A highly conserved residue unlocks thermostability in β-glucuronidases

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Protein Chemistry

Background:

• Beta-glucuronidase enzymes are vital for producing pharmaceuticals.
• Low thermal stability limits their industrial use.
• Directed evolution and screening can enhance enzyme properties.

Purpose of the Study:

• Identify thermostability-enhancing mutations in beta-glucuronidase.
• Characterize the structural and kinetic basis of improved thermal stability.
• Provide a strategy for robust enzyme industrial applications.

Main Methods:

• Screening workflow and directed evolution to identify mutants.
• Site-directed mutagenesis of conserved residues.
• Kinetic parameter characterization.
• Molecular dynamics simulations and structural analysis.

Main Results:

• Identified a critical "N" residue in the "WNFADF" motif regulating thermostability.
• N→T substitution increased melting temperature by 6.6-12.61 °C and half-life by 4.26-33.6-fold.
• Observed the typical activity-stability trade-off.
• N→T variants showed reduced local flexibility and enhanced compactness.

Conclusions:

• The "WNFADF" motif and its conserved "N" residue are key to beta-glucuronidase thermostability.
• N→T mutation offers a robust strategy for enhancing enzyme stability.
• Mechanistic insights gained can guide future enzyme engineering for industrial biocatalysis.