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Sample records for prenyl diphosphate synthase

  1. Coenzyme Q10 supplementation rescues renal disease in Pdss2kd/kd mice with mutations in prenyl diphosphate synthase subunit 2.

    Science.gov (United States)

    Saiki, Ryoichi; Lunceford, Adam L; Shi, Yuchen; Marbois, Beth; King, Rhonda; Pachuski, Justin; Kawamukai, Makoto; Gasser, David L; Clarke, Catherine F

    2008-11-01

    Homozygous mice carrying kd (kidney disease) mutations in the gene encoding prenyl diphosphate synthase subunit 2 (Pdss2kd/kd) develop interstitial nephritis and eventually die from end-stage renal disease. The PDSS2 polypeptide in concert with PDSS1 synthesizes the polyisoprenyl tail of coenzyme Q (Q or ubiquinone), a lipid quinone required for mitochondrial respiratory electron transport. We have shown that a deficiency in Q content is evident in Pdss2kd/kd mouse kidney lipid extracts by 40 days of age and thus precedes the onset of proteinuria and kidney disease by several weeks. The presence of the kd (V117M) mutation in PDSS2 does not prevent its association with PDSS1. However, heterologous expression of the kd mutant form of PDSS2 together with PDSS1 in Escherichia coli recapitulates the Q deficiency observed in the Pdss2kd/kd mouse. Dietary supplementation with Q10 provides a dramatic rescue of both proteinuria and interstitial nephritis in the Pdss2kd/kd mutant mice. The results presented suggest that Q may be acting as a potent lipid-soluble antioxidant, rather than by boosting kidney mitochondrial respiration. Such Q10 supplementation may have profound and beneficial effects in treatment of certain forms of focal segmental glomerulosclerosis that mirror the renal disease of the Pdss2kd/kd mouse.

  2. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  3. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  4. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

    Science.gov (United States)

    Bergstrom, J D; Bostedor, R G; Masarachia, P J; Reszka, A A; Rodan, G

    2000-01-01

    Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

  5. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  6. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  7. An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases.

    Science.gov (United States)

    Dozier, Jonathan K; Distefano, Mark D

    2012-02-01

    Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone, and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays either use radiolabeled substrates and are discontinuous or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format, and that it can reproduce IC(50) values for several previously reported FDPS inhibitors. This new method offers a simple, safe, and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target.

  8. Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa.

    Science.gov (United States)

    Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

    2010-06-01

    Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum.

  9. Two Eucommia farnesyl diphosphate synthases exhibit distinct enzymatic properties leading to end product preferences.

    Science.gov (United States)

    Kajiura, Hiroyuki; Suzuki, Nobuaki; Tokumoto, Yuji; Yoshizawa, Takuya; Takeno, Shinya; Fujiyama, Kazuhito; Kaneko, Yoshinobu; Matsumura, Hiroyoshi; Nakazawa, Yoshihisa

    2017-08-01

    Farnesyl diphosphate synthase (FPS) is an essential enzyme in the biosynthesis of prenyl precursors for the production of primary and secondary metabolites, including sterols, dolichols, carotenoids and ubiquinones, and for the modification of proteins. Here we identified and characterized two FPSs (EuFPS1 and EuFPS2) from the plant Eucommia ulmoides. The EuFPSs had seven highly conserved prenyltransferase-specific domains that are critical for activity. Complementation and biochemical analyses using bacterially produced recombinant EuFPS isoforms showed that the EuFPSs had FPP synthesis activities both in vivo and in vitro. In addition to the typical reaction mechanisms of FPS, EuFPSs utilized farnesyl diphosphate (FPP) as an allylic substrate and participated in further elongation of the isoprenyl chain, resulting in the synthesis of geranylgeranyl diphosphate. However, despite the high amino acid similarities between the two EuFPS isozymes, their specific activities, substrate preferences, and final reaction products were different. The use of dimethylallyl diphosphate (DMAPP) as an allylic substrate highlighted the differences between the two enzymes: depending on the pH, the metal ion cofactor, and the cofactor concentration, EuFPS2 accumulated geranyl diphosphate as an intermediate product at a constant rate, whereas EuFPS1 synthesized little geranyl diphosphate. The reaction kinetics of the EuFPSs demonstrated that isopentenyl diphosphate and DMAPP were used both as substrates and as inhibitors of EuFPS activity. Taken together, the results indicate that the biosynthesis of FPP is highly regulated by various factors indispensable for EuFPS reactions in plants. Copyright © 2017. Published by Elsevier B.V.

  10. A new motif for inhibitors of geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Foust, Benjamin J; Allen, Cheryl; Holstein, Sarah A; Wiemer, David F

    2016-08-15

    The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure-function relationship which is dependent on the nature of the alkyl group at the α-carbon.

  11. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...... PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate...

  12. Bacillus caldolyticus prs gene encoding phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-1-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  13. Rv0989c encodes a novel (E)-geranyl diphosphate synthase facilitating decaprenyl diphosphate biosynthesis in Mycobacterium tuberculosis.

    Science.gov (United States)

    Mann, Francis M; Thomas, Jill A; Peters, Reuben J

    2011-02-04

    Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the "missing" GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from Mtb, resolving previous discrepancies between biochemical and genetic studies of cell wall biosynthesis.

  14. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    Science.gov (United States)

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.

  15. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemolsynthase activity

    NARCIS (Netherlands)

    Yang, T.; Gao, L.; Hu, H.; Stoopen, G.M.; Wang, C.; Jongsma, M.A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme inthe biosynthesis of pyrethrins, the most widely used plant-derivedpesticide.CDScatalyzes c1’-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP).

  16. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemolsynthase activity

    NARCIS (Netherlands)

    Yang, T.; Gao, L.; Hu, H.; Stoopen, G.M.; Wang, C.; Jongsma, M.A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme inthe biosynthesis of pyrethrins, the most widely used plant-derivedpesticide.CDScatalyzes c1’-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP).

  17. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  18. Molecular cloning and catalytic activity of a membrane-bound prenyl diphosphate phosphatase from Croton stellatopilosus Ohba.

    Science.gov (United States)

    Nualkaew, Natsajee; Guennewich, Nils; Springob, Karin; Klamrak, Anuwatchakit; De-Eknamkul, Wanchai; Kutchan, Toni M

    2013-07-01

    Geranylgeraniol (GGOH), a bioactive acyclic diterpene with apoptotic induction activity, is the immediate precursor of the commercial anti-peptic, plaunotol (18-hydroxy geranylgeraniol), which is found in Croton stellatopilosus (Ohba). From this plant, a cDNA encoding a prenyl diphosphate phosphatase (CsPDP), which catalyses the dephosphorylation of geranylgeranyl diphosphate (GGPP) to GGOH, was isolated using a PCR approach. The full-length cDNA contained 888bp and encoded a 33.6 kDa protein (295 amino acids) that was phylogenetically grouped into the phosphatidic acid phosphatase (PAP) enzyme family. The deduced amino acid sequence showed 6 hydrophobic transmembrane regions with 57-85% homology to the sequences of other plant PAPs. The recombinant CsPDP and its 4 truncated constructs exhibited decreasing dephosphorylation activities relative to the lengths of the N-terminal deletions. While the full-length CsPDP successfully performed the two sequential monodephosphorylation steps on GGPP to form GGOH, the larger N-terminal deletion in the truncated enzymes appeared to specifically decrease the catalytic efficiency of the second monodephosphorylation step. The information presented here on the CsPDP cDNA and factors affecting the dephosphorylation activity of its recombinant protein may eventually lead to the discovery of the specific GGPP phosphatase gene and enzyme that are involved in the formation of GGOH in the biosynthetic pathway of plaunotol in C. stellatopilosus.

