Shaping Substrate Selectivity in a Broad-Spectrum Metallo-β-Lactamase.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Metallo-β-lactamases (MBLs) are critical carbapenemase enzymes produced by bacterial pathogens, posing significant challenges in antimicrobial drug development.
• Understanding how MBLs recognize and process substrates is crucial for designing effective inhibitors, but this knowledge remains incomplete.
• B2 MBLs exhibit a narrower substrate profile compared to broad-spectrum B1 MBLs, a difference often linked to specific sequence insertions near the active site.

Purpose of the Study:

• To investigate the functional role of sequence insertions in modulating the substrate specificity of MBLs.
• To explore the potential for engineering MBLs with altered substrate profiles by manipulating these sequence insertions.

Main Methods:

• Comparative analysis of naturally occurring sequence insertions in the B2 enzyme Sfh-I and the B1 enzyme SPM-1.
• Engineering of a chimeric MBL by replacing the SPM-1 sequence insertion with that of Sfh-I.
• Biochemical characterization of the wild-type and engineered MBL variants to determine substrate profiles.

Main Results:

• The engineered chimeric MBL, containing the Sfh-I insertion within the SPM-1 scaffold, exhibited selective cephalosporinase activity.
• This finding demonstrates that sequence insertions play a key role in determining MBL substrate specificity.
• The study highlights that the stable MBL scaffold permits extensive modular engineering, expanding functional possibilities beyond natural evolution.

Conclusions:

• Sequence insertions are critical determinants of MBL substrate specificity, allowing for fine-tuning of enzyme activity.
• The modular nature of the MBL scaffold enables significant protein engineering for novel enzymatic functions.
• These insights can inform the development of new strategies for combating antibiotic resistance by targeting MBL enzymes.