Heavy riboflavin synthase from Bacillus subtilis. Quaternary structure and reaggregation

Area of Science:

• Enzymology
• Structural Biology
• Biochemistry

Background:

• Riboflavin synthase is a crucial enzyme in riboflavin biosynthesis.
• The native enzyme from Bacillus subtilis is a large, complex protein with distinct subunits.

Purpose of the Study:

• To elucidate the quaternary structure and assembly of Bacillus subtilis riboflavin synthase.
• To investigate the role of alpha and beta subunits in enzyme stability and function.

Main Methods:

• Simplified enzyme purification procedure.
• Protein dissociation and reaggregation studies at varying pH.
• Electron microscopy for structural visualization.
• Immunochemical methods to assess subunit accessibility.
• Sedimentation velocity analysis to determine molecular weight and aggregation states.

Main Results:

• The native enzyme (10^6 Mr) comprises approximately 3 alpha and 60 beta subunits.
• Enzyme dissociation occurs at pH > 7, reversible in phosphate buffer.
• A substrate analogue enhances stability and can induce reaggregation of isolated beta subunits.
• Reaggregated molecules (96 x 10^4 Mr) lack alpha subunits but retain structural similarity to the native enzyme.
• Electron microscopy reveals spherical particles and hollow vesicles, suggesting a capsid-like structure.

Conclusions:

• The native riboflavin synthase likely possesses a central core of alpha subunits surrounded by a beta subunit capsid.
• Beta subunits can self-assemble into functional structures in the presence of a ligand.
• Understanding the enzyme's quaternary structure provides insights into its catalytic mechanism and stability.