Avibactam (AVI) Inactivation of PER-2 and CTX-M-151 Extended-Spectrum beta-Lactamases (ESBLs)

P. POWER; M. RUGGIERO; B. GHIGLIONE; M. M. RODRIGUEZ; K. M. PAPP-WALLACE; G. GUTKIND; Y. ISHII; R. A. BONOMO; S. KLINKE

Congreso; ASM Microbe 2017; 2017

Avibactam (AVI) Inactivation of PER-2 and CTX-M-151 Extended-Spectrum β-Lactamases (ESBLs)P. Power1, B. Ghiglione1, M. Ruggiero1, M. M. Rodríguez1, K. M. Papp-Wallace2, G. Gutkind1, Y. Ishii3, R. A. Bonomo2, S. Klinke41 Laboratorio de Resistencia Bacteriana, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.2 Cleveland VAMC, Cleveland, OH, USA.3 Department of Microbiology and Infectious Diseases, Toho University, Tokyo, Japan4 Fundación Instituto Leloir, Buenos Aires, ArgentinaBackground: AVI is a reversible diazabicyclo[3.2.1]octanone, DBO, β-lactamase inhibitor (BLI) that inactivates class A and C β-lactamases. As the diversity of class A and C β-lactamases is ever increasing, understanding the mechanistic and structural basis of inhibition by AVI can give insight into future resistance development. PER-2 β-lactamase possesses unique structural features that enlarge the active site entrance by 2-fold vs. other class A β-lactamases. Uniquely, an H-bonding (HB) network is also observed and may play a role in their different catalytic profiles. CTX-M-151 possesses 70% amino acid identity with closest CTX-M β-lactamases. It demonstrates a higher catalytic efficiency towards cefepime, and 5X higher KI value of clavulanic acid, compared to CTX-M-9. Our goal was to provide insights into the ability of AVI to inhibit these singular β-lactamases and to probe the mechanism of inhibition. Methods: Crystals of PER-2 and CTX-M-151 in complex with AVI were generated. X-ray diffraction was measured at PLABEM (Argentina) and Soleil synchrotron (France). Indexing, integration and scaling were performed with XDS, and the structures were solved with Phaser. Refinement (Phenix) and model building (Coot) were finally performed. Results: The structure of CTX-M-151 in complex with AVI was refined at 1.3 Å. Here, AVI adopted an equivalent position in the active site of CTX-M-151 as CTX-M-15, showing the carbonyl oxygen pointing towards the oxyanion hole and close HB interactions with K73, N104, S130, N132, T235, and S237. The structure of PER-2/AVI complex refined at 2.4 Å showed 4 monomers/asu. In contrast, we observed that the W105 side chain moved >5 Å towards the active site upon binding of AVI; this is accompanied by a ~4-Å shift of T104 side chain that forms a new HB with H170. This residue, belonging to the inverted -loop, lies in a favored position by HB with T104, N132 and E166 carbonyl revealed. AVI also formed the same other HB interactions as CTX-M-151. Conclusion: Insights into the structures of both PER-2 and CTX-M-151 in complex with AVI reveal distinctive interactions of AVI with residues in the active site of these two ESBLs. In the case of PER-2, the significant rearrangements and movement of residues such as W105 and T104 upon binding of AVI is unprecedented compared to other class A enzymes.