Domain structure of riboflavin synthase

Riboflavin synthase of Escherichia coli is a homotrimer of 23.4 kDa subunit s catalyzing the formation of the carbocyclic ring of the vitamin, riboflav in, by dismutation of 6,7-dimethyl-8-ribityllumazine. Intramolecular sequen ce similarity suggested that each subunit folds into two topologically simi lar domains. In order to test this hypothesis, sequence segments comprising amino-acid residues 1-97 or 101-213 were expressed in recombinant E. coli strains. The recombinant N-terminal domain forms a homodimer that can bind riboflavin, 6,7-dimethyl-8-ribityllumazine and trifluoromethyl-substituted 8-ribityllumazine derivatives as shown by absorbance, circular dichroism, a nd NMR. spectroscopy. Most notably, the recombinant domain dimer displays t he same diastercoselectivity for ligands as the full length protein. The mi nimum N-terminal peptide segment required for ligand binding comprises amin o-acid residues 1-87. The recombinant C-terminal domain comprising amino-ac id residues 101-213 is relatively unstable and was shown not to bind ribofl avin but to differentiate between certain diastereomeric trifluoromethyl-8- ribityllumazine derivatives. The data show that a single domain comprises t he intact binding site for one substrate molecule. The enzyme-catalyzed dis mutation requires two substrate molecules to be bound in close proximity, a nd each active site of the enzyme appears to be located at the interface of an N-terminal and C-terminal domain.