Heavy riboflavin synthase from Bacillus subtilis. Quaternary structure and reaggregation.

Heavy riboflavin synthase of Bacillus subtilis was purified by a simplified procedure. The enzyme is a complex protein containing about 3 alpha-subunits (23.5 X 10(3) Mr) and 60 beta-subunits (16 X 10(3) Mr). The 10(6) Mr protein dissociates upon exposure to pH values above neutrality. Phosphate ions increase the stability at neutral pH. The dissociation induced by exposure of the enzyme to elevated pH is reversible in phosphate buffer at neutral pH. The stability of the enzyme at elevated pH values is greatly enhanced by the substrate analogue, 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione. Electron micrographs of negatively stained enzyme specimens show spherical particles with a diameter of 15.6 nm. Various immunochemical methods show that the alpha-subunits are not accessible to antibodies in the native molecule. The native enzyme is not precipitated by anti-alpha-subunit serum, and riboflavin synthase activity is not inhibited by the serum. However, these tests become positive at pH values that lead to dissociation of the enzyme. Subsequent to dissociation of the native enzyme at elevated pH values, the beta-subunits form high molecular weight aggregates. These aggregates form a complex mixture of different molecular species, which sediment at velocities of about 48 S and 70 S. The average molecular weight was approximately 5.6 X 10(6). Homogeneous preparations have not been obtained. Electron micrographs show hollow, spherical vesicles with diameters of about 29 nm. The substrate analogue 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione can induce the reaggregation of isolated beta-subunits with formation of smaller molecules, which are structurally similar to native riboflavin synthase. A homogeneous preparation of reaggregated molecules was obtained by renaturation of beta-subunits from 6.4 M-urea in the presence of the ligand. The sedimentation velocity of this aggregate is about 7% smaller than that of the native enzyme. The molecular weight is 96 X 10(4). Electron micrographs show spherical particles with a diameter of about 17.4 nm. Inspection of the micrographs tentatively suggests the presence of a central cavity. It appears likely that these molecules, which are devoid of alpha-subunits, have the same number and spatial arrangement of beta-subunits as the native enzyme. All data are consistent with the hypothesis that the native enzyme consists of a central core of alpha-subunits surrounded by a capsid-like arrangement of beta-subunits. The number of beta-subunits and the shape of the protein suggest a capsid-like arrangement of beta-subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Riboflavin synthases of Bacillus subtilis. Purification and properties.

A variety of Bacillus and Clostridium strains were found to contain two forms of riboflavin synthase which can be easily separated by density gradient centrifugation. The fast sedimenting species accounts for 12 to 44% of the total riboflavin synthase activity in the strains analyzed. Both riboflavin synthases were purified to apparent homogeneity from cell extracts of a genetically derepressed mutant of Bacillus subtilis. The specific activities of the pure proteins were 50,000 nmol mg-1 h-1 (light enzyme) and 2,000 nmol mg-1 h-1 (heavy enzyme). The sedimentation velocities (S20,w) were 4.1 and 26.5 S, respectively. Light riboflavin synthase showed a molecular weight of 70,000 in sedimentation equilibrium experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of about 23,500. Thus the enzyme appears to consist of three identical subunits (alpha type). Heavy riboflavin synthase has a molecular weight of 1,000,000 as shown by sedimentation equilibrium analysis. The protein appears to consist of 2 or 3 alpha subunits and approximately 60 beta subunits. A fragment apparently identical with light riboflavin synthase can be obtained from the heavy enzyme by mild dissociating treatment.