- Rapid preparation of isotopolog libraries by in vivo transformation of 13C-glucose. Studies on 6,7-dimethyl-8-ribitylluinazine, a biosynthetic precursor of vitamin B2
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An Escherichia coli strain engineered for expression of the ribABGH genes of Bacillus subtilis was shown to produce 100 mg of the riboflavin precursor 6,7-dimethyl-8-ribityllumazine per liter of minimal medium. Growth of the recombinant strain in medium supplemented with [U-13C6] glucose and/or 15NH4Cl as single sources of carbon and/or nitrogen afforded 6,7-dimethyl-8-ribityllumazine universally labeled with 13C and/or 15N. The yield of [U-13C 13]-6,7-dimethyl-8-ribityllumazine based on [U-13C 6]glucose was 25 mg/g. Fermentation with [1-13C 1]-, [2-13C1]-, or [3-13C 1]glucose afforded mixtures of 6,7-dimethyl-8-ribityllumazine isotopologs, predominantly with 13C enrichment of single carbon atoms. The isotope-labeled samples enabled a comprehensive NMR analysis of 6,7-dimethyl-8-ribityllumazine. Isotopolog libraries of a wide variety of microbial metabolites can be produced by the same experimental approach.
- Illarionov, Boris,Fischer, Markus,Lee, Chan Yong,Bacher, Adelbert,Eisenreich, Wolfgang
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- Biosynthesis of riboflavin. Single turnover kinetic analysis of 6,7-dimethyl-8-ribityllumazine synthase
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6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate, affording the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine. Single turnover experiments monitored by multiwavelength photometry were performed with the recombinant lumazine synthase of Bacillus subtilis. Mixing of the enzyme with the pyrimidine type substrate is conducive to a hypsochromic shift as well as a decrease in absorbance of the heterocyclic substrate; the rate constant for that reaction is 0.02 s-1 μM-1. Rapid mixing of the complex between enzyme and pyrimidine type substrate with the second substrate, 3,4-dihydroxy-2-butanone 4-phosphate, is followed by the appearance of an early optical transient characterized by an absorption maxima at 330 nm of low intensity which was tentatively assigned as a Schiff base intermediate. The subsequent elimination of phosphate affords a transient with intense absorption maxima at 455 and 282 nm, suggesting an intermediate with an extended system of conjugated double bonds. The subsequent formation of the enzyme product, 6,7-dimethyl-8-ribityllumazine, is the rate-determining step.
- Schramek, Nicholas,Haase, Ilka,Fischer, Markus,Bacher, Adelbert
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- Rate limitations in the lumazine synthase mechanism
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Lumazine synthase has a slow rate of catalysis: steady-state k(cat) 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 k(cat) 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 (pre-steady 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 pK(a) 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 k(cat) 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. (C) 2000 Academic Press.
- Zheng, Ya-Jun,Viitanen, Paul V.,Jordan, Douglas B.
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- 63. Isolierung und Struktur von Pteridinen (Lumazinen) aus Russula sp. (Taeublinge; Basidiomycetes)
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Extensive chromatographic separations and chemical and spectroscopic investigations have led to the isolation and identification of several water-soluble pteridines from Russula sp., the so-called russupteridines, namely: 1-(5-amino-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)amino-1-deoxy-D-ribitol (1; a pro-lumazine; first identification in a basidiomycete); 1-deoxy-1-(6-methyl-2,4,7-trioxo-1,2,3,4,7,8-hexahydropteridin-8-yl)-D-ribitol (3) and 1-deoxy-1-(2,4,7-trioxo-1,2,3,4,7,8-hexahydropteridin-8-yl)-D-ribitol (4); both compounds found for the first time in higher fungi; they belong to the components with the strongest violet-blue fluorescence in Russula sp.; riboflavine (6; now recognized as an important yellow colorant in a great many of Russula sp.); russupteridine-yellow 1 ( = 1-(6-amino-7-(N-formylimino)-2,4-dioxo-1,2,3,4,7,8-hexahydropteridin-8-yl)-1-deoxy-D-ribitol; 5; a component with very strong fluorescence; the first derivative of the novel 6,7-diamino-lumazine); russupteridine-yellow IV ( = 1-deoxy-1-(2,6,8-trioxo-2,4,5,6,7,8-hexahydro-1H-imidazolopteridin-4-yl)-D-ribitol (7).Two further yellow russupteridines (yellow II and yellow V) with very strong fluorescence have been isolated and characterized.
