THE LUMAZINE SYNTHASE RIBOFLAVIN SYNTHASE COMPLEX OF BACILLUS-SUBTILIS - X-RAY STRUCTURE-ANALYSIS OF HOLLOW RECONSTITUTED BETA-SUBUNIT CAPSIDS

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a tripler of a subunits. An X-ray structure of 0.32 nn resolution has been obtained for the icosahedral capsid of the native alpha(3) beta(60) complex [Ladenstein, R., Schneider, M., Huber, R., Bartunik, H, D., Wilson, K., Schott, K. and Bacher, A. (1988) J. Mol. Biol. 203, 1045-1070]. beta subunits were isolated after denaturation of the alpha(3) beta(60) complex and were subsequently reconstituted in a ligand-driven reaction yielding artifactual, hollow beta(60) capsids with icosahedral symmetry. Hexagonal crystals (space group P6(3)22 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 beta(60) capsids and the native alpha(3) beta(60) complex are isomorphous. Orientation and translation parameters of the beta-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 beta-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 beta subunits in the artifactual beta(60) capsid are similar to the native alpha(3) beta(60) enzyme. Structural features of the substrate-binding site and the binding of the substrate-analogous analogous ligand 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione are dis cussed. 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.