  19. Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; McGuire, James N

    2004-01-01

    The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms...... competitive with respect to ATP. A predicted structure of the B. caldolyticus enzyme based on homology modelling with the structure of B. subtilis 5-phospho-alpha-D-ribosyl 1-diphosphate synthase shows 92% of the amino acid differences to be on solvent exposed surfaces in the hexameric structure....

  20. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    Energy Technology Data Exchange (ETDEWEB)

    Aripirala, Srinivas [Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States); Gonzalez-Pacanowska, Dolores [López-Neyra Institute of Parasitology and Biomedicine, 18001 Granada (Spain); Oldfield, Eric [University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Kaiser, Marcel [University of Basel, Petersplatz 1, CH-4003 Basel (Switzerland); Amzel, L. Mario, E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Gabelli, Sandra B., E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States)

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  1. A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum.

    Science.gov (United States)

    Liu, Yan; Luo, Shi-Hong; Schmidt, Axel; Wang, Guo-Dong; Sun, Gui-Ling; Grant, Marcus; Kuang, Ce; Yang, Min-Jie; Jing, Shu-Xi; Li, Chun-Huan; Schneider, Bernd; Gershenzon, Jonathan; Li, Sheng-Hong

    2016-03-01

    Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-D-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms.

  2. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    Science.gov (United States)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions. PMID:21602811

  3. Structure and Mechanism of the Farnesyl Diphosphate Synthase from Trypanosoma cruzi: Implications for Drug Design

    Energy Technology Data Exchange (ETDEWEB)

    Gabelli,S.; McLellan, J.; Montalvetti, A.; Oldfield, E.; Docampo, R.; Amzel, L.

    2006-01-01

    Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C{sub 5} alcohols (isopentenyl and dimethylallyl) to form C{sub 10} and C{sub 15} diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.

  4. Metal ions control product specificity of isoprenyl diphosphate synthases in the insect terpenoid pathway

    Science.gov (United States)

    Frick, Sindy; Nagel, Raimund; Schmidt, Axel; Bodemann, René R.; Rahfeld, Peter; Pauls, Gerhard; Brandt, Wolfgang; Gershenzon, Jonathan; Boland, Wilhelm; Burse, Antje

    2013-01-01

    Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co2+ or Mn2+ as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg2+. In reaction with Co2+, PcIDS1 has a Km of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg2+, PcIDS1 has a Km of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co2+, Mn2+, or Mg2+ in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism. PMID:23440195

  5. Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae, and the role of isoprenyl diphosphate synthases.

    Science.gov (United States)

    Keeling, Christopher I; Chiu, Christine C; Aw, Tidiane; Li, Maria; Henderson, Hannah; Tittiger, Claus; Weng, Hong-Biao; Blomquist, Gary J; Bohlmann, Joerg

    2013-11-19

    The mountain pine beetle (Dendroctonus ponderosae Hopkins) is the most destructive pest of western North American pine forests. Adult males produce frontalin, an eight-carbon antiaggregation pheromone, via the mevalonate pathway, as part of several pheromones that initiate and modulate the mass attack of host trees. Frontalin acts as a pheromone, attractant, or kairomone in most Dendroctonus species, other insects, and even elephants. 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to originate from 10-carbon geranyl diphosphate (GPP), 15-carbon farnesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate (GGPP) via a dioxygenase- or cytochrome P450-mediated carbon-carbon bond cleavage. To investigate the role of isoprenyl diphosphate synthases in pheromone biosynthesis, we characterized a bifunctional GPP/FPP synthase and a GGPP synthase in the mountain pine beetle. The ratio of GPP to FPP produced by the GPP/FPP synthase was highly dependent on the ratio of the substrates isopentenyl diphosphate and dimethylallyl diphosphate used in the assay. Transcript levels in various tissues and life stages suggested that GGPP rather than GPP or FPP is used as a precursor to frontalin. Reduction of transcript levels by RNA interference of the isoprenyl diphosphate synthases identified GGPP synthase as having the largest effect on frontalin production, suggesting that frontalin is derived from a 20-carbon isoprenoid precursor rather than from the 10- or 15-carbon precursors.

  6. Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium

    OpenAIRE

    Rivera, Susan B.; Swedlund, Bradley D.; King, Gretchen J.; Bell, Russell N.; Hussey, Charles E.; Shattuck-Eidens, Donna M.; Wrobel, Wislawa M.; Peiser, Galen D.; Poulter, C. Dale

    2001-01-01

    Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1′-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPa...

  7. Expression pattern of the coparyl diphosphate synthase gene in developing rice anthers.

    Science.gov (United States)

    Fukuda, Ari; Nemoto, Keisuke; Chono, Makiko; Yamaguchi, Shinjiro; Nakajima, Masatoshi; Yamagishi, Junko; Maekawa, Masahiko; Yamaguchi, Isomaro

    2004-08-01

    Rice anthers contain high concentrations of gibberellins A(4) and A(7). To understand their physiological roles, we examined the site of their biosynthesis by analyzing the expression pattern of a gene (OsCPS) encoding coparyl diphosphate synthase in developing rice flowers. Expression was apparent in the anthers 1-2 days before flowering, and CPS mRNA accumulated in the maturing pollen.

  8. Role of Arginine-304 in the Diphosphate-Triggered Active Site Closure Mechanism of Trichodiene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Vedula,L.; Cane, D.; Christianson, D.

    2005-01-01

    The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PPi) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PPi bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg{sup 2+}{sub 3-}PP{sub i}, in which R304 donates hydrogen bonds to PP{sub i} and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP{sub i} in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP{sub i} and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg{sup 2+}{sub 3-}PP{sub i} binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP{sub i} leaving group.

  9. Adaptive evolution of the chrysanthemyl diphosphate synthase gene involved in irregular monoterpene metabolism

    Directory of Open Access Journals (Sweden)

    Liu Ping-Li

    2012-11-01

    Full Text Available Abstract Background Chrysanthemyl diphosphate synthase (CDS is a key enzyme in biosynthetic pathways producing pyrethrins and irregular monoterpenes. These compounds are confined to plants of the tribe Anthemideae of the Asteraceae, and play an important role in defending the plants against herbivorous insects. It has been proposed that the CDS genes arose from duplication of the farnesyl diphosphate synthase (FDS gene and have different function from FDSs. However, the duplication time toward the origin of CDS and the evolutionary force behind the functional divergence of the CDS gene are still unknown. Results Two duplication events were detected in the evolutionary history of the FDS gene family in the Asteraceae, and the second duplication led to the origin of CDS. CDS occurred after the divergence of the tribe Mutisieae from other tribes of Asteraceae but before the birth of the Anthemideae tribe. After its origin, CDS accumulated four mutations in sites homologous to the substrate-binding and catalysis sites of FDS. Of these, two sites were involved in the binding of the nucleophilic substrate isopentenyl diphosphate in FDS. Maximum likelihood analyses showed that some sites in CDS were under positive selection and were scattered throughout primary sequences, whereas in the three-dimensional structure model they clustered in the large central cavity. Conclusion Positive selection associated with gene duplication played a major role in the evolution of CDS.