- Iten, Peter Xaver von,Maerki-Danzig, Hana,Koch, Herbert,Eugster, Conrad Hans
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- The effect of MR1 ligand glyco-analogues on mucosal-associated invariant T (MAIT) cell activation
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Mucosal-associated invariant T (MAIT) cells are a subset of recently identified innate-like T lymphocytes that appear to play an important role in many pathologies ranging from viral and bacterial infection, to autoimmune disorders and cancer. MAIT cells are activated via the presentation of ligands by MR1 on antigen presenting cells to the MAIT T cell receptor (TCR), however few studies have explored the effects of systematic changes to the ligand structure on MR1 binding and MAIT cell activation. Herein, we report on the first study into the effects of changes to the sugar motif in the known MAIT cell agonists 7-hydroxy-6-methyl-8-d-ribityllumazine (RL-6-Me-7-OH) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU). Tetramer staining of MAIT cells revealed that the absence of the 2′-hydroxy group on the sugar backbone of lumazines improved MR1-MAIT TCR binding, which could be rationalised using computational docking studies. Although none of the lumazines activated MAIT cells, all 5-OP-RU analogues showed significant MAIT cell activation, with several analogues exhibiting comparable activity to 5-OP-RU. Docking studies with the 5-OP-RU analogues revealed different interactions between the sugar backbone and MR1 and the MAIT TCR compared to those observed for the lumazines and confirmed the importance of the 2′-hydroxy group for ligand binding and activity. Taken together, this information will assist in the development of future potent agonists and antagonists of MAIT cells.
- Braganza, Chriselle D.,Shibata, Kensuke,Fujiwara, Aisa,Motozono, Chihiro,Sonoda, Koh-Hei,Yamasaki, Sho,Stocker, Bridget L.,Timmer, Mattie S. M.
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supporting information
p. 8992 - 9000
(2019/10/28)
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- O-nucleoside, S-nucleoside, and N-nucleoside probes of lumazine synthase and riboflavin synthase
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Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin, while riboflavin synthase catalyzes the last step. O-Nucleoside, S-nucleoside, and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine synthase and riboflavin synthase inhibitors, while the C-nucleosides were the least potent. A series of molecular dynamics simulations followed by free energy calculations using the Poisson-Boltzmann/surface area (MM-PBSA) method were carried out in order to rationalize the results of ligand binding to lumazine synthase, and the results provide insight into the dynamics of ligand binding as well as the molecular forces stabilizing the intermediates in the enzyme-catalyzed reaction.
- Talukdar, Arindam,Zhao, Yujie,Lv, Wei,Bacher, Adelbert,Illarionov, Boris,Fischer, Markus,Cushman, Mark
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experimental part
p. 6239 - 6261
(2012/09/25)
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- Virtual screening, selection and development of a benzindolone structural scaffold for inhibition of lumazine synthase
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Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a Ki of 70 μM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (Ki 38 μM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve π-π stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.
- Talukdar, Arindam,Morgunova, Ekaterina,Duan, Jianxin,Meining, Winfried,Foloppe, Nicolas,Nilsson, Lennart,Bacher, Adelbert,Illarionov, Boris,Fischer, Markus,Ladenstein, Rudolf,Cushman, Mark
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experimental part
p. 3518 - 3534
(2010/08/05)
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- A new series of N-[2,4-dioxo-6-D-ribitylamino-1,2,3,4-tetrahydropyrimidin- 5-yl]oxalamic acid derivatives as inhibitors of lumazine synthase and riboflavin synthase: Design, synthesis, biochemical evaluation, crystallography, and mechanistic implications
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(Figure Presented) The penultimate step in the biosynthesis of riboflavin is catalyzed by lumazine synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine synthase, Bacillus subtilis lumazine synthase, and Schizosaccharomyces pombe lumazine synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli riboflavin synthase with a Ki of 1.3 nM. The crystal structure of M. tuberculosis lumazine synthase in complex with one of the inhibitors provides a model of the conformation of the intermediate occurring immediately after phosphate elimination, supporting a mechanism in which phosphate elimination occurs before a conformational change of the Schiff base intermediate toward a cyclic structure.
