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Mimicking natural evolution in metallo-beta-lactamases through second-shell ligand mutations
Pablo E Tomatis1, Rodolfo M Rasia, Lorenzo Segovia
1Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina.
Summary
Metallo-beta-lactamases (MBLs) evolved enhanced cephalexin hydrolysis. Remote mutations altered active sites, expanding substrate range without losing function, mimicking natural evolution.
Area of Science:
- Enzymology
- Molecular Evolution
- Antimicrobial Resistance
Background:
- Metallo-beta-lactamases (MBLs) are crucial in antibiotic resistance.
- Their broad substrate profile confers resistance to most beta-lactam antibiotics.
- Understanding MBL evolution is key to combating resistance.
Purpose of the Study:
- To investigate the evolutionary potential of MBLs.
- To explore how directed evolution impacts MBL hydrolytic efficiency.
- To understand the role of remote mutations in enzyme adaptation.
Main Methods:
- Directed evolution of Bacillus cereus MBL (BcII).
- Systematic analysis of hydrolytic profiles and substrate binding.
- Characterization of active-site features and mutations.
Main Results:
- Evolved BcII showed increased hydrolytic efficiency toward cephalexin.
- Remote mutations reshaped the active site for improved cephalosporin hydrolysis.
- Mutations lowered activation energy, not substrate affinity.
- One mutation identified in pathogenic bacteria contributes to increased hydrolytic profile.
Conclusions:
- MBLs can expand substrate spectrum without losing inherent capabilities.
- Directed evolution effectively mimics natural mutation processes.
- Second-shell mutations tune metal-ligand strength and catalytic efficiency.
- Altered Zn(II) ion positioning affects substrate binding in the active site.