Domain structure of riboflavin synthase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Riboflavin synthase catalyzes riboflavin synthesis via substrate dismutation.
• Escherichia coli riboflavin synthase is a homotrimer with potential domain similarity.

Purpose of the Study:

• To investigate the domain structure and function of Escherichia coli riboflavin synthase.
• To identify the ligand-binding and catalytic domains within the enzyme subunit.

Main Methods:

• Recombinant expression of N-terminal (1-97) and C-terminal (101-213) domains.
• Ligand binding studies using absorbance, circular dichroism, and NMR spectroscopy.
• Analysis of domain stability and substrate/ligand interaction specificity.

Main Results:

• The N-terminal domain dimer binds riboflavin and lumazine derivatives with native diastereoselectivity.
• A minimum N-terminal peptide (residues 1-87) is sufficient for ligand binding.
• The C-terminal domain is unstable, does not bind riboflavin, but differentiates lumazine derivatives.

Conclusions:

• A single N-terminal domain contains the binding site for one substrate molecule.
• The enzyme's active site is located at the interface of N-terminal and C-terminal domains.
• Substrate binding in proximity is essential for the dismutation reaction.