Lumazine Synthase Research Articles

Investigation of the binding of epimer A of the covalent hydrate of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine to a recombinant F22W Bacillus subtilis lumazine synthase mutant by (15)N[(19)F] REDOR NMR.

The two epimeric covalent hydrates A and B of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine are metabolically stable analogues of hypothetical intermediates proposed in the reactions catalyzed by riboflavin synthase and lumazine synthase. To confirm the stereochemical assignments previously based solely on results for epimer B, a (15)N[(19)F] REDOR NMR study was performed on the complex formed from epimer A and a recombinant, uniformly (15)N-labeled F22W mutant of Bacillus subtilis lumazine synthase. The results indicate that the fluorines of the ligands are closer to the side chain nitrogens of Arg127 and farther away from the side chain nitrogens of Lys135 in epimer B than in epimer A. These results are consistent with the assignment of the earlier 7R configuration of epimer A and the 7S configuration of epimer B.

Design, synthesis, and evaluation of 9-D-ribityl-1,3,7-trihydro-2,6,8-purinetrione, a potent inhibitor of riboflavin synthase and lumazine synthase.

Reduction of 5-nitro-6-D-ribitylaminouracil (9) afforded 5-amino-6-D-ribitylaminouracil (1), which reacted with ethyl chloroformate to yield 5-ethylcarbamoyl-6-D-ribitylaminouracil (12). The latter compound was cyclized to 9-D-ribityl-1,3,7-trihydropurine-2,6,8-trione (13), which was found to be a relatively potent inhibitor of both Escherichia coli riboflavin synthase (K(i) 0.61 microM) and Bacillus subtilis lumazine synthase (K(i) 46 microM). Molecular modeling of the lumazine synthase-inhibitor complex indicated the possibility for hydrogen bonding between the Lys135 epsilon-amino group of the enzyme and both the 8-keto group and the 4'-hydroxyl group of the ligand. A bisubstrate analogue of the riboflavin synthase-catalyzed reaction, 1,4-bis[1-(9-D-ribityl-1,3,7-trihydropurine-2,6,8-trionyl)]butane (18), was also synthesized using a similar route and was found to be inactive as an inhibitor of both riboflavin synthase and lumazine synthase.

Design of Biofunctional Assemblies on Solids through Recombinant Spherical Bacterial Protein Lumazine Synthase

The stepwise design of large protein assemblies such as actin-myosin complexes on solid supports is presented. The functional entities are anchored to the solid surface or solid-supported membranes through lumazine synthase, a 15 nm icosahedral capsid which can be functionalized by recombinant coupling of different linkers to the protomer, as shown in the cartoon. Separating the protein assemblies from the solid by 15 nm spaces avoids protein denaturing by nonspecific adsorption.

Design of Biofunctional Assemblies on Solids through Recombinant Spherical Bacterial Protein Lumazine Synthase

Design of Biofunctional Assemblies on Solids through Recombinant Spherical Bacterial Protein Lumazine Synthase

19F NMR ligand perturbation studies on 6,7-bis(trifluoromethyl)-8-ribityllumazine-7-hydrates and the lumazine synthase complex of Bacillus subtilis. Site-directed mutagenesis changes the mechanism and the stereoselectivity of the catalyzed haloform-type reaction.

