M. Nukaga, S. T. Lefurgy, M. D. Barnes, K. M. Papp-Wallace and R. A. Bonomo,
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Background: AVI is a diazabicyclooctane (DBO) inhibitor that is paired with ceftazidime for the treatment of serious Gram negative infections. The chemical structure of AVI closely resembles portions of the cephem bicyclic ring system, and AVI was shown to bond covalently and reversibly to many serine β-lactamases. In rare cases, AVI is hydrolyzed. Knowledge of the interactions of AVI with different target enzymes is required to anticipate future resistance threats. Here, we probed the atomic structure of AVI with P99, GC1 and FOX-4, three representative class C β-lactamases with unique hydrolytic properties, in order to understand how diverse active site topologies affected AVI inhibition. Methods: P99 and GC1 β-lactamases from Enterobacter cloacae were crystallized in space group P21212 with one molecule in the asymmetric unit by vapor diffusion method and FOX-4 was P212121 with two molecules. Refinements were performed by SHELX (GC1 and P99) or PHENIX (FOX-4).Results: AVI complexes were obtained by soaking with 1 mM AVI for 60 min. Diffraction data of P99, GC1 and FOX-4 β-lactamases complexes with AVI were collected and resolved to 1.15, 1.15 and 1.5 Å resolution, respectively. From the current models (~1.5 Å), the binding of P99 and FOX-4 seems to be similar to AmpC (Pseudomonas aeruginosa):AVI complex reported previously. On the other hand, the GC1:AVI complex appears more flexible: two alternate conformations of sulfonate group and at least two conformations for Ω loop region.Conclusions: AVI bound to P99, FOX-4, and GC1 reveals similar features compared to the previous P. aeruginosa AmpC structure. Again, the movement of the Tyr150 recapitulates a key finding important in acylation/deacylation chemistry. Interestingly, the extended spectrum class C β-lactamase, GC1, discloses alternate conformations of AVI. Taken together, these findings suggest that differences in the active-site volume/flexibility can be overcome by the steric properties of small molecule DBO inhibitors, specifically AVI. This key feature may explain in part activity of AVI against many class A and C enzymes.
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