  10. A New Farnesyl Diphosphate Synthase Gene from Taxus media Rehder: Cloning, Characterization and Functional Complementation

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hua Liao; Min Chen; Yi-Fu Gong; Zhu-Gang Li; Kai-Jing Zuo; Peng Wang; Feng Tan; Xiao-Fen Sun; Ke-Xuan Tang

    2006-01-01

    Farnesyl diphosphate synthase (FPS; EC 2.5.1.10) catalyzes the production of 15-carbon farnesyl diphosphate which is a branch-point intermediate for many terpenoids. This reaction is considered to be a ratelimiting step in terpenoid biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl diphosphate synthase from a gymnosperm plant species, Taxus media Rehder,designated as TmFPS1. The full-length cDNA of TmFPS1 (GenBank accession number: AY461811) was 1 464bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptide with a calculated molecular weight of 40.3 kDa and a theoretical pl of 5.07. Bioinformatic analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetic analysis showed that farnesyl diphosphate synthases can be divided into two groups: one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homologybased structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature is the arrangement of 13 core helices around a large central cavity in which the catalytic reaction takes place. Our bioinformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPS gene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, including the needles, stems and roots of T. media. Subsequently, functional complementation with TmFPS1 in a FPS-deficient mutant yeast demonstrated that TmFPS1 did encode farnesyl diphosphate synthase, which rescued the yeast mutant.This study will be helpful in future investigations aiming at understanding the detailed role of FPS in terpenoid biosynthesis flux control at the molecular genetic level.

  11. Additional diterpenes from Physcomitrella patens synthesized by copalyl diphosphate/kaurene synthase (PpCPS/KS).

    Science.gov (United States)

    Zhan, Xin; Bach, Søren Spanner; Hansen, Nikolaj Lervad; Lunde, Christina; Simonsen, Henrik Toft

    2015-11-01

    The bifunctional diterpene synthase, copalyl diphosphate/kaurene synthase from the moss Physcomitrella patens (PpCPS/KS), catalyses the formation of at least four diterpenes, including ent-beyerene, ent-sandaracopimaradiene, ent-kaur-16-ene, and 16-hydroxy-ent-kaurene. The enzymatic activity has been confirmed through generation of a targeted PpCPS/KS knock-out mutant in P. patens via homologous recombination, through transient expression of PpCPS/KS in Nicotiana benthamiana, and expression of PpCPS/KS in E. coli. GC-MS analysis of the knock-out mutant shows that it lacks the diterpenoids, supporting that all are products of PpCPS/KS as observed in N. benthamiana and E. coli. These results provide additional knowledge of the mechanism of this bifunctional diterpene synthase, and are in line with proposed reaction mechanisms in kaurene biosynthesis.

  12. Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Ignea, Codruta; Trikka, Fotini A; Nikolaidis, Alexandros K; Georgantea, Panagiota; Ioannou, Efstathia; Loupassaki, Sofia; Kefalas, Panagiotis; Kanellis, Angelos K; Roussis, Vassilios; Makris, Antonios M; Kampranis, Sotirios C

    2015-01-01

    Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and β-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms.

  13. Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

    Directory of Open Access Journals (Sweden)

    Jianrong Wang

    2014-12-01

    Full Text Available Poria cocos (P. cocos has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%. The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP from geranyl diphosphate (GPP and isopentenyl diphosphate (IPP. Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

  14. Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poria cocos.

    Science.gov (United States)

    Wang, Jianrong; Li, Yangyuan; Liu, Danni

    2014-12-02

    Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

  15. Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Piotr Szymczyk

    2016-09-01

    Full Text Available The promoter, 5' UTR, and 34-nt 5' fragments of protein encoding region of the Salvia miltiorrhiza copalyl diphosphate synthase gene were cloned and characterized. No tandem repeats, miRNA binding sites, or CpNpG islands were observed in the promoter, 5' UTR, or protein encoding fragments. The entire isolated promoter and 5' UTR is 2235 bp long and contains repetitions of many cis-active elements, recognized by homologous transcription factors, found in Arabidopsis thaliana and other plant species. A pyrimidine-rich fragment with only 6 non-pyrimidine bases was localized in the 33-nt stretch from nt 2185 to 2217 in the 5' UTR. The observed cis-active sequences are potential binding sites for trans-factors that could regulate spatio-temporal CPS gene expression in response to biotic and abiotic stress conditions. Obtained results are initially verified by in silico and co-expression studies based on A. thaliana microarray data. The quantitative RT-PCR analysis confirmed that the entire 2269-bp copalyl diphosphate synthase gene fragment has the promoter activity. Quantitative RT-PCR analysis was used to study changes in CPS promoter activity occurring in response to the application of four selected biotic and abiotic regulatory factors; auxin, gibberellin, salicylic acid, and high-salt concentration.

  16. Geosmin biosynthesis. Streptomyces coelicolor germacradienol/germacrene D synthase converts farnesyl diphosphate to geosmin.

    Science.gov (United States)

    Jiang, Jiaoyang; He, Xiaofei; Cane, David E

    2006-06-28

    Geosmin is responsible for the characteristic odor of moist soil. Incubation of recombinant germacradienol synthase, encoded by the SCO6073 (SC9B1.20) gene of the Gram-positive soil bacterium Streptomyces coelicolor, with farnesyl diphosphate (2, FPP) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (74%), (-)-(7S)-germacrene D (4) (10%), geosmin (1) (13%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (3%). Individual incubations of recombinant germacradienol synthase with [1,1-2H2]FPP (2a), (1R)-[1-2H]-FPP (2b), and (1S)-[1-2H]-FPP (2c), as well as with FPP (2) in D2O, and GC-MS analysis of the resulting deuterated products supported a mechanism of geosmin formation involving proton-initiated cyclization and retro-Prins fragmentation of the initially formed germacradienol to give intermediate 5, followed by protonation of 5, 1,2-hydride shift, and capture of water.

  17. Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii

    DEFF Research Database (Denmark)

    Kadziola, Anders; Jepsen, Clemens H; Johansson, Eva;

    2005-01-01

    The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and....... The properties of M.jannaschii PRPP synthase differ widely from previously characterised PRPP synthases by its tetrameric quaternary structure and the simultaneous phosphate ion-activation and lack of allosteric inhibition, and, thus, constitute a novel class of PRPP synthases....

  18. The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis.

    Science.gov (United States)

    Thabet, Insaf; Guirimand, Grégory; Courdavault, Vincent; Papon, Nicolas; Godet, Stéphanie; Dutilleul, Christelle; Bouzid, Sadok; Giglioli-Guivarc'h, Nathalie; Clastre, Marc; Simkin, Andrew J

    2011-11-15

    Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.

