PMID:11488927
| Citation |
Eberhardt, S, Zingler, N, Kemter, K, Richter, G, Cushman, M and Bacher, A (2001) Domain structure of riboflavin synthase. Eur. J. Biochem. 268:4315-23 |
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| 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. |
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| Keywords |
Amino Acid Sequence; Binding Sites; Chromatography, High Pressure Liquid; Circular Dichroism; Dimerization; Escherichia coli/enzymology; Ligands; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Sequence Data; Peptides/chemistry; Plasmids/metabolism; Protein Structure, Tertiary; Pteridines/chemistry; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Riboflavin/chemistry; Riboflavin Synthase/chemistry; Riboflavin Synthase/isolation & purification; Sequence Homology, Amino Acid; Spectrophotometry; Ultracentrifugation |
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