An Ion-Pair Induced Intermediate Complex Captured in Class D Carbapenemase Reveals Chloride Ion as a Janus Effector...

Area of Science:

• Biochemistry
• Enzyme kinetics
• Structural biology

Background:

• Antibiotic-resistant Enterobacterales producing OXA-48-like carbapenemases contribute to high mortality.
• The catalytic mechanism of OXA-48 is known, but the origin of its biphasic kinetics remains unclear.

Purpose of the Study:

• To elucidate the molecular basis of OXA-48-like enzyme biphasic kinetics.
• To investigate the role of chloride ions in OXA-48 enzyme activity and antibiotic efficacy.

Main Methods:

• Isothermal titration calorimetry (ITC) to monitor the complete reaction course.
• Structural investigation of enzyme-inhibitor complexes.
• Site-directed mutagenesis and mathematical simulation.

Main Results:

• Selective chloride binding, not lysine decarbamylation, drives biphasic kinetics.
• A chloride ion stabilizes an inactive acyl intermediate via interaction with a conserved arginine.
• Chloride acts as a 'Janus effector,' allosterically activating the burst phase and inhibiting the steady state.
• Chloride-induced kinetics affect antibiotic efficacy and aid in differentiating carbapenemase classes.

Conclusions:

• Chloride is a key modulator of OXA-48-like enzyme activity, influencing both catalysis and antibiotic efficacy.
• Understanding chloride's role is crucial for predicting antibiotic pharmacokinetics and effectiveness in vivo.
• This finding expands the known roles of chloride in enzyme modulation and has implications for clinical diagnostics.