  19. Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase.

    Science.gov (United States)

    Turner, Glenn W; Croteau, Rodney

    2004-12-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.

  20. Isolation and functional analysis of two Cistus creticus cDNAs encoding geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Pateraki, Irene; Kanellis, Angelos K

    2008-05-01

    Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called "ladanum", which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5-1.0cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus.

  1. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP.

    Science.gov (United States)

    Andersen, Rune W; Leggio, Leila Lo; Hove-Jensen, Bjarne; Kadziola, Anders

    2015-03-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg(2+)-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sulphate precipitation and the structure of S. solfataricus PRPP synthase was determined at 2.8 Å resolution. A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion.

  2. Specific partial reduction of geranylgeranyl diphosphate by an enzyme from the thermoacidophilic archaeon Sulfolobus acidocaldarius yields a reactive prenyl donor, not a dead-end product.

    Science.gov (United States)

    Sato, Sho; Murakami, Motomichi; Yoshimura, Tohru; Hemmi, Hisashi

    2008-06-01

    Geranylgeranyl reductase from Sulfolobus acidocaldarius was shown to catalyze the reduction of geranylgeranyl groups in the precursors of archaeal membrane lipids, generally reducing all four double bonds. However, when geranylgeranyl diphosphate was subjected to the reductase reaction, only three of the four double bonds were reduced. Mass spectrometry and acid hydrolysis indicated that the allylic double bond was preserved in the partially reduced product derived from geranylgeranyl diphosphate. Thus, the reaction product was shown to be phytyl diphosphate, which is a substrate for archaeal prenyltransferases, unlike the completely reduced compound phytanyl diphosphate.

  3. Structure of the ent-Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase.

    Science.gov (United States)

    Rudolf, Jeffrey D; Dong, Liao-Bin; Cao, Hongnan; Hatzos-Skintges, Catherine; Osipiuk, Jerzy; Endres, Michael; Chang, Chin-Yuan; Ma, Ming; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2016-08-31

    Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three α-helical domains (αβγ), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (α) and type II TSs (βγ). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtmT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 Å, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg(2+)-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.

  4. Molecular cloning and characterization of three isoprenyl diphosphate synthase genes from alfalfa.

    Science.gov (United States)

    Sun, Yan; Long, Ruicai; Kang, Junmei; Zhang, Tiejun; Zhang, Ze; Zhou, He; Yang, Qingchuan

    2013-02-01

    Isoprenoid is the precursor for the biosynthesis of saponins, abscisic acid, gibberellins, chlorophylls and many other products in plants. Saponins are an important group of bioactive plant natural products. The alfalfa (Medicago sativa L.) saponins are glycosides of different triterpene aglycones and possess many biological activities. We isolated three genes (MsFPPS, MsGPPS and MsGGPPS) encoding isoprenyl diphosphate synthases (IDS) from alfalfa via a homology-based PCR approach. The enzyme activity assay of purified recombined MsFPPS and MsGGPPS expressed in Escherichia coli indicated that they all had IDS activity. Expression analysis of the three genes in different alfalfa tissues using real time PCR displayed that they were expressed in all tissues although they had a different expression patterns. MsFPPS and MsGPS displayed a significant increase in transcript level in response to methyl jasmonate, but the transcript level of MsGGPPS decreased obviously. To elucidate the functions of the three IDSs, their overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants was applied and analyzed. The T(0) transgenic plants of MsFPPS showed high levels of squalene content when compared with control. However, no differences were detected in T(0) transgenic plants of MsGPPS and MsGGPPS. In addition, the overexpression of MsFPPS induced senescence response in transgenic plant leaves. This result may indicate that MsFPPS performs a role not only in phytosterol and triterpene biosynthesis, but also in growth regulation.

  5. Biosynthesis of the psychotropic plant diterpene salvinorin A: Discovery and characterization of the Salvia divinorum clerodienyl diphosphate synthase.

    Science.gov (United States)

    Pelot, Kyle A; Mitchell, Rod; Kwon, Moonhyuk; Hagelthorn, David M; Wardman, Jacob F; Chiang, Angela; Bohlmann, Jörg; Ro, Dae-Kyun; Zerbe, Philipp

    2017-03-01

    Salvia divinorum commonly known as diviner's sage, is an ethnomedicinal plant of the mint family (Lamiaceae). Salvia divinorum is rich in clerodane-type diterpenoids, which accumulate predominantly in leaf glandular trichomes. The main bioactive metabolite, salvinorin A, is the first non-nitrogenous natural compound known to function as an opioid-receptor agonist, and is undergoing clinical trials for potential use in treating neuropsychiatric diseases and drug addictions. We report here the discovery and functional characterization of two S. divinorum diterpene synthases (diTPSs), the ent-copalyl diphosphate (ent-CPP) synthase SdCPS1, and the clerodienyl diphosphate (CLPP) synthase SdCPS2. Mining of leaf- and trichome-specific transcriptomes revealed five diTPSs, two of which are class II diTPSs (SdCPS1-2) and three are class I enzymes (SdKSL1-3). Of the class II diTPSs, transient expression in Nicotiana benthamiana identified SdCPS1 as an ent-CPP synthase, which is prevalent in roots and, together with SdKSL1, exhibits a possible dual role in general and specialized metabolism. In vivo co-expression and in vitro assays combined with nuclear magnetic resonance (NMR) analysis identified SdCPS2 as a CLPP synthase. A role of SdCPS2 in catalyzing the committed step in salvinorin A biosynthesis is supported by its biochemical function, trichome-specific expression and absence of additional class II diTPSs in S. divinorum. Structure-guided mutagenesis revealed four catalytic residues that enabled the re-programming of SdCPS2 activity to afford four distinct products, thus advancing our understanding of how neo-functionalization events have shaped the array of different class II diTPS functions in plants, and may promote synthetic biology platforms for a broader spectrum of diterpenoid bioproducts.

  6. Cloning and functional analysis of the second geranylgeranyl diphosphate synthase gene influencing helvolic acid biosynthesis in Metarhizium anisopliae.

    Science.gov (United States)

    Singkaravanit, Suthitar; Kinoshita, Hiroshi; Ihara, Fumio; Nihira, Takuya

    2010-07-01

    A gene (ggs2) having high similarity to the geranylgeranyl diphosphate synthase (GGPP synthase) gene was cloned from Metarhizium anisopliae NAFF635007. The ggs2 gene (1,239-bp open reading frame with no intron) encoded a protein of 412 amino acids, and the transcription occurred only after late log-phase during the growth. Gene disruption of ggs2, performed to clarify the function in M. anisopliae, resulted in decreased GGPP synthase activity together with a slight delay of sporulation. An high performance liquid chromatography (HPLC) comparison of compound profiles between the wild-type strain and the disruptant revealed that a compound was abolished by the ggs2 disruption. Purification and structural elucidation by 1H-NMR and mass spectrometry analyses revealed that the lost compound is helvolic acid. Furthermore, the pathogenicity assay against two species of insect larvae revealed that the ggs2-disruptant possessed much weaker toxicity than the wild-type strain. Based on these results, it was concluded that ggs2 encodes the GGPP synthase influencing the biosynthesis of secondary metabolites in various species, including helvolic acid in M. anisopliae. To the best of our knowledge, this is the first report to identify a GGPP synthase gene related to secondary metabolism in entomopathogenic fungi.

