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The lumazine synthase/riboflavin synthase complex of Bacillus subtilis. X-ray structure analysis of hollow reconstituted beta-subunit capsids.
Ladenstein R, Ritsert K, Huber R, Richter G, Bacher A.

Center for Structural Biochemistry, Karolinska Institute, Huddinge, Sweden.

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a triplet
of alpha subunits. An X-ray structure of 0.32 nm 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. & 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 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.

PMID: 8055941 [PubMed - indexed for MEDLINE]