The riboflavin synthase/lumazine synthase complex of Bacillus subtilis catalyzes the last two steps in riboflavin biosynthesis. The protein comprises a capsid of 60 beta subunits with lumazine synthase activity and a core of three alpha subunits with riboflavin synthase activity. The beta subunits catalyze the formation of 6,7-dimethyl-8-ribityllumazine (3) from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) and 3,4-dihydroxy-2-butanone 4-phosphate (2). Complexes of recombinant lumazine synthase (beta(60) capsids) with 6-trifluoromethyl-7-oxo-8-ribityllumazine (10) as well as 7S- or 7R-6,7-bistrifluoromethyl-8-ribityllumazine hydrate (11) were studied by (19)F NMR spectroscopy. Despite the large molecular weight of approximately 960 kDa of the protein, spectra with separated signals of free and bound ligand could be obtained. An unusually large shift difference of 8 ppm was observed between the 7-trifluoromethyl signals of free and bound ligand for epimer B of 11 and the enzyme. The signal is sensitive to the replacement of amino acid residues F22 and H88. Lumazine synthase catalyzes the elimination of the 7-trifluoromethyl group of R-diastereomer epimer A in a haloform-like reaction. The elimination reaction is also catalyzed by F22 mutants. The H88R mutant displays an opposite stereoselectivity for epimer B and a greatly enhanced reaction rate. From a model of the epimers in the active site of the protein, the main function of the side chain of F22 seems to be to keep the substrate ring in the correct position. H88 is in a position suited to act as proton acceptor in both the physiological as well as the haloform reaction. A different mechanism of the haloform-reaction is proposed in the case of the H88R mutant, initiated by hydrogen bonding of the 7-trifluorormethyl group and the guanidinium group of the arginine residue.

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

An open reading frame optimized for expression of 6,7-dimethyl-8-ribityllumazine 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 Å 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 Å. 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 Å). The extremely thermostable lumazine synthase from A. aeolicus has a melting temperature of 119.9°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.

Biosynthesis of riboflavin. The reaction catalyzed by 6,7-dimethyl-8-ribityllumazine synthase can proceed without enzymatic catalysis under physiological conditions.

6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of the vitamin, riboflavin. The biosynthetic formation of the lumazine by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate is catalyzed by the enzyme, lumazine synthase. We show that the condensation reaction can proceed without enzyme catalysis in dilute aqueous solution at room temperature and neutral pH. The reaction rate is proportional to e (pH). The activation energy of the uncatalyzed reaction is E(a) = 46.3 kJ mol(-)(1). The regioselectivity of the uncatalyzed reaction increases with pH and temperature (70% at 65 degrees C and pH 7.75). The data suggest partitioning of the uncatalyzed reaction via two different reaction pathways. The value of k(cat)/k(uncat) may be indicative for an entropy driven process for the enzyme-catalyzed reaction.

Synthesis of Nanophase Iron Oxide in Lumazine Synthase Capsids.

An enzyme-based bio-nanoreactor: By acting as a mineralization template, lumazine synthase, a 60-subunit enzyme complex which has a hollow porous shell, can fabricate nanocrystalline iron oxide. Fe ions can permeate the capsid through hydrophilic funnel-shaped channels lined with glutamic acid residues and become encapsulated in the cavity as FeIII oxide. The capsid increases in size from 15 to 30 nm in diameter through formation of a higher order structure as the concentration of FeIII increases.

Synthesis of Nanophase Iron Oxide in Lumazine Synthase Capsids

Synthesis of Nanophase Iron Oxide in Lumazine Synthase Capsids

Synthesis of Nanophase Iron Oxide in Lumazine Synthase Capsids

Synthesis of Nanophase Iron Oxide in Lumazine Synthase Capsids

Structural, functional and immunological studies on a polymeric bacterial protein.

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-A resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

The atomic structure of pentameric lumazine synthase from Saccharomyces cerevisiae at 1.85 Å resolution reveals the binding mode of a phosphonate intermediate analogue

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 P41212 with lattice dimensions 82.9 Å ∗ 82.9 Å ∗ 300.2 Å. X-ray diffraction data collected under cryogenic conditions were complete to 1.85 Å resolution. The structure of the enzyme in complex with the intermediate analogue, 5-(6-d-ribitylamino-2,4-dihydroxypyrimidine-5-yl)-1-pentyl-phosphonic 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.