  7. Molecular cloning and functional expression analysis of a new gene encoding geranylgeranyl diphosphate synthase from hazel (Corylus avellana L. Gasaway).

    Science.gov (United States)

    Wang, Yechun; Miao, Zhiqi; Tang, Kexuan

    2010-10-01

    Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes such as taxol. Herein, a full-length cDNA encoding GGPPS (designated as CgGGPPS) was cloned and characterized from hazel (Corylus avellana L. Gasaway), a taxol-producing angiosperms. The full-length cDNA of CgGGPPS was 1515 bp with a 1122 bp open reading frame (ORF) encoding a 373 amino acid polypeptide. The CgGGPPS genomic DNA sequence was also obtained, revealing CgGGPPS gene was not interrupted by an intron. Southern blot analysis indicated that CgGGPPS belonged to a small gene family. Tissue expression pattern analysis indicated that CgGGPPS expressed the highest in leaves. RT-PCR analysis indicated that CgGGPPS expression could be induced by exogenous methyl jasmonate acid. Furthermore, carotenoid accumulation was observed in Escherichia coli carrying pACCAR25ΔcrtE plasmid carrying CgGGPPS. The result revealed that cDNA encoded a functional GGPP synthase.

  8. Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.

    Science.gov (United States)

    Misra, Rajesh Chandra; Garg, Anchal; Roy, Sudeep; Chanotiya, Chandan Singh; Vasudev, Prema G; Ghosh, Sumit

    2015-11-01

    Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance.

  9. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae: combinatorial expression of the five PRS genes in Escherichia coli.

    Science.gov (United States)

    Hove-Jensen, Bjarne

    2004-09-24

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no detectable PRPP synthase activity. In contrast PRPP could be detected in growing cells containing PRS1 PRS2, PRS1 PRS3, PRS5 PRS2, or PRS5 PRS4. These apparent conflicting results indicate that, apart from the PRS1 PRS3-specified enzyme, PRS-specified enzyme is functional in vivo but unstable when released from the cell. Certain combinations of three PRS genes appeared to produce an enzyme that is stable in vitro. Thus, extracts of strains harboring PRS1 PRS2 PRS5, PRS1 PRS4 PRS5, or PRS2 PRS4 PRS5 as well as extracts of strains harboring combinations with PRS1 PRS3 contained readily assayable PRPP synthase activity. The data indicate that although certain pairwise combinations of subunits produce an active enzyme, yeast PRPP synthase requires at least three different subunits to be stable in vitro. The activity of PRPP synthases containing subunits 1 and 3 or subunits 1, 2, and 5 was found to be dependent on Pi, to be temperature-sensitive, and inhibited by ADP.

  10. Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts...

  11. Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chuan-Hsiang; Gabelli, Sandra B.; Oldfield, Eric; Amzel, L. Mario (UIUC); (JHU-MED)

    2010-11-15

    Bisphosphonates (BPs) are a class of compounds that have been used extensively in the treatment of osteoporosis and malignancy-related hypercalcemia. Some of these compounds act through inhibition of farnesyl diphosphate synthase (FPPS), a key enzyme in the synthesis of isoprenoids. Recently, nitrogen-containing bisphosphonates (N-BPs) used in bone resorption therapy have been shown to be active against Trypanosoma cruzi, the parasite that causes American trypanosomiasis (Chagas disease), suggesting that they may be used as anti-trypanosomal agents. The crystal structures of TcFPPS in complex with substrate (isopentenyl diphosphate, IPP) and five N-BP inhibitors show that the C-1 hydroxyl and the nitrogen-containing groups of the inhibitors alter the binding of IPP and the conformation of two TcFPPS residues, Tyr94 and Gln167. Isothermal titration calorimetry experiments suggest that binding of the first N-BPs to the homodimeric TcFPPS changes the binding properties of the second site. This mechanism of binding of N-BPs to TcFPPS is different to that reported for the binding of the same compounds to human FPPS.

  12. Characterization of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) gene from Ginkgo biloba.

    Science.gov (United States)

    Kim, Sang-Min; Kim, Soo-Un

    2010-02-01

    Diterpene trilactone ginkgolides, one of the major constituents of Ginkgo biloba extract, have shown interesting bioactivities including platelet-activating factor antagonistic activity. 1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), converting 2-C-methyl-d-erythritol-2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate, is the penultimate enzyme of the seven-step 2-C-methyl-d-erythritol 4-phosphate pathway that supplies building blocks for plant isoprenoids of plastid origin such as ginkgolides and carotenoids. Here, we report on the isolation and characterization of the full-length cDNA encoding HDS (GbHDS, GenBank accession number: DQ251630) from G. biloba. Full-length cDNA of GbHDS, 2,763 bp long, contained an ORF of 2,226 bp encoding a protein composed of 741 amino acids. The theoretical molecular weight and pI of the deduced mature GbHDS of 679 amino acid residues are 75.6 kDa and 5.5, respectively. From 2 weeks after initiation of the culture onward, transcription level of this gene in the ginkgo embryo roots increased to about two times higher than that in the leaves. GbHDS was predicted to possess chloroplast transit peptide of 62 amino acid residues, suggesting its putative localization in the plastids. The transient gene expression in Arabidopsis protoplasts confirmed that the transit peptide was capable of delivering the GbHDS protein from the cytosol into the chloroplasts. The isolation and characterization of GbHDS gene enabled us to further understand the role of GbHDS in the terpenoid biosynthesis in G. biloba.

  13. Organization of Monoterpene Biosynthesis in Mentha. Immunocytochemical Localizations of Geranyl Diphosphate Synthase, Limonene-6-Hydroxylase, Isopiperitenol Dehydrogenase, and Pulegone Reductase1

    Science.gov (United States)

    Turner, Glenn W.; Croteau, Rodney

    2004-01-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (−)-(4S)-limonene-6-hydroxylase, peppermint (−)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (−)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (−)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity. PMID:15542490

  14. Molecular cloning and functional characterization of Catharanthus roseus hydroxymethylbutenyl 4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway.

    Science.gov (United States)

    Ginis, Olivia; Courdavault, Vincent; Melin, Céline; Lanoue, Arnaud; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Courtois, Martine; Oudin, Audrey

    2012-05-01

    The Madagascar periwinkle produces monoterpenoid indole alkaloids (MIA) of high interest due to their therapeutical values. The terpenoid moiety of MIA is derived from the methyl erythritol phosphate (MEP) and seco-iridoid pathways. These pathways are regarded as the limiting branch for MIA biosynthesis in C. roseus cell and tissue cultures. In previous studies, we demonstrated a coordinated regulation at the transcriptional and spatial levels of genes from both pathways. We report here on the isolation of the 5'-flanking region (1,049 bp) of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene from the MEP pathway. To investigate promoter transcriptional activities, the HDS promoter was fused to GUS reporter gene. Agrobacterium-mediated transformation of young tobacco leaves revealed that the cloned HDS promoter displays a tissue-specific GUS staining restricted to the vascular region of the leaves and limited to a part of the vein that encompasses the phloem in agreement with the previous localization of HDS transcripts in C. roseus aerial organs. Further functional characterizations in stably or transiently transformed C. roseus cells allowed us to identify the region that can be consider as the minimal promoter and to demonstrate the induction of HDS promoter by several hormonal signals (auxin, cytokinin, methyljasmonate and ethylene) leading to MIA production. These results, and the bioinformatic analysis of the HDS 5'-region, suggest that the HDS promoter harbours a number of cis-elements binding specific transcription factors that would regulate the flux of terpenoid precursors involved in MIA biosynthesis.

