Klebsiella pneumoniae Carbapenemase Variant 44 Acquires Ceftazidime-Avibactam Resistance by Altering the Conformation of Active-Site Loops

Authors

Zhizeng Sun
Hanfeng Lin
Liya Hu
Neetu Neetu
Banumathi Sankaran
Jin Wang
B V Venkataram Prasad
Timothy Palzkill

Publication Date

1-1-2024

Journal

Journal of Biological Chemistry

DOI

10.1016/j.jbc.2023.105493

PMID

38000656

PMCID

PMC10716778

PubMedCentral® Posted Date

11-23-2023

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Humans, Amino Acids, Anti-Bacterial Agents, Bacterial Proteins, beta-Lactamases, Ceftazidime, Klebsiella Infections, Klebsiella pneumoniae, Drug Resistance, Bacterial, Crystallography, X-Ray, Catalytic Domain, Protein Structure, Tertiary, Models, Molecular, antibiotics, antibiotic resistance, bacteria, crystal structure, enzyme catalysis, enzyme kinetics, enzyme inhibitor, molecular dynamics, molecular evolution

Abstract

Klebsiella pneumoniae carbapenemase 2 (KPC-2) is an important source of drug resistance as it can hydrolyze and inactivate virtually all β-lactam antibiotics. KPC-2 is potently inhibited by avibactam via formation of a reversible carbamyl linkage of the inhibitor with the catalytic serine of the enzyme. However, the use of avibactam in combination with ceftazidime (CAZ-AVI) has led to the emergence of CAZ-AVI-resistant variants of KPC-2 in clinical settings. One such variant, KPC-44, bears a 15 amino acid duplication in one of the active-site loops (270-loop). Here, we show that the KPC-44 variant exhibits higher catalytic efficiency in hydrolyzing ceftazidime, lower efficiency toward imipenem and meropenem, and a similar efficiency in hydrolyzing ampicillin, than the WT KPC-2 enzyme. In addition, the KPC-44 variant enzyme exhibits 12-fold lower AVI carbamylation efficiency than the KPC-2 enzyme. An X-ray crystal structure of KPC-44 showed that the 15 amino acid duplication results in an extended and partially disordered 270-loop and also changes the conformation of the adjacent 240-loop, which in turn has altered interactions with the active-site omega loop. Furthermore, a structure of KPC-44 with avibactam revealed that formation of the covalent complex results in further disorder in the 270-loop, suggesting that rearrangement of the 270-loop of KPC-44 facilitates AVI carbamylation. These results suggest that the duplication of 15 amino acids in the KPC-44 enzyme leads to resistance to CAZ-AVI by modulating the stability and conformation of the 270-, 240-, and omega-loops.

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