Riboflavin synthases of Bacillus subtilis. Purification and amino acid sequence of the alpha subunit.
Bacillus subtilis has two different riboflavin synthases characterized by the subunit structures alpha3 (light enzyme) and alpha3beta60 (heavy enzyme). The light enzyme was purified by a novel procedure with increased yield and excellent reproducibility. The proposed trimer structure was confirmed by cross-linking experiments with dimethyl suberimidate. Fragments of alpha subunits were prepared by cleavage with cyanogen bromide, trypsin, protease Lys-C, and Staphylococcus aureus protease V8, respectively. Sequences were determined by automated liquid or gas phase Edman degradation. The complete sequence (202 amino acids) was established by direct sequencing of the N terminus and sequencing of overlapping peptides. The sequence shows marked internal homology between the NH2-terminal and COOH-terminal half encompassing 26 identical positions and 23 conservative replacements. This suggests that the protomer forms two structurally similar domains. Since it is known that the enzyme has two binding sites per subunit for the substrate 6,7-dimethyl-8-ribityllumazine, it appears likely that each of the homologous protein domains provides one binding site. The stereochemical features of the enzyme mechanism and the structural relation of the alpha trimer to the beta60 capsid of heavy riboflavin synthase suggest that the six domains corresponding to the alpha subunit trimer are related by pseudo 32 symmetry.
Schott
K
K
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Garching, Federal Republic of Germany.
Kellermann
J
J
Lottspeich
F
F
Bacher
A
A
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D010450:Endopeptidases; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010446:Peptide Fragments; D012258:Riboflavin Synthase / Q000302:isolation & purification*; D012697:Serine Endopeptidases; D014166:Transferases / Q000302:isolation & purification*; D014357:Trypsin
D046911:Macromolecular Substances; D010446:Peptide Fragments; D014166:Transferases; D012258:Riboflavin Synthase; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D003488:Cyanogen Bromide