Arginine Modulates Carbapenem Deactivation by OXA-24/40 in Acinetobacter baumannii

The resistance of Gram-negative bacteria to beta-lactam antibiotics stems mainly from beta-lactamase proteins that hydrolytically deactivate the beta-lactams. Of particular concern are the beta-lactamases that can deactivate a class of beta-lactams known as carbapenems. Carbapenems are among the few anti-infectives that can treat multi-drug resistant bacterial infections. Revealing the mechanisms of their deactivation by beta-lactamases is a necessary step for preserving their therapeutic value. Here, we present NMR investigations of OXA-24/40, a carbapenem-hydrolyzing Class D beta-lactamase (CHDL) expressed in the gram-negative pathogen, Acinetobacter baumannii. Using rapid data acquisition methods, we were able to study the real-time deactivation of the carbapenem known as doripenem by OXA-24/40. Our results indicate that OXA-24/40 has two deactivation mechanisms: canonical hydrolytic cleavage, and a distinct mechanism that produces a beta-lactone product that has weak affinity for the OXA-24/40 active site. The mechanisms issue from distinct active site environments poised either for hydrolysis or beta-lactone formation. Mutagenesis reveals that R261, a conserved active site arginine, stabilizes the active site environment enabling beta-lactone formation. Our results have implications not only for OXA-24/40, but the larger family of CHDLs now challenging clinical settings on a global scale. (C) 2021 Elsevier Ltd. All rights reserved.

Automated summary

This study reveals the real-time deactivation mechanisms of carbapenem-hydrolyzing beta-lactamase OXA-24/40 in Acinetobacter baumannii using NMR. The enzyme was found to have two deactivation mechanisms stemming from distinct active site environments, with mutagenesis showing that a conserved active site arginine stabilizes the environment for beta-lactone formation. These results have implications for the broader family of carbapenem-hydrolyzing beta-lactamases in clinical settings globally.