- Zhang, Yanlei,Illarionov, Boris,Morgunova, Ekaterina,Jin, Guangyi,Bacher, Adelbert,Fischer, Markus,Ladenstein, Rudolf,Cushman, Mark
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p. 2715 - 2724
(2008/09/19)
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- A new series of 3-alkyl phosphate derivatives of 4,5,6,7-tetrahydro-1-D- ribityl-1H-pyrazolo[3,4-d]pyrimidinedione as inhibitors of lumazine synthase: Design, synthesis, and evaluation
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(Chemical Equation Presented) Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin. A homologous series of three pyrazolopyrimidine analogues of a hypothetical intermediate in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. The key steps of the synthesis were C-5 deprotonation of 4-chloro-2,6-dimethoxypyrimidine, acylation of the resulting anion, and conversion of the product to a pyrazolopyrimidine with hydrazine. Alkylation of the pyrazolopyrimidine with a substituted ribityl iodide and deprotection of the ribityl chain afforded the final set of three products. All three compounds were extremely potent inhibitors of the lumazine synthases of Mycobacterium tuberculosis, Magnaporthe grisea, Candida albicans, and Schizosaccharomyces pombe lumazine synthase, with inhibition constants in the low nanomolar to subnanomolar range. Molecular modeling of one of the homologues bound to Mycobacterium tuberculosis lumazine synthase suggests that both the hypothetical intermediate in the lumazine synthase-catalyzed reaction pathway and the metabolically stable analogues bind similarly.
- Zhang, Yanlei,Jin, Guangyi,Illarionov, Boris,Bacher, Adelbert,Fischer, Markus,Cushman, Mark
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p. 7176 - 7184
(2008/02/12)
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- Incorporation of an amide into 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors results in an unexpected reversal of selectivity for riboflavin synthase vs lumazine synthase
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Several analogues of a hypothetical intermediate in the reaction catalyzed by lumazine synthase were synthesized and tested as inhibitors of both Bacillus subtilis lumazine synthase and Escherichia coli riboflavin synthase. The new compounds were designed by replacement of a two-carbon fragment of several 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors with an amide linkage that was envisioned as an analogue of a Schiff base moiety of a hypothetical intermediate in the enzyme-catalyzed reaction. The incorporation of the amide group led to an unexpected reversal in selectivity for inhibition of lumazine synthase vs riboflavin synthase. Whereas the parent 5-phosphonoalkyl-6-D-ribitylaminopyrimidinediones were lumazine synthase inhibitors and did not inhibit riboflavin synthase, the amide-containing derivatives inhibited riboflavin synthase and were only very weak or inactive as lumazine synthase inhibitors. Molecular modeling of inhibitor-lumazine synthase complexes did not reveal a structural basis for these unexpected findings. However, molecular modeling of one of the inhibitors with E. coli riboflavin synthase demonstrated that the active site of the enzyme could readily accommodate two ligand molecules.
- Cushman, Mark,Yang, Donglai,Mihalic, Jeffrey T.,Chen, Jinhua,Gerhardt, Stefan,Huber, Robert,Fischer, Markus,Kis, Klaus,Bacher, Adelbert
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p. 6871 - 6877
(2007/10/03)
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- Biosynthesis of riboflavin. The reaction catalyzed by 6,7-dimethyl-8-ribityllumazine synthase can proceed without enzymatic catalysis under physiological conditions
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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 Ea = 46.3 kJ mol-1. The regioselectivity of the uncatalyzed reaction increases with pH and temperature (70% at 65 °C and pH 7.75). The data suggest partitioning of the uncatalyzed reaction via two different reaction pathways. The value of kcat/kuncat may be indicative for an entropy driven process for the enzyme-catalyzed reaction.
- Kis,Kugelbrey,Bacher
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p. 2555 - 2559
(2007/10/03)
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