 accepted 4 February 2011 doi The metallo-b-lactamase MBL GOB-1 was expressed via a T7 expression system in Escherichia coli BL21 DE3 . The MBL was purified to homogeneity and shown to exhibit a broad substrate profile hydrolyzing all the tested p-lactam compounds efficiently. The GOB enzymes are unique among MBLs due to the presence of a glutamine residue at position 116 a zinc-binding residue in all known class B1 and B3 MBL structures. Here we produced and studied the Q116A Q116N and Q116H mutants. The substrate profiles were similar for each mutant but with significantly reduced activity compared with that of the wild-type. In contrast to the Q116H enzyme which bound two zinc ions just like the wild-type only one zinc ion is present in Q116A and Q116N. These results suggest that the Q116 residue plays a role in the .