  15. Bioinformatic and molecular analysis of hydroxymethylbutenyl diphosphate synthase (GCPE) gene expression during carotenoid accumulation in ripening tomato fruit.

    Science.gov (United States)

    Rodríguez-Concepción, Manuel; Querol, Jordi; Lois, Luisa María; Imperial, Santiago; Boronat, Albert

    2003-07-01

    Carotenoids are plastidic isoprenoid pigments of great biological and biotechnological interest. The precursors for carotenoid production are synthesized through the recently elucidated methylerythritol phosphate (MEP) pathway. Here we have identified a tomato ( Lycopersicon esculentum Mill.) cDNA sequence encoding a full-length protein with homology to the MEP pathway enzyme hydroxymethylbutenyl 4-diphosphate synthase (HDS, also called GCPE). Comparison with other plant and bacterial HDS sequences showed that the plant enzymes contain a plastid-targeting N-terminal sequence and two highly conserved plant-specific domains in the mature protein with no homology to any other sequence in the databases. The ubiquitous distribution of HDS-encoding expressed sequence tags (ESTs) in the tomato collections suggests that the corresponding gene is likely expressed throughout the plant. The role of HDS in controlling the supply of precursors for carotenoid biosynthesis was estimated from the bioinformatic and molecular analysis of transcript abundance in different stages of fruit development. No significant changes in HDS gene expression were deduced from the statistical analysis of EST distribution during fruit ripening, when an active MEP pathway is required to support a massive accumulation of carotenoids. RNA blot experiments confirmed that similar transcript levels were present in both the wild-type and carotenoid-depleted yellow ripe ( r) mutant fruit independent of the stage of development and the carotenoid composition of the fruit. Together, our results are consistent with a non-limiting role for HDS in carotenoid biosynthesis during tomato fruit ripening.

  16. Arabidopsis GERANYLGERANYL DIPHOSPHATE SYNTHASE 11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids.

    Science.gov (United States)

    Ruiz-Sola, M Águila; Coman, Diana; Beck, Gilles; Barja, M Victoria; Colinas, Maite; Graf, Alexander; Welsch, Ralf; Rütimann, Philipp; Bühlmann, Peter; Bigler, Laurent; Gruissem, Wilhelm; Rodríguez-Concepción, Manuel; Vranová, Eva

    2016-01-01

    Most plastid isoprenoids, including photosynthesis-related metabolites such as carotenoids and the side chain of chlorophylls, tocopherols (vitamin E), phylloquinones (vitamin K), and plastoquinones, derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. Seven out of 10 functional GGPPS isozymes in Arabidopsis thaliana reside in plastids. We aimed to address the function of different GGPPS paralogues for plastid isoprenoid biosynthesis. We constructed a gene co-expression network (GCN) using GGPPS paralogues as guide genes and genes from the upstream and downstream pathways as query genes. Furthermore, knock-out and/or knock-down ggpps mutants were generated and their growth and metabolic phenotypes were analyzed. Also, interacting protein partners of GGPPS11 were searched for. Our data showed that GGPPS11, encoding the only plastid isozyme essential for plant development, functions as a hub gene among GGPPS paralogues and is required for the production of all major groups of plastid isoprenoids. Furthermore, we showed that the GGPPS11 protein physically interacts with enzymes that use GGPP for the production of carotenoids, chlorophylls, tocopherols, phylloquinone, and plastoquinone. GGPPS11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids. Both gene co-expression and protein-protein interaction likely contribute to the channeling of GGPP by GGPPS11. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  17. Exploring diterpene metabolism in non-model species: transcriptome-enabled discovery and functional characterization of labda-7,13E-dienyl diphosphate synthase from Grindelia robusta.

    Science.gov (United States)

    Zerbe, Philipp; Rodriguez, Selina M; Mafu, Sibongile; Chiang, Angela; Sandhu, Harpreet K; O'Neil-Johnson, Mark; Starks, Courtney M; Bohlmann, Jörg

    2015-09-01

    Grindelia robusta or gumweed, is a medicinal herb of the sunflower family that forms a diverse suite of diterpenoid natural products. Its major constituents, grindelic acid and related grindelane diterpenoids accumulate in a resinous exudate covering the plants' surfaces, most prominently the unopened composite flower. Recent studies demonstrated potential pharmaceutical applications for grindelic acid and its synthetic derivatives. Mining of the previously published transcriptome of G. robusta flower tissue identified two additional diterpene synthases (diTPSs). We report the in vitro and in vivo functional characterization of an ent-kaurene synthase of general metabolism (GrTPS4) and a class II diTPS (GrTPS2) of specialized metabolism that converts geranylgeranyl diphosphate (GGPP) into labda-7,13E-dienyl diphosphate as verified by nuclear magnetic resonance (NMR) analysis. Tissue-specific transcript abundance of GrTPS2 in leaves and flowers accompanied by the presence of an endocyclic 7,13 double bond in labda-7,13E-dienyl diphosphate suggest that GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of labda-7,13E-dienyl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Together with a recently identified functional diTPS pair of G. robusta producing manoyl oxide, GrTPS2 lays the biosynthetic foundation of the diverse array of labdane-related diterpenoids in the genus Grindelia. Knowledge of these natural diterpenoid metabolic pathways paves the way for developing biotechnology approaches toward producing grindelic acid and related bioproducts.

  18. A type-B response regulator drives the expression of the hydroxymethylbutenyl diphosphate synthase gene in periwinkle.

    Science.gov (United States)

    Ginis, Olivia; Oudin, Audrey; Guirimand, Grégory; Chebbi, Mouadh; Courdavault, Vincent; Glévarec, Gaëlle; Papon, Nicolas; Crèche, Joel; Courtois, Martine

    2012-10-15

    In plant cytokinin (CK) signaling, type-B response regulators (RRs) act as major players in orchestrating the transcriptome changes in response to CK. However, their direct targets are poorly known. The identification of putative type-ARR1 motifs located within the promoter of the CK-responsive hydroxyl methyl butenyl diphosphate synthase (HDS) gene from the methyl erythritol phosphate (MEP) pathway prompted us to investigate the ability of a previously isolated periwinkle type-B RR (CrRR5) that presents high homologies with ARR1 to interact with the promoter. Electrophoretic mobility shift assays (EMSAs) demonstrated that the CrRR5 DNA-binding domain binds specifically type-ARR1 motifs within the HDS promoter. We also established through yellow fluorescent protein (YFP) imaging the targeting of CrRR5 into cell nucleus in accordance with its putative function of transcription factor. In transient assays performed on periwinkle cells cultivated with CK, overexpression of the full-length CrRR5 or a truncated CrRR5 engineering a constitutive active form (35S:ΔDDK) did not affect the HDS promoter activity that reached a threshold. By contrast, in absence of CK, overexpression of CrRR5ΔDDK enhanced promoter activity up to the threshold level observed in cells grown with CK. Our results strongly suggest that CrRR5 directly transactivates the HDS promoter. CrRR5 is the first identified transcription factor mediating the CK signaling that targets a gene from the MEP pathway involved in isoprenoid metabolism. Moreover, CrRR5 could play a role in a regulatory mechanism controlling CK homeostasis in periwinkle cells.

