Publications

• Biosynthesis of riboflavin: structure and mechanism of lumazine synthase.

  Bacher A., Fischer M., Kis K., Kugelbrey K., Mortl S., Scheuring J., Weinkauf S., Eberhardt S., Schmidt-Base K., Huber R., Ritsert K., Cushman M., Ladenstein R.

  Biochem Soc Trans 24:89-94(1996) [PubMed] [EuropePMC]

• Biosynthesis of riboflavin. Lumazine synthase of Escherichia coli.

  Moertl S., Fischer M., Richter G., Tack J., Weinkauf S., Bacher A.

  A gene located at 443 kilobases on the Escherichia coli chromosome (subsequently designated ribE) was expressed in a recombinant E. coli strain and was shown to code for the enzyme 6, 7-dimethyl-8-ribityllumazine synthase. The recombinant enzyme was purified to homogeneity. The protein is an icosa ... >> More

  A gene located at 443 kilobases on the Escherichia coli chromosome (subsequently designated ribE) was expressed in a recombinant E. coli strain and was shown to code for the enzyme 6, 7-dimethyl-8-ribityllumazine synthase. The recombinant enzyme was purified to homogeneity. The protein is an icosahedral capsid of 60 subunits with a mass of about 1 MDa as shown by hydrodynamic studies and by electron microscopy. In contrast to the icosahedral lumazine synthase-riboflavin synthase complex of Bacillus subtilis, the lumazine synthase of E. coli is not physically associated with another enzyme of the riboflavin pathway, and the core of the icosahedral capsid is empty. The RIB4 gene of Saccharomyces cerevisiae was also expressed to a high level (about 40% of cellular protein) in E. coli. The recombinant protein is a pentamer of 90 kDa. An insertion of 4 amino acids into helix alpha4 is likely to hinder the formation of an icosahedral capsid by the yeast protein. The kinetic properties of lumazine synthase of E. coli, B. subtilis, and S. cerevisiae are similar. << Less

  J. Biol. Chem. 271:33201-33207(1996) [PubMed] [EuropePMC]

• X-ray structure analysis and crystallographic refinement of lumazine synthase from the hyperthermophile Aquifex aeolicus at 1.6 A resolution: determinants of thermostability revealed from structural comparisons.

  Zhang X., Meining W., Fischer M., Bacher A., Ladenstein R.

  An open reading frame optimized for expression of 6,7-dimethyl-8-ribityl-lumazine synthase of the hyperthermophilic bacterium Aquifex aeolicus in Escherichia coli was synthesized and expressed in a recombinant E. coli strain to a level of around 15 %. The recombinant protein was purified by heat-t ... >> More

  An open reading frame optimized for expression of 6,7-dimethyl-8-ribityl-lumazine synthase of the hyperthermophilic bacterium Aquifex aeolicus in Escherichia coli was synthesized and expressed in a recombinant E. coli strain to a level of around 15 %. The recombinant protein was purified by heat-treatment and gel-filtration. The protein was crystallized in the cubic space group I23 with the cell dimensions a = b = c = 180.8 A, and diffraction data were collected to 1.6 A resolution. The structure was solved by molecular replacement using lumazine synthase from Bacillus subtilis as search model. The structure of the A. aeolicus enzyme was refined to a resolution of 1.6 A. The spherical protein consists of 60 identical subunits with strict icosahedral 532 symmetry. The subunit fold is closely related to that of the B. subtilis enzyme (rmsd 0.80 A). The extremely thermostable lumazine synthase from A. aeolicus has a melting temperature of 119.9 degrees C. Compared to other icosahedral and pentameric lumazine synthases, the A. aeolicus enzyme has the largest accessible surface presented by charged residues and the smallest surface presented by hydrophobic residues. It also has the largest number of ion-pairs per subunit. Two ion-pair networks involving two, respectively three, stacking arginine residues assume a distinct role in linking adjacent subunits. The findings indicate the influence of the optimization of hydrophobic and ionic contacts in gaining thermostability. << Less

  J. Mol. Biol. 306:1099-1114(2001) [PubMed] [EuropePMC]

• Crystal structure of lumazine synthase from Mycobacterium tuberculosis as a target for rational drug design: binding mode of a new class of purinetrione inhibitors.

