The Structural Role of N170 in Substrate-Assisted Deacylation in KPC-2 β-Lactamase

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• Klebsiella pneumoniae carbapenemase 2 (KPC-2) is a key enzyme conferring resistance to beta-lactam antibiotics.
• Ceftazidime-avibactam is a critical treatment for infections caused by KPC-producing Gram-negative bacteria.
• Clinical mutations in KPC-2, especially in the omega loop, are linked to reduced susceptibility to ceftazidime-avibactam.

Purpose of the Study:

• To investigate the structural mechanisms by which KPC-2 variants confer resistance to ceftazidime-avibactam.
• To elucidate the role of specific amino acid substitutions (R164, D179) in the omega loop on enzyme structure and function.
• To understand the impact of these mutations on drug binding and catalytic activity.

Main Methods:

• Accelerated rare-event sampling well-tempered metadynamics simulations were employed.
• Detailed structural analysis of R164 and D179 variants was performed.
• The role of N170 conformation in drug interaction and catalysis was examined.

Main Results:

• D179 substitutions induce significant omega loop disorder, more so than R164 mutations.
• The conformation of N170 was found to be critical for drug binding and deacylation.
• KPC-2 D179 variants may utilize substrate-assisted catalysis, involving the aminothiazole ring for enhanced hydrolysis.
• Wild-type N170 conformation shifts contribute to reduced deacylation and altered enzymatic activity.

Conclusions:

• Specific KPC-2 omega loop mutations, particularly D179, lead to structural changes that confer ceftazidime-avibactam resistance.
• The N170 residue plays a crucial, previously underappreciated role in KPC-2's interaction with avibactam and its catalytic efficiency.
• Understanding these structural dynamics provides insights into antibiotic resistance mechanisms and potential therapeutic strategies.