Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

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.

X. Zhang, P. Konarev, D. Svergun, I. Haase, M. Fischer, A. Bacher, R. Ladenstein, W. Meining Karolinska Institutet, Center for Structural Biochemistry, SE-14157 Huddinge, Sweden Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany EMBL Hamburg Outstation, Notkestrasse 85, 22603 Hamburg, Federal Republic of Germany The penultimate step in the biosynthesis of riboflavin is catalyzed by lumazine synthase (LS), a homomeric enzyme consisting of subunits with a molecular mass of 16-17 kDa. The LSs from Saccharomyces cerevisiae, Saccharomyces cerevisiae, Magnaporthe grisea, and Schizosaccharomyces pombe appear in nature in form of pentamers, the enzymes from Bacillus subtilis, Aquifex aeolicus, Spinacia oleracea, and Escherichia coli assemble to icosahedral capsids with a diameter of about 160 Å [1-3,6-8], and Brucella abortus LS was found to form a dimer of pentamers [4,5]. Moreover, the B. subtilis LS forms larger capsids with hitherto unknown molecular structure [1]. In B. subtilis the LS capsid encloses a trimer of riboflavin synthase, which catalyses the formation of riboflavin from lumazine. The nature of various forms of LS was studied using x-ray crystallography, electron microscopy, ultracentrifugation, and gel electrophoresis. Solutions containing larger capsids appear to be polydisperse and there was indication that the assembly is dependent on pH and buffer type [1]. The insertion of 4 extra residues in lumazine synthase from A. aeolicus as well as the replacement of Arg127 by Thr in the B. subtilis enzyme leads to the formation of large capsids with a diameter of more than 280 Å (unpublished results). Whether the ability of the enzyme to appear in various forms has a biological function is to date unknown. In order to explore factors influencing the appearance of the enzyme, we have commenced a combined study by electron microscopy and SAXS. In this study we investigate the assembly of wildtype enzymes from B. subtilis and A. aeolicus and selected mutants of enzyme solutions in varying buffers and at varying pH’s. In order to complete an earlier series of SAXS experiments we collected x-ray scattering data of the Arg127Thr mutant of lumazine synthase of B. subtilis in phosphate (pH 6.0-8.0) and Tris buffer (pH 7.0 – 9.0), of the IDEA insertion mutant of the A. aeolicus lumazine synthase in Tris (pH 7.0 – 9.0) and the B. subtilis wildtype mutant in borate (pH 7.0 – 10.0). The scattering curves are shown in Fig 1.

The protein shells of the bifunctional Lumazine/Riboflavin synthase complex found in bacteria, archaea and plants show some similarity to the assembly of small spherical viruses. Sixty lumazine synthase subunits form a T = 1 icosahedral capsid, which instead of nucleic acids in the central core, contains a trimer of riboflavin synthase. Lumazine synthases from fungi, yeasts and some bacteria, however, exist only in pentameric form. Capsid formation in icosahedral lumazine synthases is dependent on the presence of certain substrate-analogous ligands, on pH and phosphate concentration. The experimental background from X-ray crystallography. X-ray small angle scattering and electron microscopy will be discussed. Different active assemblies of the enzyme are observed in vivo and in vitro. There is experimental evidence for the formation of large capsids, obtained spontaneously or after certain mutations to the sequence of the lumazine synthase subunit. Those presumably metastable T = 3 capsids can be reassembled into T = 1 capsids by ligand-driven reassembly in vitro. Cryo electron microscopy of the IDEA mutant of Aquifex aeolicus LS surprisingly showed large icosahedral 180 subunit capsids (T = 3) with a diameter of similar to 290 angstrom. The pentamers in this structure assumed an expanded conformation including a widened central channel. This feature led us to suggest a model for assembly-controlled catalysis, which relates the LS/RS complex, on a microscopic scale, to form and function of a biochemical reactor.

6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of riboflavin, which, as a coenzyme, plays a vital role in the electron transfer process for energy production in all cellular organisms. The enzymes involved in lumazine biosynthesis have been studied in considerable detail. However, the conclusive mechanism of the reaction catalyzed by lumazine synthase has remained unclear. Here, we report four crystal structures of the enzyme from the hyperthermophilic bacterium Aquifex aeolicus in complex with different inhibitor compounds. The structures were refined at resolutions of 1.72 A, 1.85 A, 2.05 A and 2.2 A, respectively. The inhibitors have been designed in order to mimic the substrate, the putative reaction intermediates and the final product. Structural comparisons of the native enzyme and the inhibitor complexes as well as the kinetic data of single-site mutants of lumazine synthase from Bacillus subtilis showed that several highly conserved residues at the active site, namely Phe22, His88, Arg127, Lys135 and Glu138 are most likely involved in catalysis. 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 cyclization is proposed.

