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

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• Gram-negative bacteria exhibit resistance to β-lactam antibiotics primarily through β-lactamase enzymes.
• Carbapenem-hydrolyzing β-lactamases (CHBLs) are a significant concern due to their ability to deactivate carbapenems, a crucial class of antibiotics for treating multi-drug resistant infections.

Purpose of the Study:

• To investigate the deactivation mechanisms of the carbapenem-hydrolyzing Class D β-lactamase (CHDL) OXA-24/40 against the carbapenem doripenem.
• To elucidate the structural and mechanistic basis for OXA-24/40's activity using real-time NMR studies.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy with rapid data acquisition methods.
• Real-time kinetic studies of doripenem deactivation by OXA-24/40.
• Site-directed mutagenesis to probe the role of key active site residues, specifically R261.

Main Results:

• OXA-24/40 employs two distinct deactivation pathways for doripenem: canonical hydrolysis and a novel mechanism yielding a β-lactone product.
• The β-lactone product exhibits weak affinity for the OXA-24/40 active site.
• The conserved active site arginine, R261, plays a critical role in stabilizing the active site environment conducive to β-lactone formation.

Conclusions:

• OXA-24/40 possesses dual carbapenem deactivation mechanisms, contributing to antibiotic resistance.
• Understanding these mechanisms, particularly the β-lactone pathway influenced by R261, is vital for developing strategies against CHDLs.
• These findings have implications for combating resistance mediated by the broader family of CHDLs.