Flavin binding to the high affinity riboflavin transporter RibU

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Membrane transporters are crucial for nutrient uptake.
• Riboflavin (vitamin B2) transport is essential for cellular function.
• Characterization of microbial transporters provides insights into biological mechanisms.

Purpose of the Study:

• To biochemically and spectroscopically characterize the purified riboflavin transporter RibU from Lactococcus lactis.
• To identify the substrate binding properties and the nature of the binding pocket.
• To investigate the role of specific residues in substrate binding.

Main Methods:

• Overexpression, solubilization, and purification of the RibU transporter.
• Mass spectrometry for co-purified substrate identification.
• Spectroscopic analysis (absorption and fluorescence) of substrate binding.
• Site-directed mutagenesis to probe residue function.

Main Results:

• RibU was purified and shown to co-purify with riboflavin.
• Riboflavin, FMN, and roseoflavin bound with high affinity (K(d) = 0.6 nM for riboflavin) and 1:1 stoichiometry; FAD did not bind.
• Spectroscopic changes indicated a hydrophobic binding pocket and riboflavin stacking with tryptophan residues.
• Mutagenesis identified Trp-68 as critical for riboflavin binding.

Conclusions:

• The study presents the first biochemical and spectroscopic characterization of a purified riboflavin transporter, RibU.
• RibU exhibits exceptionally high affinity for riboflavin, involving a hydrophobic binding pocket and tryptophan stacking.
• The findings provide insights into the structural and mechanistic aspects of flavin transport in bacteria.