Crystal structure of CTX-M-151 extended-spectrum beta-lactamase in complex with Avibactam (AVI) (Póster)

KRISZTINA M. PAPP-WALLACE; ROBERT A. BONOMO; BÁRBARA GHIGLIONE; YOSHIKAZU ISHII; PABLO POWER; MARÍA M. RODRÍGUEZ; GABRIEL GUTKIND; SEBASTIÁN KLINKE

Santo Stefano di Sessanio

Congreso; 13th Beta-Lactamase Meeting; 2017

University of L'Aquila

Background: AVI is a reversible diazabicyclooctane, DBO, beta-lactamase inhibitor (BLI) that inactivates class A and C beta-lactamases. As the diversity of class A and C beta-lactamases is ever increasing, understanding the mechanistic and structural basis of inhibition by AVI can give insight into future resistance development. CTX-M-151 possesses 70% amino acid identity with closest CTX-M beta-lactamases, demonstrates a higher catalytic efficiency towards cefepime, and is more difficult to inhibit with clavulanic acid compared to CTX-M-9. We have observed that resistance to inactivation by clavulanic acid is also accompanied by high inhibition constants. Beta-lactamases resistant to inhibition by clavulanic acid (e.g., SHV-1 and KPC-2 S130 G variants) can also be less responsive to AVI.  Our goal was to provide insights into the ability of AVI to inhibit the CTX-M-151 beta-lactamase and to probe the mechanism of inhibition.Methods: Crystals of CTX-M-151 in complex with AVI were obtained by the hanging drop vapor diffusion method at 20 °C. The AVI adduct was obtained by soaking the crystals of the apo-CTX-M-15 in 7-15 mM AVI in the mother liquor for 24 h. X-ray diffraction was measured in a Bruker D8 QUEST microfocus source (Argentina) and at the Soleil synchrotron (France) under cryogenic conditions (100 K). Indexing, integration and scaling were performed with XDS, and the structure was solved by molecular replacement with Phaser. Refinement and model building were performed with Phenix, and Coot, respectively. The model was visualized with PyMol.  Results: The structure of CTX-M-151 in complex with AVI was refined at 1.3 A (Rfree < 0.21). Despite significant differences in amino acid primary sequence, AVI adopts a chair conformation and is in equivalent position in the active site of CTX-M-151 and CTX-M-15.  The AVI carbonyl oxygen points towards the oxyanion hole and close hydrogen bonding interactions are observed with K73, N104, S130, N132, T235, and S237. In both structures, the deacylating water molecule is hydrogen bonded to E166. Noteworthy, AVI Ki for CTX-M-151 was 10-fold higher than for CTX-M-15.Conclusions: Insights into the structure of CTX-M-151 in complex with AVI reveal similar interactions of AVI with residues in the active site of this singular ESBL, which support the efficacy of AVI towards a wide range of serine-beta-lactamases, including enzymes with low homology within the same family like CTX-M-151.