Structural studies of lumazine synthases : Thermostability, catalytic mechanism and molecular assembly

Abstract: Riboflavin, also known as vitamin B2, is biosynthesized in plants, bacteria, archaea and fungi. The primary biological function of riboflavin is related to its existence as a component of the two coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which play an important role for electron transfer in energy metabolism. This project is mainly focused on structural studies of lumazine synthase (LS) from the hyperthermophilic bacterium Aquifex aeolicus (LSAQ). The enzyme is involved in the penultimate step of biosynthesis of riboflavin. The aim of this study is to gain insights into the structural basis of thermostability, catalytic mechanism as well as the molecular assembly of the enzyme. Methods used for these studies include X-ray crystallography, electron microscopy (EM), small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). Lumazine synthase from the hyperthermophile A. aeolicus displays dramatic stability against high temperature. The calorimetric melting profile indicates an apparent melting temperature (TM) of 120 degrees centigrade. The factors that determine the thermostability of A. aeolicus LS were revealed by structural comparisons (Paper I, 2001). In the second last step of riboflavin biosynthesis, lumazine synthase catalyzes the formation of 67-dimethyl-8-ribityllumazine, which is subsequently converted to riboflavin. In light of the structural studies of the enzyme in complexes with inhibitors (four complex structures were studied in this work), which were designed to mimic the substrates, reaction intermediate and the product at different stages of the reaction, a structural model of the catalytic process, which illustrates binding of substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclisation, was proposed (Paper II, 2003). Lumazine synthase assumes at least four assembly forms, namely the virus-like icosahedral capsid with a diameter of about 160 A, the pentameric form, the stacking pentamers and larger capsids with a diameter of about 300 A (metamorphosis of the enzyme is reviewed in Appendix A). The pH and/or buffer dependence of the assembly states of LS from B. subtilis, A. aeolicus and a designed mutant LS from A. aeolicus (structure determined by cryo-EM in manuscript IV) were studied using small angle x-ray scattering (SAXS) and cryo-EM. The results indicate that multiple assembly states are a general feature of lumazine synthases. Furthermore, the catalytic function of the enzyme is closely correlated with the assembly state (Manuscript, III). Sequence alignment revealed that an insertion of 1-4 residues after Gly138 is unique for the pentameric lumazine synthases. Structural comparisons and modeling studies suggested that this insertion may inhibit the formation of icosahedral capsids. The structure of lumazine synthase from A. aeolicus with a four-residue's insertion (IDEA) is studied by cryoEM. It is shown that the mutant forms large capsids with a diameter of 292 A. The analysis of the subunit interactions indicated that the assembly of the mutant does not follow the theory of "quasi-equivalence", because the contact surfaces are non-equivalent. Compared to that of the wild type enzyme, the pentamer of the mutant is widened. The expanded pentameric structure provides a model for an alternative conformation of the LS pentamer as it could also be formed during the catalytic reaction in the T=1 capsid (Manuscript IV)