The solution structure of the N-terminal domain of riboflavin synthase

The structure of the amino-terminal domain of Escherichia coli riboflavin s ynthase (RiSy) has been determined by NMR spectroscopy with riboflavin as a bound ligand. RiSy is functional as a 75 kDa homotrimer, each subunit of w hich consists of two domains which share very similar sequences and structu res. The N-terminal domain (RiSy-N; 97 residues) forms a 20 kDa homodimer i n solution which binds riboflavin with high affinity. The structure feature s a six-stranded antiparallel beta -barrel with a Greek-key fold, both ends of which are closed by an alpha -helix. One riboflavin molecule is bound p er monomer in a site at one end of the barrel which is comprised of element s of both monomers. The structure and ligand binding are similar to that of the FAD binding domains of ferrodoxin reductase family proteins. The struc ture provides insights into the structure of the whole enzyme, the organisa tion of the functional trimer and the mechanism of riboflavin synthesis. C4 8 from the N-terminal domain is identified as the free cysteine implicated in a nucleophilic role in the synthesis mechanism, while H102 from the C-te rminal domains is also likely to play a key role. Both are invariant in all known riboflavin synthase sequences. (C) 2001 Academic Press.