Molecular insights into the oxidative perturbation of VIM-2 metallo-β-lactamase: Active site remodeling restores imipenem susceptibility in Pseudomonas aeruginosa
Microbial Pathogenesis, cilt.214, 2026 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 214
- Basım Tarihi: 2026
- Doi Numarası: 10.1016/j.micpath.2026.108411
- Dergi Adı: Microbial Pathogenesis
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, EMBASE, Environment Index, MEDLINE
- Anahtar Kelimeler: Active-site remodeling, Hydrogen peroxide, Molecular dynamics, Pseudomonas aeruginosa, VIM-2 metallo-β-lactamase
- Süleyman Demirel Üniversitesi Adresli: Evet
Özet
BackgroundThe global escalation of carbapenem-resistant Pseudomonas aeruginosa (CRPA), primarily mediated by VIM-2 metallo-β-lactamase (MBL), necessitates innovative adjuvant strategies. This study investigates the potential of sub-inhibitory hydrogen peroxide (H2O2) to restore imipenem (IMP) susceptibility by targeting the structural and catalytic vulnerability of VIM-2.MethodsEighty-six clinical isolates were screened, and a high-resistance/high-biofilm isolate (Pa13) was selected for mechanistic depth. The potentiation and near-synergistic additive effects of sub-MIC H2O2 and IMP were evaluated via checkerboard and biofilm assays. To resolve the molecular underpinnings, we utilized 100 ns Molecular Dynamics (MD) simulations and LC-MS/MS analysis of purified VIM-2 to identify site-specific oxidative modifications. Enzyme kinetics (Km and Vmax) were measured to distinguish between global denaturation and targeted catalytic impairment.ResultsSub-inhibitory H2O2 significantly reduced IMP MICs (4-to-8-fold) and impaired biofilm formation across multiple isolates. Kinetic analysis revealed a dramatic 66% collapse in Vmax while Km remained relatively stable, suggesting a localized disruption of the catalytic machinery. LC-MS/MS identified Cys221 as a primary target for oxidative conversion to sulfenic and sulfinic derivatives. MD simulations confirmed that Cys221 oxidation preserves IMP recruitment consistent with stable Km and docking scores but triggers active-site remodeling that disrupts the geometric alignment essential for turnover, effectively decoupling substrate recognition from enzymatic catalysis.ConclusionThese findings identify Cys221 as a critical “redox switch” within the VIM-2 active site. Site-specific oxidative perturbation effectively silences the enzyme's resistance function without requiring global protein denaturation. This research provides a robust structural rationale for developing redox-based adjuvants to combat MBL-mediated resistance in CRPA.