  19. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  20. The alteration of protein prenylation induces cardiomyocyte hypertrophy through Rheb-mTORC1 signalling and leads to chronic heart failure.

    Science.gov (United States)

    Xu, Na; Guan, Shan; Chen, Zhong; Yu, Yang; Xie, Jun; Pan, Fei-Yan; Zhao, Ning-Wei; Liu, Li; Yang, Zhong-Zhou; Gao, Xiang; Xu, Biao; Li, Chao-Jun

    2015-04-01

    G protein-regulated cell function is crucial for cardiomyocytes, and any deregulation of its gene expression or protein modification can lead to pathological cardiac hypertrophy. Herein, we report that protein prenylation, a lipidic modification of G proteins that facilitates their association with the cell membrane, might control the process of cardiomyocyte hypertrophy. We found that geranylgeranyl diphosphate synthase (GGPPS), a key enzyme involved in protein prenylation, played a critical role in postnatal heart growth by regulating cardiomyocyte size. Cardiac-specific knockout of GGPPS in mice led to spontaneous cardiac hypertrophy, beginning from week 4, accompanied by the persistent enlargement of cardiomyocytes. This hypertrophic effect occurred by altered prenylation of G proteins. Evaluation of the prenylation, membrane association and hydrophobicity showed that Rheb was hyperactivated and increased mTORC1 signalling pathway after GGPPS deletion. Protein farnesylation or mTORC1 inhibition blocked GGPPS knockdown-induced mTORC1 activation and suppressed the larger neonatal rat ventricle myocyte size and cardiomyocyte hypertrophy in vivo, demonstrating a central role of the FPP-Rheb-mTORC1 axis for GGPPS deficiency-induced cardiomyocyte hypertrophy. The sustained cardiomyocyte hypertrophy progressively provoked cardiac decompensation and dysfunction, ultimately causing heart failure and adult death. Importantly, GGPPS was down-regulated in the hypertrophic hearts of mice subjected to transverse aortic constriction (TAC) and in failing human hearts. Moreover, HPLC-MS/MS detection revealed that the myocardial farnesyl diphosphate (FPP):geranylgeranyl diphosphate (GGPP) ratio was enhanced after pressure overload. Our observations conclude that the alteration of protein prenylation promotes cardiomyocyte hypertrophic growth, which acts as a potential cause for pathogenesis of heart failure and may provide a new molecular target for hypertrophic heart disease

  1. Structure of the ent -Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Rudolf, Jeffrey D.; Dong, Liao-Bin; Cao, Hongnan; Hatzos-Skintges, Catherine; Osipiuk, Jerzy; Endres, Michael; Chang, Chin-Yuan; Ma, Ming; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

    2016-08-31

    Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three alpha-helical domains (alpha beta gamma), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (alpha) and type II TSs (beta gamma). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtnaT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 angstrom, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg2+-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.

  2. Association of farnesyl diphosphate synthase polymorphisms and response to alendronate treatment in Chinese postmenopausal women with osteoporosis

    Institute of Scientific and Technical Information of China (English)

    Liu Yi; Liu Haijuan; Li Mei; Zhou Peiran; Xing Xiaoping; Xia Weibo; Zhang Zhenlin

    2014-01-01

    Background Genetic factors are important in the pathogenesis of osteoporosis,but less is known about the genetic determinants of osteoporosis treatment.We aimed to explore the association between the gene polymorphisms of key enzyme farnesyl diphosphate synthase (FDPS) in mevalonate signaling pathway of osteoclast and response to alendronate therapy in osteoporotic postmenopausal women in China.Methods The study group comprised 639 postmenopausal women aged (62.2±7.0) years with osteoporosis or osteopenia who had been randomly assigned to low dose group (70 mg/2w) or standard dose group (70 mg/w) of alendronate in this 1-year study.We identified allelic variant of the FDPS gene using the polymerase chain reaction and restriction enzyme Faul.Before and after treatment,serum levels of calcium,phosphate,alkaline phosphatase (ALP),cross linked C-telopeptide of type Ⅰ collagen (β-CTX) were detected.Bone mineral density (BMD) at lumbar spine and proximal femur was measured.The association was analyzed between the polymorphisms of FDPS gene and the changes of BMD,bone turnover biomarkers after the treatment.Results The FDPS rs2297480 polymorphisms were associated with baseline BMD at femoral neck,and patients with CC genotype had significantly higher baseline femoral neck BMD ((733.6±84.1) mg/cm2) than those with AC genotypes ((703.0±86.9) mg/cm2) and AA genotypes ((649.8±62.4) mg/cm2) (P <0.01).No significant difference in BMD at lumbar spine was observed among different genotypes of FDPS.The percentage change of serum ALP level was significantly lower in patients with CC genotype (-22.9%) than that in those with AC genotype (-24.1%) and AA genotype (-29.8%) of FDPS after 12 months of alendronate treatment (P <0.05).Neither percentage change of BMD nor β-CTX level after alendronate treatment had association with FDPS genotype.Conclusions FDPS gene was probably a candidate gene to predict femoral neck BMD at baseline.FDPS gene alleles could predict

  3. Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses1[OPEN

    Science.gov (United States)

    Andrade, Paola; Caudepón, Daniel; Arró, Montserrat

    2016-01-01

    Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. PMID

  4. Identification and functional characterization of monofunctional ent-copalyl diphosphate and ent-kaurene synthases in white spruce reveal different patterns for diterpene synthase evolution for primary and secondary metabolism in gymnosperms.

    Science.gov (United States)

    Keeling, Christopher I; Dullat, Harpreet K; Yuen, Mack; Ralph, Steven G; Jancsik, Sharon; Bohlmann, Jörg

    2010-03-01

    The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.

  5. Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms1[W][OA

    Science.gov (United States)

    Keeling, Christopher I.; Dullat, Harpreet K.; Yuen, Mack; Ralph, Steven G.; Jancsik, Sharon; Bohlmann, Jörg

    2010-01-01

    The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms. PMID:20044448

  6. Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

    Science.gov (United States)

    Chung, Jinhwa A.; Wollack, James W.; Hovlid, Marisa L.; Okesli, Ayse; Chen, Yan; Mueller, Joachim D.; Distefano, Mark D.; Taton, T. Andrew

    2009-01-01

    Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their non-prenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography media that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and non-natural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction—farnesylation of a mCherry-CAAX fusion construct—was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a non-natural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate, as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation. PMID:18834849

  7. A copal-8-ol diphosphate synthase from the angiosperm Cistus creticus subsp. creticus is a putative key enzyme for the formation of pharmacologically active, oxygen-containing labdane-type diterpenes.