  Morgunova E., Meining W., Illarionov B., Haase I., Jin G., Bacher A., Cushman M., Fischer M., Ladenstein R.

  The enzymes involved in the biosynthesis of riboflavin represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with enzymes of the mammalian metabolism. Lumazine synthase catalyzes the penultimate st ... >> More

  The enzymes involved in the biosynthesis of riboflavin represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with enzymes of the mammalian metabolism. Lumazine synthase catalyzes the penultimate step in the riboflavin biosynthesis pathway. A number of substituted purinetrione compounds represent a new class of highly specific inhibitors of lumazine synthase from Mycobacterium tuberculosis. To develop potent antibiotics for the treatment of tuberculosis, we have determined the structure of lumazine synthase from M. tuberculosis in complex with two purinetrione inhibitors and have studied binding via isothermal titration calorimetry. The structures were determined by molecular replacement using lumazine synthase from Saccharomyces cerevisiae as a search model and refined at 2 and 2.3 A resolution. The R-factors were 14.7 and 17.4%, respectively, and the R(free) values were 19.3 and 26.3%, respectively. The enzyme was found to be a pentamer consisting of five subunits related by 5-fold local symmetry. The comparison of the active site architecture with the active site of previously determined lumazine synthase structures reveals a largely conserved topology with the exception of residues Gln141 and Glu136, which participate in different charge-charge interactions in the core space of the active site. The impact of structural changes in the active site on the altered binding and catalytic properties of the enzyme is discussed. Isothermal titration calorimetry measurements indicate highly specific binding of the purinetrione inhibitors to the M. tuberculosis enzyme with dissociation constants in micromolar range. << Less

  Biochemistry 44:2746-2758(2005) [PubMed] [EuropePMC]

• Biosynthesis of riboflavin. Studies on the reaction mechanism of 6,7-dimethyl-8-ribityllumazine synthase.

  Kis K., Volk R., Bacher A.

  The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits surrounding a core of 3 alpha subunits. The beta subunits catalyze the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with (3S)-3,4-dihydroxy-2-butanone u ... >> More

  The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits surrounding a core of 3 alpha subunits. The beta subunits catalyze the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with (3S)-3,4-dihydroxy-2-butanone under formation of 6,7-dimethyl-8-ribityllumazine. This intermediate is converted to riboflavin by the alpha subunits via an unusual dismutation yielding 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione as second product. (3R)- and (3S)-3,4-dihydroxy-2-butanone 4-phosphate were synthesized. Both enantiomers can serve as substrate for 6,7-dimethyl-8-ribityllumazine synthase. The reaction rate of the natural S-enantiomer is about 6-fold higher than that of the R-enantiomer. The Km value for (3S)-3,4-dihydroxy-2-butanone 4-phosphate is 130 microM, and the Km value for the pyrimidine substrate is 5 microM. Diacetyl and 3,4-dihydroxy-2-butanone 3-phosphate do not serve as substrates for lumazine synthase. The enzyme-catalyzed condensation of the carbohydrate with the pyrimidine is strictly regiospecific. The enzyme does not catalyze the exchange of protons between (3S)-3,4-dihydroxy-2-butanone 4-phosphate and solvent water in the absence of the pyrimidine cosubstrate. A reaction mechanism starting with the formation of a Schiff base followed by elimination of phosphate and cyclization is proposed. The lumazine synthase activities of the native enzyme complex and of reconstituted, hollow beta 60 capsids are virtually identical (about 12,000 nmol mg-1 h-1). << Less

  Biochemistry 34:2883-2892(1995) [PubMed] [EuropePMC]