19F-NMR applied to understand the beta-lactone inhibitory effect on OXA-143

Denize Favaro (CUNY – Advanced Science Research Center, USA)

LinkedIn: @DenizeFavaro, X: @DenizeFavaro

Abstract: In 2018, a mechanism distinct from the formation of the inactive hydrolyzed β-lactam was described for 1-methyl-carbapenem hydrolysis by OXA-48, OXA-10, and OXA-23, leading to β-lactone formation. Previous studies have demonstrated that the S configuration at β-lactone C-2 can weakly inhibit these enzymes, and that the lactone-to-hydrolyzed product ratio can vary depending on the residues surrounding the active site. In this study, we used 1D-1H, 15N/1H-TROSY, and 1D-19F Nuclear Magnetic Resonance methods to demonstrate that OXA-143 can also hydrolyze the lactone product, resulting in lactone/hydrolyzed meropenem ratios that are highly dependent on the enzyme/antibiotic ratio, consistent with a reversible covalent inhibition mechanism.
Furthermore, using the cysteine variant OXA-143(D224C) labeled with 3-bromo-1,1,1-trifluoropropan-2-one (BTFA) and 19F-NMR, we identified the enzyme’s conformational states when bound to the substrate and the (S)- lactone, without the need for physical separation of the products. Additionally, the hMER binds weakly to the active site after the lactone is fully hydrolyzed.
Finally, the only mutant to show relevant differences in lactone-to-hydrolyzed product ratio was R261S – almost no lactone formation.