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Studies on the Lumazine Synthase/Riboflavin Synthase Complex ofBacillus subtilis: Crystal Structure Analysis of Reconstituted, Icosahedral β-subunit Capsids with Bound Substrate Analogue Inhibitor at 2.4 Å Resolution

1995; Elsevier BV; Volume: 253; Issue: 1 Linguagem: Inglês

10.1006/jmbi.1995.0542

1089-8638

K. Ritsert, Robert Huber, Vito Türk, Rudolf Ladenstein, Karen Schmidt‐Bäse, Adelbert Bacher,

Biochemical and Molecular Research

The lumazine synthase/riboflavin synthase ofBacillus subtilisis a bifunctional enzyme complex catalysing the formation of riboflavin from 5-amino-6-(d-ribitylamino)-2,4(1H,3H)-pyrimidinedione andl-3,4-dihydroxy-2-butanone-4-phosphatevia6,7-dimethyl-8-ribityllumazine. The complex is composed of 3α (riboflavin synthase) subunits and 60 β (lumazine synthase) subunits and has a relative mass of 1 MDa. The 60 β subunits are arranged in an icosahedral capsid enclosing the α trimer in the central core. The protein was crystallised, and an X-ray structure of the icosahedral capsid was obtained at 3.3 Å resolution with a crystallographicR-factor of 0.33. Hollow, icosahedral capsids consisting of 60 β subunits can be obtained by inhibitor-driven renaturation of isolated β subunits. They catalyse the formation of 6,7-dimethyl-8-ribityllumazine at the same rate as the native α3β60complex and can be crystallised in two different hexagonal and one monoclinic form. Crystallographic intensity data of the monoclinic crystals to a resolution of 2.4 Å were obtained using synchrotron radiation and an image plate detector system. The orientation of the icosahedral molecules in the monoclinic cell was deduced by real space vector search procedures from a 3.5 Å Patterson map. Phases were calculated from the model of the α3β60protein and were extended by cyclic averaging exploring the 30-fold redundancy of the electron density. The 2.4 Å map allowed us to refine the existing atomic model of lumazine synthase. The refined model includes 154 amino acid residues, one inhibitor molecule, 58 water molecules and one phosphate ion. Applying non-crystallographic-symmetry restraints the crystallographicR-factor is 16.7% for 100,092 reflections between 10 and 2.4 Å. The chain folding of the β subunits is closely similar to the native α3β60enzyme. The lumazine synthase bears resemblance to the sugar binding proteins. The significantly higher resolution compared to the α3β60structure determination allows a detailed description of the substrate analogue binding site. The environment of the 5-nitro-6-(d-ribitylamino)-2,4(1H,3H)-pyrimidinedione inhibitor is particularly rigid, and the chain segments involved in forming the active site are highly conserved for lumazine synthases of different species. A residual density feature in the final map is interpreted as a bound phosphate which mimics the binding of the second substrate. We discuss the reaction mechanism on this structural basis.