Role of Ambler Position 104 in Defining Substrate Specificity in the KPC Family of β-Lactamases

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• Carbapenem-resistant Gram-negative bacteria, particularly Klebsiella pneumoniae carbapenemases (KPCs), present a significant clinical threat.
• The α3-α4 loop's residue 104 in KPC enzymes is crucial for active-site structure and substrate recognition.

Purpose of the Study:

• To investigate the impact of amino acid substitutions at KPC-3's residue 104 on antibiotic susceptibility, enzyme kinetics, and structural dynamics.
• To understand how modifications at position 104 affect resistance to beta-lactams and beta-lactamase inhibitors.

Main Methods:

• Systematic evaluation of 19 amino acid substitutions at position 104 in KPC-3.
• Assays for beta-lactam and inhibitor susceptibility in E. coli.
• Steady-state enzyme kinetics.
• Molecular dynamics simulations and deep-learning analyses.

Main Results:

• P104K and P104R variants showed increased resistance to ceftazidime, ceftazidime/avibactam, ceftazidime/relebactam, and cefiderocol.
• Carbapenem susceptibility was largely unaffected by these substitutions.
• Kinetics revealed enhanced hydrolysis of ceftazidime and cefiderocol by the variants.
• Structural analyses indicated altered W105 orientation, expanded active-site volume, and increased flexibility.

Conclusions:

• Residue 104 plays a critical role in determining substrate specificity and inhibitor susceptibility in KPC enzymes.
• Modifications at position 104 can enhance resistance to advanced antibiotics by altering enzyme structure and dynamics.
• Findings have implications for developing new antimicrobial strategies and understanding resistance mechanisms.