Enhancing recombinant urate oxidase stability and catalytic function with TMAO osmolyte: integrated experimental and...

Area of Science:

• Biochemistry
• Protein Stabilization
• Enzyme Kinetics

Background:

• Pharmaceutical enzymes like urate oxidase (UOX) exhibit poor thermal stability in aqueous solutions.
• Compatible osmolytes, such as trimethylamine N-oxide (TMAO), are known protein stabilizers, but their mechanisms are not fully understood.
• Understanding osmolyte-protein interactions is crucial for improving enzyme applications.

Purpose of the Study:

• To investigate the molecular mechanisms by which TMAO enhances the catalytic efficiency and stability of urate oxidase (UOX).
• To evaluate the kinetic, thermodynamic, and structural effects of TMAO on UOX using integrated experimental and computational methods.

Main Methods:

• Enzyme kinetics assays to measure catalytic activity.
• Thermodynamic studies (ΔH°, ΔS°, ΔG°) to analyze UOX-TMAO interactions.
• Fluorescence spectroscopy to detect structural changes.
• Molecular dynamics (MD) simulations for structural compactness and stability analysis.
• Molecular docking to predict binding sites and interactions.

Main Results:

• TMAO significantly enhanced UOX's enzymatic activity.
• Thermodynamic data indicated spontaneous, favorable binding of TMAO to UOX via van der Waals forces and hydrogen bonding.
• Fluorescence spectroscopy revealed structural modifications in UOX upon TMAO binding.
• MD simulations showed increased structural compactness, enhanced stability, and preserved active site integrity in UOX with TMAO.
• Molecular docking confirmed favorable non-covalent interactions between TMAO and UOX.

Conclusions:

• TMAO effectively improves the stability and catalytic function of urate oxidase.
• The stabilizing effect of TMAO on UOX is attributed to increased structural compactness and preservation of the active site.
• This study provides atomic-level insights into osmolyte-mediated enzyme stabilization, relevant for pharmaceutical and biotechnological applications.