Bacterial Phosphorylation Suppresses Carbapenemase Activity of the Class-D β-Lactamase OXA-24/40 from <i>Acinetobacter baumannii</i>

The resistance of Gram-negative bacteria to β-lactam antibiotics is mostly due to deactivation of the antibiotics by bacterial enzymes, β-lactamases. Disclosing the factors regulating β-lactamase activity is vital for developing therapies to combat multidrug-resistant pathogens, such as <i>Acinetobacter baumannii</i>. Recent <i>A. baumannii</i> studies have revealed post-translational phosphorylation of serine β-lactamases at the active site serine. However, the functional consequences of such phosphorylation are unclear. We have taken the first steps to define these consequences through studies of OXA-24/40, a carbapenem-hydrolyzing class D β-lactamase in <i>A. baumannii</i>. We generated OXA-24/40 phosphorylated at its active site serine, S81, and explored its effects via NMR and MS. Phosphorylation inhibits carbapenemase activity by altering the active site conformation and impeding the carboxylation of an active site lysine, a requirement for class D β-lactamase activity. The inhibition varies with the carbapenem side chain properties. Phosphorylation-induced chemical shift perturbations extend beyond the active site, suggesting allosteric effects. Our findings offer the first atomic-level insights into the functional consequences of serine phosphorylation of class D β-lactamases.