Divergence in macromolecular assembly: X-ray crystallographic structure analysis of lumazine synthase from Brucella abortus

We have determined the three-dimensional structure of 6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) from Brucella abortus, the infectious organism of the disease brucellosis in animals. This enzyme catalyses the formation of 6,7-dimethyl-8-ribityllumazine, the penultimate product in the synthesis of riboflavin. The three-dimensional X-ray crystal structure of the enzyme from B. abortus has been solved and refined at 2.7 Å resolution to a final R-value of 0.18 (Rfree=0.23). The macromolecular assembly of the enzyme differs from that of the enzyme from Bacillus subtilis, the only other lumazine synthase structure known. While the protein from B. subtilis assembles into a 60 subunit icosahedral capsid built from 12 pentameric units, the enzyme from B. abortus is pentameric in its crystalline form. Nonetheless, the active sites of the two enzymes are virtually identical indicating inhibitors to theses enzymes could be effective pharmaceuticals across a broad species range. Furthermore, we compare the structures of the enzyme from B. subtilis and B. abortus and describe the C teminus structure which accounts for the differences in quaternary structure.

Rate Limitations in the Lumazine Synthase Mechanism

Lumazine synthase has a slow rate of catalysis: steady-state kcat values for the Escherichia coli, Magnaporthe grisea, and spinach enzymes are 0.024, 0.052, and 0.023 s−1, respectively, at pH 7.5 and 25°C. Following the formation of an imine connecting the two substrates 3,4-dihydroxy-2-butanone 4-phosphate and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine, there is a chemically difficult isomerization. Calculated estimates of the free energy barrier for the isomerization are equal to or greater than 15 kcal/mol at 25°C. Free energies calculated from the steady-state kcat values at 25°C for the E. coli, M. grisea, and spinach enzymes are 19.7, 19.2, and 19.7 kcal/mol, respectively. The single-turnover rate (presteady state) at pH 7.5 and 25°C for the M. grisea enzyme is 140-fold greater than the steady-state rate and it has a free energy barrier of 16.3 kcal/mol. In the pre-steady state the M. grisea enzyme has a pKa of 5.8, plausibly reporting the proposed general base of catalysis (His127). The M. grisea enzyme has an off rate of 0.37 s−1 for its product, 6,7-dimethyl-8-ribityllumazine, approximately 7-fold higher than kcat and 20-fold lower than the single-turnover rate. The off rate for the product orthophosphate is about 1 s−1. Thus, for the M. grisea enzyme at pH 7.5 and 25°C, product dissociation is significantly rate limiting to the steady-state rate of catalysis, whereas the isomerization step limits the single turnover rate. The spinach and E. coli enzymes display a significant lag in pre-steady state, suggesting that substrate association is significantly rate limiting for these catalysts. Temperature studies on the enzyme-catalyzed rates for the three enzymes indicate a dominating enthalpic term.

Electron microscopic observations on protein crystallization: adsorption layers, aggregates and crystal defects

Transmission electron microscopy of freeze-etched and heavy-metal-decorated large protein complexes is capable of portraying their molecular symmetries and orientations. The technique in combination with image analysis has been applied to study the rotational order of individual lumazine synthase molecules either in crystals or adsorbed on solid substrates. On crystal surfaces rotational disorder was mainly observed for the molecules at and in the vicinity of relief perturbations. Molecules along surface steps and in the vicinity of vacancies showed no noticeable deviation from their translational and rotational order dictated by the lattice. Adsorption of lumazine synthase on mica led to layers with different degrees of coverage. Single, isolated molecules showed a slight preferential orientation with a hydrophobic region of their surface in contact with the substrate. In continuous, monomolecular adsorption layers microclusters have been observed in which the neighboring molecules were oriented in the same way with respect to the substrate. These microclusters may act as nuclei for the second layer which revealed a perfect crystalline order as do the consecutive layers. Thus, the multilayer structures grown on mica present real three-dimensional crystals except for the first layer contacting the substrate.

ChemInform Abstract: Investigation of the Binding of Fluorolumazines to the 1‐MDa Capsid of Lumazine Synthase by 15N{19F} REDOR NMR.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The 18-kDa cytoplasmic protein of Brucella species --an antigen useful for diagnosis--is a lumazine synthase.