6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyses the penultimate step in the biosynthesis of riboflavin. In Bacillus subtilis, 60 lumazine synthase subunits form an icosahedral capsid enclosing a homotrimeric riboflavin synthase unit. The ribH gene specifying the lumazine synthase subunit can be expressed in high yield. All amino acid residues exposed at the surface of the active site cavity were modified by PCR assisted mutagenesis. Polar amino acid residues in direct contact with the enzyme substrates, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, could be replaced with relative impunity with regard to the catalytic properties. Only the replacement of Arg127, which forms a salt bridge with the phosphate group of 3,4-dihydroxy-2-butanone 4-phosphate, reduced the catalytic rate by more than one order of magnitude. Replacement of His88, which is believed to assist in proton transfer reactions, reduced the catalytic activity by about one order of magnitude. Surprisingly, the activation enthalpy ΔH‡ of the lumazine synthase reaction exceeds that of the uncatalysed reaction. On the other hand, the free energy of activation ΔG‡ of the uncatalysed reaction is characterised by a large entropic term (TΔS‡) of −37.8kJmol−1, whereas the entropy of activation (TΔS‡) of the enzyme-catalysed reaction is −6.7kJmol−1. This suggests that the rate enhancement by the enzyme is predominantly achieved by establishing a favourable topological relation of the two substrates, whereas acid/base catalysis may play a secondary role.

Serine/Threonine Protein Phosphatases are important in many cellular processes including protein synthesis, signal transduction glycogen metabolism in muscle and immunosuppression.Many prokaryotic organisms produce phosphatase inhibitors and can be toxic to humans if ingested.The surface of Ser/Thr Phosphatases contain an inhibitor-binding loop which is independent of the catalytic mechanism of the enzyme, but appears to be important in inhibitor activity.How this loop determines inhibitor-specificity between the phosphatases is unknown.There is speculation that the loop may move to accommodate different inhibitors, giving each phosphatase its own response to specific inhibitors.We have solved the structures of Protein Phosphatase-1 (PP1) bound to two different natural product inhibitors, okadaic acid and microcystin-LA(2H).Both of these inhibitors bind in a similar manner to the phosphatase, exhibiting analogous interactions with the inhibitor-binding loop.Importantly, the inhibitor-binding loop is in almost identical positions in both structures.A similar structure using a mutant PP1 with the inhibitor-binding loop from calcineurin substituted in reveals repositioning of only specific amino acid side-chains, but identical positioning of the backbone of the loop.These results indicate that inhibitor specificity in Ser/Thr Phosphatases is most likely only due to specific inhibitor-enzyme interactions within the inhibitorbinding loop and not structural rearrangements of the loop.

During vacuum condensation of metals on frozen proteins, nanoclusters are preferentially formed at specific surface sites (decoration). Understanding the nature of metal/protein interaction is of interest for structure analysis and is also important in the fields of biocompatibility and sensor development. Studies on the interaction between metal and distinct areas on the protein which enhance or impede the probability for cluster formation require information on the structural details of the protein's surface underlying the metal clusters. On three enzyme complexes, lumazine synthase from Bacillus subtilis, proteasome from Thermoplasma acidophilum and GTP cyclohydrolase I from Escherichia coli, the decoration sites as determined by electron microscopy (EM) were correlated with their atomic surface structures as obtained by X-ray crystallography. In all three cases, decoration of the same protein results in different cluster distributions for gold and silver. Gold decorates surface areas consisting of polar but uncharged residues and with rough relief whereas silver clusters are preferentially formed on top of protein pores outlined by charged and hydrophilic residues and filled with frozen buffer under the experimental conditions. A common quality of both metals is that they strictly avoid condensation on hydrophobic sites lacking polar and charged residues. The results open ways to analyse the binding mechanism of nanoclusters to small specific sites on the surface of hydrated biomacromolecules by non-microscopic, physical-chemical methods. Understanding the mechanism may lead to advanced decoration techniques resulting in fewer background clusters. This would improve the analysis of single molecules with regard to their symmetries and their orientation in the adsorbed state and in precrystalline assemblies as well as facilitate the detection of point defects in crystals caused by misorientation or by impurities.

