Unraveling the Molecular Evolution and Structural Landscape of Klebsiella pneumoniae Carbapenemase Variants
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Abstract
Mutations in the blaKPC gene significantly influence the effectiveness of antibiotics and combination therapies used to combat Klebsiella pneumoniae resistance. This study aimed to analyze genetic variations, physicochemical properties, and structural characteristics between mutant and wild-type class A β-lactamase KPC enzymes responsible for ceftazidime–avibactam resistance. Strains with the most significant mutations relative to the wild-type were identified based on physicochemical changes, haplotype networks, and structural conformations. The binding mechanisms between antibiotics and KPC enzymes were also evaluated to determine the role of conserved residues in enzymatic interactions and catalytic activity. Two wild types and 260 KPC variant sequences were retrieved from the NCBI database. ProtParam analysis indicated that all KPC variants exhibited stable physicochemical characteristics. Motif analysis using MEME revealed 15 conserved motifs across all variants. Phylogenetic tree reconstruction with MEGA 10 and iTOL, combined with haplotype analysis using DnaSP, identified KPC-2 and KPC-3 as ancestral variants. Protein modeling with AlphaFold and structural superimposition in PyMOL showed conformational shifts in the Ω-loop, 240-loop, and 270-loop regions. Molecular docking in PyRx demonstrated that ceftazidime acted as the strongest inhibitor against several KPC variants, which was supported by visualization using Discovery Studio 2025. Variants such as KPC-9, KPC-117, KPC-135, KPC-201, and KPC-258 showed the highest divergence, whereas those in Haplotype 1 remained closest to the wild-type. Mutations predominantly occurred within loop regions, while the protein core remained conserved, suggesting selective adaptation under antibiotic pressure. Further in vitro and in vivo validation is recommended to confirm in silico predictions and improve future therapeutic design.
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