Previous studies have shown that the detection of antibodies to an 18-kDa cytoplasmic protein of Brucella spp. is useful for the diagnosis of human and animal brucellosis. This protein has now been expressed in recombinant form in Escherichia coli. The recombinant protein is soluble only under reducing conditions, but alkylation with iodoacetamide renders it soluble in non-reducing media. As shown by gel exclusion chromatography, this soluble form arranges in pentamers of 90 kDa. The reactivity of human and animal sera against the recombinant protein was similar to that found with the native protein present in brucella cytoplasmic fraction, suggesting that the recombinant protein is correctly folded. The protein has low but significant homology (30%) with lumazine synthases involved in bacterial riboflavin biosynthesis, which also arrange as pentamers. Biological tests on the crude extract of the recombinant bacteria and on the purified recombinant protein showed that the biological activity of the Brucella spp. 18-kDa protein is that of lumazine synthase. Preliminary crystallographic analysis showed that the Brucella spp. lumazine synthase arranges in icosahedric capsids similar to those formed by the lumazine synthases of other bacteria. The high immunogenicity of this protein, potentially useful for the design of acellular vaccines, could be explained by this polymeric arrangement.

Investigation of the Binding of Fluorolumazines to the 1-MDa Capsid of Lumazine Synthase by 15N{19F} REDOR NMR

The 15N{19F} REDOR NMR spectra of two fluorolumazines complexed to the 1-MDa β60 capsid of lumazine synthase have been obtained at 20.3 and 50.7 MHz. Distances from CF3 groups of the ligands to six side- and main-chain nitrogens have been measured. These distances were used in combination with the X-ray crystal coordinates of wild-type lumazine synthase, complexed to a related substrate ligand, in a series of distance-restrained molecular dynamics simulations. The result is a model of the binding site of lumazine synthase that has sufficient detail to predict the absolute configuration at C-7 of complexed 7-hydroxy-8-d-ribityl-6,7-bis(trifluoromethyl)-7,8-dihydropteridine-2,4(1H,3H)-dione, a fluorinated analogue of an unstable, hypothetical intermediate in the reactions catalyzed by both lumazine synthase and riboflavin synthase.

Plant Riboflavin Biosynthesis: CLONING, CHLOROPLAST LOCALIZATION, EXPRESSION, PURIFICATION, AND PARTIAL CHARACTERIZATION OF SPINACH LUMAZINE SYNTHASE

Lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis, has been cloned from three higher plants (spinach, tobacco, and arabidopsis) through functional complementation of an Escherichia coli auxotroph. Whereas the three plant proteins exhibit some structural similarities to known microbial homologs, they uniquely possess N-terminal polypeptide extensions that resemble typical chloroplast transit peptides. In vitro protein import assays with intact chloroplasts and immunolocalization experiments verify that higher plant lumazine synthase is synthesized in the cytosol as a larger molecular weight precursor protein, which is post-translationally imported into chloroplasts where it is proteolytically cleaved to its mature size. The authentic spinach enzyme is estimated to constitute <0.02% of the total chloroplast protein. Recombinant "mature" spinach lumazine synthase is expressed in E. coli at levels exceeding 30% of the total soluble protein and is readily purified to homogeneity using a simple two-step procedure. Apparent V(max) and K(m) values obtained with the purified plant protein are similar to those reported for microbial lumazine synthases. Electron microscopy and hydrodynamic studies reveal that native plant lumazine synthase is a hollow capsid-like structure comprised of 60 identical 16.5-kDa subunits, resembling its icosahedral counterparts in E. coli and Bacillus subtilis.

ChemInform Abstract: Design and Synthesis of 6‐(6‐D‐Ribitylamino‐2,4‐dihydroxypyrimidin‐5‐yl)‐1‐hexylphosphonic Acid, a Potent Inhibitor of Lumazine Synthase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.