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The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are attractive targets for drugs against various diseases, including malaria. We describe herein the structure-based design, synthesis, conformational analysis, and biological evaluation of several 8-brominated or 8-aminated adenosine derivatives with different substituents at C(5′), targeting the ATP-adenine binding site of the IspE protein from the non-mevalonate pathway. An exhaustive conformational analysis of the adenosine derivatives both in solution and in the solid state confirmed the desired syn orientation of the adenine moiety. Despite this favorable pre-organization for binding to the cofactor pocket, biological evaluation of the inhibitors showed only a very modest inhibitory activity. [ABSTRACT FROM AUTHOR]

We investigated the lumazine proteinfrom Photobacteriumleiognathiin complex with its biologically active cofactor,6,7-dimethyl-8-ribityllumazine, at different redox states and comparedthe results with samples containing a riboflavin cofactor. Using anaerobicphotoreduction, we were able to record optical absorption kineticsfrom both cofactors in similar protein environments. It could be demonstratedthat the protein is able to stabilize a neutral ribolumazine radicalwith ∼35% yield. The ribolumazine radical state was furtherinvestigated by W-band continuous-wave EPR and X-band pulsed ENDORspectroscopy. Here, both the principal values of the g-tensor and an almost complete mapping of the proton hyperfine couplings(hfcs) could be obtained. Remarkably, the g-tensor’sprincipal components are similar to those of the respective riboflavin-containingprotein; however, the proton hfcs show noticeable differences. Comparingtime-resolved optical absorption and fluorescence data from ribolumazine-containingsamples, solely fluorescence but no signs of any intermediate radicalor a triplet state could be identified. This is in contrast to lumazineprotein samples containing the riboflavin cofactor, for which a highyield of the photogenerated triplet state and some excited flavinradical could be detected using time-resolved spectroscopy. Theseresults clearly demonstrate that ribolumazine is a redox-active moleculeand could, in principle, be employed as a cofactor in other enzymaticreactions. [ABSTRACT FROM AUTHOR]

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are potential new targets for the development of selective drugs for the treatment of important infectious diseases. This pathway is used by major human pathogens, such as Plasmodium falciparum and Mycobacterium tuberculosis, but not by humans. The fourth enzyme in the pathway is the kinase IspE, and we report here the development and biological evaluation of new ligands for this enzyme from Escherichia coli and Aquifex aeolicus species as model systems for the pathogenic enzymes. The study focuses on analysis of the methylerythritol pocket of the 4-diphosphocytidyl-2- C-methyl- D-erythritol binding site. A series of 5-substituted 1-(thiolan-2-yl)cytosines with increasingly polar substituents were synthesized, opting for possible water-replacements in that sub-pocket as well as a high water-solubility of the ligands. In vitro studies showed IC50 values in the micromolar range against E. coli IspE, but, unexpectedly, no inhibition against A. aeolicus IspE within the measurement range of the biological tests. [ABSTRACT FROM AUTHOR]

Specific inhibition of enzymes of the non-mevalonatepathway isa promising strategy for the development of novel antiplasmodial drugs.α-Aryl-substituted β-oxa isosteres of fosmidomycin witha reverse orientation of the hydroxamic acid group were synthesizedand evaluated for their inhibitory activity against recombinant 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) of Plasmodium falciparumand for their in vitroantiplasmodial activity against chloroquine-sensitive and resistantstrains of P. falciparum. The mostactive derivative inhibits IspC protein of P. falciparum(PfIspC) with an IC50value of 12 nMand shows potent in vitroantiplasmodial activity.In addition, lipophilic ester prodrugs demonstrated improved P. falciparumgrowth inhibition in vitro. [ABSTRACT FROM AUTHOR]

The plant blue light receptor phototropin comprises a protein kinase domain and two FMN-binding LOV domains (LOV1 and LOV2). Blue light irradiation of recombinant LOV domains is conducive to the addition of a cysteinyl thiolate group to carbon 4a of the FMN chromophore, and spontaneous cleavage of that photoadduct completes the photocycle of the receptor. The present study is based on 13C NMR signal modulation observed after reconstitution of LOV domains of different origins with random libraries of 13C-labeled FMN isotopologues. Using this approach, all 13C signals of FMN bound to LOV1 and LOV2 domains of Avena sativa and to the LOV2 domain of the fern, Adiantum capillus-veneris, could be unequivocally assigned under dark and under blue light irradiation conditions. 13C Chemical shifts of FMN are shown to be differently modulated by complexation with the LOV domains under study, indicating slight differences in the binding interactions of FMN and the apoproteins. [ABSTRACT FROM AUTHOR]

Air oxidation of 1,3,6,8-tetrahydroxy-2,7-naphthyridine afforded 2,5,8,11-tetraaza-5,11-dihydro-4,10-dihydroxyperylene-1,3,6,7,9,12-hexaone. X-ray crystallography of the product revealed that it exists in the meso form in the solid state. The mechanism of product formation most likely involves oxidative phenolic coupling and oxidation. The product proved to be a competitive inhibitor of Schizosaccharomyces pombe lumazine synthase with a Ki of 66 ± 13 µM in Tris buffer and 22 ± 4 µin phosphate buffer, This is significantly more potent than the reactant (Ki 350 ± 76 µM, competitive inhibition), which had previously been identified as a lumazine synthase inhibitor by high-throughput screening. Ab initio calculations indicate that the meso form is slightly less stable than the enantiomeric form, and that the two forms interconvert rapidly at room temperature. [ABSTRACT FROM AUTHOR]

