Characterization of KPC‐2 Backbone Dynamics Upon Avibactam Binding via NMR Spectroscopic Methods

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Antibiotic resistance is a continual threat in the clinic. One of the resistance modes bacteria employ is the expression of the beta‐lactamases that target most of the clinically relevant antibiotics, such as penicillin. The enzyme hydrolyzes the β‐lactam bond, which renders the antibiotic ineffective. Targeting β‐lactamases with small molecule inhibitors can restore efficacy of current generation antibiotics. Klebsiella pneumoniae carbapenemase (KPC) presents a global threat to public health as it confers multidrug resistance, including resistance to last resort antibiotics. Avibactam is the only FDA approved inhibitor for KPC are on the market, however some variants of KPC are showing increased resistance to avibactam. Futhermore, avibactam can be slowly degraded by KPC‐2 through a de‐sulfonation mechanism. A conserved omega‐loop near the active site of KPC‐2 has been implicated in substrate binding and is of particular interest as a target for future inhibitors. We report the first backbone dynamic characterization of a Class A carbapenemase in the presence and absence of an inhibitor. Backbone dynamic parameters for KPC‐2 obtained through the use of 3D HNCO based T1, T2, and heteronuclear NOE relaxation experiments. Chemical shift perturbations were obtained by comparing avibactam bound and apo states. H/D exchange experiments were also conducted to examine relative rates of exchange in avibactam bound and apo KPC‐2 in order to estimate slow timescale dynamics. The binding of avibactam caused an increase in R1 relaxation rates, suggesting avibactam can increase the dynamic flexibility of KPC‐2. The changes in the R2 relaxation rates suggest that the binding of avibactam might make the Ω‐loop slightly more rigid. These results could be used as a benchmark in measuring interactions with the Ω‐loop, interactions with KPC‐2, or interactions with other β‐lactamases.Support or Funding InformationNIH R35 GM128595This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.