The lumazine synthase/riboflavin synthase complex of Bacillus subtilis

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Abstract
The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 β subunits enclosing a triplet of α subunits. An X‐ray structure of 0.32 nm resolution has been obtained for the icosahedral capsid of the native α3β60 complex [Ladenstein, R., Schneider, M., Huber, R., Bartunik, H. D., Wilson, K., Schott, K. & Bacher, A. (1988) J. Mol. Biol. 203, 1045–1070]. β subunits were isolated after denaturation of the α3β60 complex and were subsequently reconstituted in a ligand‐driven reaction yielding artifactual, hollow β60 capsids with icosahedral symmetry. Hexagonal crystals (space group P6322) of the reconstituted capsids diffracted X‐rays to a resolution of 0.32 nm. Crystallographic intensity data were obtained using synchrotron radiation. Freeze‐etched electron‐microscopic images and rotation function calculations showed that the hexagonal crystal. forms of the artifactual β60 capsids and the native α3β60 complex are isomorphous. Orientation and translation parameters of the β‐subunit model were refined by XPLOR rigid‐body refinement. The electron‐density map was improved by cyclic icosahedral averaging and phase extension from 0.5–0.32 nm resolution. The β‐subunit structure was partially refined by energy minimization and crystallographic refinement (XPLOR) assuming strict icosahedral symmetry (final R factor 30.9% for data at 0.8–0.32 nm resolution). The topology and chain folding of the β subunits in the artifactual β60 capsid are similar to the native α3β60 enzyme. Structural features of the substrate‐binding site and the binding of the substrate‐analogous ligand 5‐nitro‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione are discussed. Ligand binding occurs at the pentamer interfaces and includes van der Waals' interactions and hydrogen bonding. The binding pocket shows a hydrophobic region which accomodates the pyrimidinedione ring and a hydrophilic region to which the ribityl side chain binds. Most amino acid residues involved in the active site are conserved as shown by sequence comparisons with the putative lumazine‐synthase genes of Escherichia coli and Photobacterium leiognathi. In the final electron‐density map, a residual density feature was tentatively assigned to a bound phosphate ion which mimics the binding of the second substrate, 3,4‐dihydroxy‐2‐butanone 4‐phosphate. This putative phosphate‐binding site involves a highly conserved amino acid sequence containing three basic residues.