Ligand binding to the class D β-lactamase OXA-66 detected by NMR after 19F-labeling of tryptophan residues.

Β-lactamases hydrolyze β-lactam antibiotics and thereby play an important role in antibiotic resistance, a major threat to public health. OXA-66, a class D β-lactamase found in Acinetobacter baumannii, has a bulky tryptophan (W222) occluding the active site. To further explore the effect of this tryptophan on binding, we biosynthetically replaced the tryptophans with 7-fluorotryptophans and carried out protein-observed 19F NMR studies on OXA-66. The resonance corresponding to W222 was assigned and titration of boronic acid inhibitor BA4 resulted in perturbation of the W222 resonance. Enzyme kinetics studies showed that 7FW OXA-66 was able to hydrolyze β-lactams and that mutation of W222 to a smaller phenylalanine resulted in an increase in activity. These studies highlight the impact of W222 in ligand binding to OXA-66.