Lumazine proteins of luminescent bacteria are paralogs of riboflavin synthase which are devoid of catalytic activity but bind the riboflavin synthase substrate, 6,7-dimethyl-8-ribityllumazine, with high affinity and are believed to serve as optical transponders for bioluminescence emission. Lumazine protein of Photobacterium leiognathi was expressed in a recombinant Escherichia coli host and was reconstituted with mixtures (random libraries) of 13C-labeled isotopologs of 6,7-dimethyl-8-ribityllumazine or riboflavin that had been prepared by biotransformation of [U-13C6]-, [1-13C1]-, [2-13C1]-, and [3-13C1]glucose. 13C NMR analysis of the protein/ligand complexes afforded the assignments of the 13C NMR chemical shifts for all carbon atoms of the protein-bound ligands by isotopolog abundance editing. The carbon atoms of the ribityl groups of both ligands studied were shifted up to 6 ppm upon binding to the protein. Chemical shift modulation of the side chain and chromophore carbon atoms due to protein/ligand interaction is discussed on the basis of the sequence similarity between lumazine protein and riboflavin synthase. [ABSTRACT FROM AUTHOR]

The dismutation of 6,7-dimethyl-8-ribityllumazine catalyzed by riboflavin synthase affords riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. A pentacyclic adduct of two 6,7-dimethyl-8-ribityllumazines has been identified earlier as a catalytically competent reaction intermediate of the Escherichia coli enzyme. Acid quenching of reaction mixtures of riboflavin synthase of Methanococcus jannaschii, a paralog of 6,7-dimethyl-8-ribityllumazine synthase devoid of similarity with riboflavin synthases of eubacteria and eukaryotes, afforded a compound whose optical absorption and NMR spectra resemble that of the pentacyclic E. coli riboflavin synthase intermediate, whereas the circular dichroism spectra of the two compounds have similar envelopes but opposite signs. Each of the compounds could serve as a catalytically competent intermediate for the enzyme by which it was produced, but not vice versa. All available data indicate that the respective pentacyclic intermediates of the M. jannaschii and E. coli enzymes are diastereomers. [ABSTRACT FROM AUTHOR]

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 15NH4C1 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-13C13]-6,7-dimethyl-8-ribityllumazine based on [U-13C6]glucose was 25 mg/g. Fermentation with [1-13C1]-, [2-13C1]-, or [3-13C1]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. [ABSTRACT FROM AUTHOR]

Background: Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs. Results: A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7- dimethyl-8-ribityllumazine at a rate of 158 nmol mg-1 min-1 with an apparent KM of 5.7 microM. 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins. Conclusion: Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7- dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism. [ABSTRACT FROM AUTHOR]

Examines the pentacyclic reaction intermediate of riboflavin synthase. Use of riboflavin synthase from Escherichia coli in the formation of riboflavin; Observation on the quenching of presteady-state reaction mixtures and trifluoroacetic acid; Utilization of nuclear magnetic resonance spectroscopy.

The human pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis resulting in over 1 million fatalities every year, despite decades of research into the development of new anti-TB compounds. Unlike most other organisms M. tuberculosis has six putative genes for epoxide hydrolases (EH) of the α/β-hydrolase family with little known about their individual substrates, suggesting functional significance for these genes to the organism. Due to their role in detoxification, M. tuberculosis EH's have been identified as potential drug targets. Here, we demonstrate epoxide hydrolase activity of M. thermoresistibile epoxide hydrolase A (Mth-EphA) and report its crystal structure in complex with the inhibitor 1,3-diphenylurea at 2.0 Å resolution. Mth-EphA displays high sequence similarity to its orthologue from M. tuberculosis and generally high structural similarity to α/β-hydrolase EHs. The structure of the inhibitor bound complex reveals the geometry of the catalytic residues and the conformation of the inhibitor. Comparison to other EHs from mycobacteria allows insight into the active site plasticity with respect to substrate specificity. We speculate that mycobacterial EHs may have a narrow substrate specificity providing a potential explanation for the genetic repertoire of epoxide hydrolase genes in M. tuberculosis. [ABSTRACT FROM AUTHOR]

A library of over 103 thousand compounds was screened for inhibitors of the IspD domain (2‐C‐methyl‐d‐erythritol 4‐phosphate cytidylyl transferase domain) of the bifunctional IspDF protein from Helicobacter pylori using a photometric assay. Around 300 compounds showed IC50 values below 100 μm, and three compounds had IC50 values below 1 μm. A few IspD inhibitors could also inhibit the IspF domain (2‐C‐Methyl‐d‐erythritol‐2,4‐cyclopyrophosphate synthase) of the IspDF protein. The most potent IspD inhibitors were tested as growth inhibitors of H. pylori. Several compounds showed inhibition of bacterial growth with IC50 in the single‐digit μm range. The most potent growth inhibitor had an IC50 value of 3.4 μm. The most potent growth inhibitor without measurable effect on eukaryotic cell viability had an IC50 value of 7.2 μm. [ABSTRACT FROM AUTHOR]