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

摘要: Riboflavin, also known as vitamin B2, is biosynthesized in plants, bacteria, archaea and fungi. The primary biological function of riboflavin related to its existence a component the two coenzymes, flavin mononucleotide (FMN) adenine dinucleotide (FAD), which play an important role for electron transfer energy metabolism. This project mainly focused on structural studies lumazine synthase (LS) from hyperthermophilic bacterium Aquifex aeolicus (LSAQ). enzyme involved penultimate step biosynthesis riboflavin. aim this study gain insights into basis thermostability, catalytic mechanism well molecular assembly enzyme. Methods used these include X-ray crystallography, microscopy (EM), small angle scattering (SAXS) differential scanning calorimetry (DSC). Lumazine hyperthermophile A. displays dramatic stability against high temperature. calorimetric melting profile indicates apparent temperature (Tm) 120 C. factors that determine thermostability LS were revealed by comparisons (Paper I, 2001). In second last biosynthesis, catalyzes formation 6-7-dimethyl-8-ribityllumazine, subsequently converted light complexes with inhibitors (four complex structures studied work), designed mimic substrates, reaction intermediate product at different stages reaction, model process, illustrates binding enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, Schiff base cyclisation, was proposed II, 2003). assumes least four forms, namely virus-like icosahedral capsid diameter about 160 A, pentameric form, stacking pentamers larger capsids 300 A (metamorphosis reviewed Appendix A). pH and/or buffer dependence states B. subtilis, mutant (structure determined cryo-EM manuscript IV) using xray cryo-EM. results indicate multiple are general feature synthases. Furthermore, closely correlated state (Manuscript, III). Sequence alignment insertion 1–4 residues after Gly138 unique Structural modeling suggested may inhibit capsids. structure four-residue’s (IDEA) It shown forms large 292 analysis subunit interactions indicated does not follow theory “quasi-equivalence”, because contact surfaces non-equivalent. Compared wild type enzyme, pentamer widened. expanded provides alternative conformation it could be formed during T=1 (Manuscript List Publications I. Zhang, X., Meining, W., Fischer, M., Bacher, & Ladenstein, R. (2001). crystallographic refinement 1.6 resolution: determinants comparisons. J. Mol. Biol. 306: 1099-1114 II. Cushman, Haase, I., (2003). structure-based catalyzed aeolicus. (328): 167-182 III. Konarev, P., Svergun, D. Xing, L., Cheng, H., W. (2005) Multiple relating assembly. Manuscript IV. Nilsson, J., Bergman, 180 violates quasi-equivalence