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 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.
Ladenstein
R
R
Center for Structural Biochemistry, Karolinska Institute, Huddinge, Sweden.
Ritsert
K
K
Huber
R
R
Richter
G
G
Bacher
A
A
D001412:Bacillus subtilis / Q000201:enzymology*; D001419:Bacteria / Q000201:enzymology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008956:Models, Chemical; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry*; D011621:Pteridines / Q000378:metabolism; D012256:Riboflavin / Q000378:metabolism; D012258:Riboflavin Synthase / Q000737:chemistry*; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism
D009097:Multienzyme Complexes; D011621:Pteridines; C040523:5-amino-6-ribitylamino-2,4-(1H,3H)pyrimidinedione; C008297:lumazine; C044822:6,7-dimethyl-8-ribityllumazine synthase; D012258:Riboflavin Synthase; D012256:Riboflavin; D014529:Uridine