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 tri pler 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) co mplex [Ladenstein, R., Schneider, M., Huber, R., Bartunik, H, D., Wils on, 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 rea ction yielding artifactual, hollow beta(60) capsids with icosahedral s ymmetry. 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-etch ed electron-microscopic images and rotation function calculations show ed that the hexagonal crystal forms of the artifactual beta(60) capsid s and the native alpha(3) beta(60) complex are isomorphous. Orientatio n 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 res olution. The beta-subunit structure was partially refined by energy mi nimization and crystallographic refinement (XPLOR) assuming strict ico sahedral symmetry (final R factor 30.9% for data at 0.8-0.32 nm resolu tion). The topology and chain folding of the beta subunits in the arti factual beta(60) capsid are similar to the native alpha(3) beta(60) en zyme. Structural features of the substrate-binding site and the bindin g 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 hydr ogen bonding. The binding pocket shows a hydrophobic region which acco modates the pyrimidinedione ring and a hydrophilic region to which the ribityl side chain binds. Most amino acid residues involved in the ac tive site are conserved as shown by sequence comparisons with the puta tive lumazine-synthase genes of Escherichia coli and Photobacterium le iognathi. In the final electron-density map, a residual density featur e was tentatively assigned to a bound phosphate ion which mimics the b inding 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.