Abstract
Bacterial production of β‐lactamases with carbapenemase activity is a global health threat. The active sites of class D carbapenemases such as OXA‐48, which is of major clinical importance, uniquely contain a carbamylated lysine residue which is essential for catalysis. Although there is significant interest in characterizing this post‐translational modification, and it is a promising inhibition target, protein carbamylation is challenging to monitor in solution. We report the use of 19F NMR spectroscopy to monitor the carbamylation state of 19F‐labelled OXA‐48. This method was used to investigate the interactions of OXA‐48 with clinically used serine β‐lactamase inhibitors, including avibactam and vaborbactam. Crystallographic studies on 19F‐labelled OXA‐48 provide a structural rationale for the sensitivity of the 19F label to active site interactions. The overall results demonstrate the use of 19F NMR to monitor reversible covalent post‐translational modifications.
Highlights
Bacterial production of b-lactamases with carbapenemase activity is a global health threat
As carbamylation is essential for class D SBL catalysis, it represents a potential target for inhibition
We proposed that the high sensitivity of 19F NMR, enhanced by the degeneracy of the three 19F nuclei in the BTFA-derived label, could overcome the limitations associated with other methods for monitoring carbamylation in solution
Summary
Bacterial production of b-lactamases with carbapenemase activity is a global health threat. Addition of sodium bicarbonate to OXA-48 L158C* and T213C* resulted in a change in the relative intensities of these two signals (Figure 1 D, S4), suggesting that they correspond to the carbamylated and uncarbamylated states of the enzyme.
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