STUDIES ON THE LUMAZINE SYNTHASE RIBOFLAVIN SYNTHASE COMPLEX OF BACILLUS-SUBTILIS - CRYSTAL-STRUCTURE ANALYSIS OF RECONSTITUTED, ICOSAHEDRAL BETA-SUBUNIT CAPSIDS WITH BOUND SUBSTRATE-ANALOG INHIBITOR AT 2.4 ANGSTROM RESOLUTION/

The lumazine synthase/riboflavin synthase of Bacillus subtilis is a bi functional enzyme complex catalysing the formation of riboflavin from mino-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and L-3,4-dihydroxy -2-butanone-4-phosphate via 6,7-dimethyl-8-ribityllumazine. The comple x is composed of 3 alpha (riboflavin synthase) subunits and 60 beta (l umazine synthase) subunits and has a relative mass of 1 MDa. The 60 be ta subunits are arranged in an icosahedral capsid enclosing the alpha trimer in the central core. The protein was crystallised, and an X-ray structure of the icosahedral capsid was obtained at 3.3 Angstrom reso lution with a crystallographic R-factor of 0.33. Hollow, icosahedral c apsids consisting of 60 beta subunits can be obtained by inhibitor-dri ven renaturation of isolated beta subunits. They catalyse the formatio n of 6,7-dimethyl-8-ribityllumazine at the same rate as the native alp ha(3) beta(60) complex and can be crystallised in two different hexago nal and one monoclinic form. Crystallographic intensity data of the mo noclinic crystals to a resolution of 2.4 Angstrom were obtained using synchrotron radiation and an image plate detector system. The orientat ion of the icosahedral molecules in the monoclinic cell was deduced by real space vector search procedures from a 3.5 Angstrom Patterson map . Phases were calculated from the model of the alpha(3) beta(60) prote in and were extended by cyclic averaging exploring the 30-fold redunda ncy of the electron density. The 2.4 Angstrom map allowed us to refine the existing atomic model of lumazine synthase. The refined model inc ludes 154 amino acid residues, one inhibitor molecule, 58 water molecu les and one phosphate ion. Applying non-crystallographic-symmetry rest raints the crystallographic R-factor is 16.7% for 100,092 reflections between 10 and 2.4 Angstrom. The chain folding of the beta subunits is closely similar to the native alpha(3) beta(60) enzyme. The lumazine synthase bears resemblance to the sugar binding proteins. The signific antly higher resolution compared to the alpha(3) beta(60) structure de termination allows a detailed description of the substrate analogue bi nding site. The environment of the itro-6-(D-ribitylamino)-2,4(1H,3H)- pyrimidinedione inhibitor is particularly rigid, and the chain segment s involved in forming the active site are highly conserved for lumazin e synthases of different species. A residual density feature in the fi nal map is interpreted as a bound phosphate which mimics the binding o f the second substrate. We discuss the reaction mechanism on this stru ctural basis. (C) 1995 Academic Press Limited