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.

Riboflavin synthase catalyzes the final step in the biosynthesis of riboflavin. Animals and humans lack this enzyme, whereas many bacteria and certain yeasts are absolutely dependent on endogenous riboflavin synthesis. Riboflavin synthase is therefore an attractive target for chemotherapy. The N-terminal domain of riboflavin synthase forms a dimer in solution and is capable of strongly binding riboflavin. It can serve as a model for the binding site of the native enzyme. Structural information obtained from this domain at high resolution will be helpful in the determination of the binding mode of riboflavin and thus for the development of antimicrobial drugs. Here, the crystallization and preliminary crystallographic analysis of the N-terminal domain of riboflavin synthase are reported. The crystals belong to the space group C222(1), with unit-cell parameters a = 50.3, b = 104.7, c = 85.3 A, alpha = beta = gamma = 90 degrees, and diffract to 2.6 A resolution.

Lumazine synthase of Saccharomyces cerevisiae is a homopentamer with a molecular weight of 90 kDa. Crystals of the recombinant enzyme with a size of up to 1.6 mm were obtained. The space group is P4(1)2(1)2 with lattice dimensions 82.9 A x 82.9 A x 300.2 A. X-ray diffraction data collected under cryogenic conditions were complete to 1.85 A resolution. The structure of the enzyme in complex with the intermediate analogue, 5-(6-D-ribitylamino-2,4-dihydroxypyrimidine-5-yl)-1-pentyl-p hosphonic acid was solved via molecular replacement using the structure of the Bacillus subtilis enzyme as search model and was refined to a final R-factor of 19.8% (Rfree: 22.5%). The conformation of the active site ligand of the enzyme mimicks that of the Schiff base intermediate of the enzyme-catalyzed reaction. The data enable the reconstruction of the reactant topology during the early steps of the catalytic reaction. Structural determinants, which are likely to be responsible for the inability of the S. cerevisiae enzyme to form icosahedral capsids, will be discussed.

Riboflavin synthase is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Recombinant riboflavin synthase of Escherichia coli and Bacillus subtilis was crystallized by the vapor diffusion method. Crystals of E. coli riboflavin synthase belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions a = 53.2 A, b = 117.6 A, c = 150.9 A, alpha = beta = gamma = 90 degrees. They diffract to better than 3.3 A resolution and have presumably one trimer in the asymmetric unit. The self rotation function indicates local 32 symmetry. Twofold local symmetry is an unexpected result in a trimeric protein. In conjunction with primary structure arguments and mechanistic considerations, we propose that the protein is a pseudohexamer where each of the peptide subunits fold into two topologically similar domains.

Conference Article| February 01 1996 Structure and mechanism of GTP cyclohydrolase I of Escherichia coli Herbert Nar; Herbert Nar ‡Max-Planck-Institut für Biochemie, Am Klopferspitz, D-82152 Martinsried, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Robert Huber; Robert Huber ‡Max-Planck-Institut für Biochemie, Am Klopferspitz, D-82152 Martinsried, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Winfried Meining; Winfried Meining #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Andreas Bracher; Andreas Bracher #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Markus Fischer; Markus Fischer #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Cornelia HÖSl; Cornelia HÖSl #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Harald Ritz; Harald Ritz #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Cornelia Schmid; Cornelia Schmid #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Sevil Weinkauf; Sevil Weinkauf #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Adelbert Bacher Adelbert Bacher #Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Federal Republic of Germany Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1996) 24 (1): 37S. https://doi.org/10.1042/bst024037s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Herbert Nar, Robert Huber, Winfried Meining, Andreas Bracher, Markus Fischer, Cornelia HÖSl, Harald Ritz, Cornelia Schmid, Sevil Weinkauf, Adelbert Bacher; Structure and mechanism of GTP cyclohydrolase I of Escherichia coli. Biochem Soc Trans 1 February 1996; 24 (1): 37S. doi: https://doi.org/10.1042/bst024037s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1996 Biochemical Society1996 Article PDF first page preview Close Modal You do not currently have access to this content.