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- Title
Domain structure of riboflavin synthase.
- Authors
Eberhardt, S; Zingler, N; Kemter, K; Richter, G; Cushman, M; Bacher, A
- Abstract
Riboflavin synthase of Escherichia coli is a homotrimer of 23.4 kDa subunits catalyzing the formation of the carbocyclic ring of the vitamin, riboflavin, by dismutation of 6,7-dimethyl-8-ribityllumazine. Intramolecular sequence similarity suggested that each subunit folds into two topologically similar 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, and NMR spectroscopy. Most notably, the recombinant domain dimer displays the same diastereoselectivity for ligands as the full length protein. The minimum N-terminal peptide segment required for ligand binding comprises amino-acid residues 1-87. The recombinant C-terminal domain comprising amino-acid residues 101-213 is relatively unstable and was shown not to bind riboflavin but to differentiate between certain diastereomeric trifluoromethyl-8-ribityllumazine derivatives. The data show that a single domain comprises the intact binding site for one substrate molecule. The enzyme-catalyzed dismutation requires two substrate molecules to be bound in close proximity, and each active site of the enzyme appears to be located at the interface of an N-terminal and C-terminal domain.
- Publication
European journal of biochemistry, 2001, Vol 268, Issue 15, p4315
- ISSN
0014-2956
- Publication type
Journal Article
- DOI
10.1046/j.1432-1327.2001.02351.x