Substrate Specificity of OXA-48 after β5-β6 Loop Replacement

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• OXA-48 carbapenemase is a significant global health threat due to its rapid spread.
• Variants of OXA-48 carbapenemase exhibit altered substrate hydrolysis profiles, often linked to modifications in the β5-β6 loop.
• Deletions in the β5-β6 loop can lead to loss of carbapenemase activity and gain of expanded-spectrum cephalosporin (ESC) hydrolysis.

Purpose of the Study:

• To investigate the role of the β5-β6 loop in the substrate selectivity of OXA-48-like enzymes.
• To engineer a hybrid enzyme with altered hydrolytic capabilities by replacing the OXA-48 β5-β6 loop with that of OXA-18.
• To elucidate the structure-function relationship of β-lactamases concerning loop conformation and substrate binding.

Main Methods:

• Enzyme engineering: replaced the β5-β6 loop of OXA-48 with the corresponding loop from OXA-18.
• Enzyme kinetics: determined kinetic parameters (kcat, Km) for substrate hydrolysis.
• MIC determination: assessed the impact of the engineered enzyme on antibiotic resistance.
• Structural analysis: employed X-ray crystallography and molecular modeling to understand loop conformation and substrate binding.

Main Results:

• The hybrid enzyme OXA-48Loop18 retained the ability to hydrolyze both carbapenems and ESCs.
• Kinetic analysis showed a lower catalytic efficiency (kcat) for carbapenem hydrolysis compared to native OXA-48, but expanded ESC hydrolysis.
• Structural studies indicated that the grafted loop adopts a conformation allowing binding of bulkier substrates, expanding the enzyme's hydrolytic profile.
• The observed changes are attributed to both amino acid sequence and backbone conformation of the β5-β6 loop.

Conclusions:

• The β5-β6 loop plays a crucial role in determining the substrate selectivity of OXA-48-like β-lactamases.
• Localized modifications, including loop grafting, can significantly alter or expand the functional capabilities of β-lactamases, highlighting their inherent plasticity.
• Understanding these structure-function relationships provides insights into the evolution of antibiotic resistance mechanisms.