Domain structure of riboflavin synthase.
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.
Eberhardt
S
S
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany.
Zingler
N
N
Kemter
K
K
Richter
G
G
Cushman
M
M
Bacher
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011621:Pteridines / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012256:Riboflavin / Q000737:chemistry; D012258:Riboflavin Synthase / Q000737:chemistry* / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014461:Ultracentrifugation
D008024:Ligands; D010455:Peptides; D011621:Pteridines; D011994:Recombinant Proteins; C087980:6-(trifluoromethyl)-7-oxo-8-ribityllumazine; D012258:Riboflavin Synthase; D012256:Riboflavin