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Heavy riboflavin synthase from Bacillus subtilis: Quaternary structure and reaggregation
Ist Teil von
Journal of molecular biology, 1986-01, Vol.187 (1), p.75-86
Ort / Verlag
Oxford: Elsevier Ltd
Erscheinungsjahr
1986
Link zum Volltext
Quelle
Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)
Beschreibungen/Notizen
Heavy riboflavin synthase of
Bacillus subtilis was purified by a simplified procedure. The enzyme is a complex protein containing about 3 α-subunits (23.5 × 10
3
M
r) and 60 β-subunits (16 × 10
3
M
r). The 10
6
M
r, 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 α-subunits are not accessible to antibodies in the native molecule. The native enzyme is not precipitated by anti-α-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 β-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 × 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 β-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 β-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 × 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 α-subunits, have the same number and spatial arrangement of β-subunits as the native enzyme.
All data are consistent with the hypothesis that the native enzyme consists of a central core of α-subunits surrounded by a capsid-like arrangement of β-subunits. The number of β-subunits and the shape of the protein suggest a capsid structure with icosahedral 532 symmetry and the triangulation number
T = 1. The 26 S aggregate of β-subunits would then represent the hollow capsid.