    Science.gov (United States)

    Falara, Vasiliki; Pichersky, Eran; Kanellis, Angelos K

    2010-09-01

    The resin of Cistus creticus subsp. creticus, a plant native to Crete, is rich in labdane-type diterpenes with significant antimicrobial and cytotoxic activities. The full-length cDNA of a putative diterpene synthase was isolated from a C. creticus trichome cDNA library. The deduced amino acid sequence of this protein is highly similar (59%-70% identical) to type B diterpene synthases from other angiosperm species that catalyze a protonation-initiated cyclization. The affinity-purified recombinant Escherichia coli-expressed protein used geranylgeranyl diphosphate as substrate and catalyzed the formation of copal-8-ol diphosphate. This diterpene synthase, therefore, was named CcCLS (for C. creticus copal-8-ol diphosphate synthase). Copal-8-ol diphosphate is likely to be an intermediate in the biosynthesis of the oxygen-containing labdane-type diterpenes that are abundant in the resin of this plant. RNA gel-blot analysis revealed that CcCLS is preferentially expressed in the trichomes, with higher transcript levels found in glands on young leaves than on fully expanded leaves, while CcCLS transcript levels increased after mechanical wounding. Chemical analyses revealed that labdane-type diterpene production followed a similar pattern, with higher concentrations in trichomes of young leaves and increased accumulation upon wounding.

  8. A Copal-8-ol Diphosphate Synthase from the Angiosperm Cistus creticus subsp. creticus Is a Putative Key Enzyme for the Formation of Pharmacologically Active, Oxygen-Containing Labdane-Type Diterpenes1[OA

    Science.gov (United States)

    Falara, Vasiliki; Pichersky, Eran; Kanellis, Angelos K.

    2010-01-01

    The resin of Cistus creticus subsp. creticus, a plant native to Crete, is rich in labdane-type diterpenes with significant antimicrobial and cytotoxic activities. The full-length cDNA of a putative diterpene synthase was isolated from a C. creticus trichome cDNA library. The deduced amino acid sequence of this protein is highly similar (59%–70% identical) to type B diterpene synthases from other angiosperm species that catalyze a protonation-initiated cyclization. The affinity-purified recombinant Escherichia coli-expressed protein used geranylgeranyl diphosphate as substrate and catalyzed the formation of copal-8-ol diphosphate. This diterpene synthase, therefore, was named CcCLS (for C. creticus copal-8-ol diphosphate synthase). Copal-8-ol diphosphate is likely to be an intermediate in the biosynthesis of the oxygen-containing labdane-type diterpenes that are abundant in the resin of this plant. RNA gel-blot analysis revealed that CcCLS is preferentially expressed in the trichomes, with higher transcript levels found in glands on young leaves than on fully expanded leaves, while CcCLS transcript levels increased after mechanical wounding. Chemical analyses revealed that labdane-type diterpene production followed a similar pattern, with higher concentrations in trichomes of young leaves and increased accumulation upon wounding. PMID:20595348

  9. Functional characterization of wheat copalyl diphosphate synthases sheds light on the early evolution of labdane-related diterpenoid metabolism in the cereals.

    Science.gov (United States)

    Wu, Yisheng; Zhou, Ke; Toyomasu, Tomonobu; Sugawara, Chizu; Oku, Madoka; Abe, Shiho; Usui, Masami; Mitsuhashi, Wataru; Chono, Makiko; Chandler, Peter M; Peters, Reuben J

    2012-12-01

    Two of the most agriculturally important cereal crop plants are wheat (Triticum aestivum) and rice (Oryza sativa). Rice has been shown to produce a number of diterpenoid natural products as phytoalexins and/or allelochemicals--specifically, labdane-related diterpenoids, whose biosynthesis proceeds via formation of an eponymous labdadienyl/copalyl diphosphate (CPP) intermediate (e.g., the ent-CPP of gibberellin phytohormone biosynthesis). Similar to rice, wheat encodes a number of CPP synthases (CPS), and the three CPS characterized to date (TaCPS1-3) all have been suggested to produce ent-CPP. However, several of the downstream diterpene synthases will only react with CPP intermediate of normal or syn, but not ent, stereochemistry, as described in the accompanying report. Investigation of additional CPS did not resolve this issue, as the only other functional synthase (TaCPS4) also produced ent-CPP. Chiral product characterization of all the TaCPS then established that TaCPS2 uniquely produces normal, rather than ent-, CPP, thus, providing a suitable substrate source for the downstream diterpene synthases. Notably, TaCPS2 is most homologous to the similarly stereochemically differentiated syn-CPP synthase from rice (OsCPS4), while the non-inducible TaCPS3 and TaCPS4 cluster with the rice OsCPS1 required for gibberellin phytohormone biosynthesis, as well as with a barley (Hordeum vulgare) CPS (HvCPS1) that also is characterized here as similarly producing ent-CPP. These results suggest that diversification of labdane-related diterpenoid metabolism beyond the ancestral gibberellins occurred early in cereal evolution, and included the type of stereochemical variation demonstrated here.

  10. Effects of Overexpression of the Endogenous Farnesyl Diphosphate Synthase on the Artemisinin Content in Artemisia annua L.

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Artemisinin Is a novel effective antimalarial drug extracted from the medicinal plant Artemisia annua L. Owing to the tight market and low yield of artemislnin, there is great interest in enhancing the production of artemisinin.In the present study, farnesyl dlphosphate synthase (FPS) was overexpressed in high-yield A. annua to increase the artemislnin content. The FPS activity in transgenic A. annua was two- to threefold greater than that in non-transgenic A. annua. The highest artemisinin content in transgenic A. annua was approximately 0.9% (dry weight), which was 34.4% higher than that in non-transgenic A. annua. The results demonstrate the regulatory role of FPS in artemisinin biosynthesis.

  11. Circadian rhythm of anti-fungal prenylated chromene in leaves of Piper aduncum.

    Science.gov (United States)

    Morandim, Andreia de A; Bergamo, Débora Cristina B; Kato, Massuo Jorge; Cavalheiro, Alberto José; Bolzani, Vanderlan da S; Furlan, Maysa

    2005-01-01

    Leaves of Piper aduncum accumulate the anti-fungal chromenes methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate (2). The enzymatic formation of 2 from dimethylallyl diphosphate and 1 was investigated using cell-free extracts of the title plant. An HPLC assay for the prenylation reaction was developed and the enzyme activity measured in the protein extracts. The prenyltransferase that catalyses the transfer of the dimethylallyl group to C-2' of 1 was soluble and required dimethylallyl diphosphate as the prenyl donor. In the leaves, the biosynthesis of the prenylated chromene 2 was time-regulated and prenyltransferase activity depended upon circadian variation. Preliminary characterisation and purification experiments on the prenyltransferase from P. aduncum have been performed.

  12. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  13. The defective phosphoribosyl diphosphate synthase in a temperature-sensitive prs-2 mutant of Escherichia coli is compensated by increased enzyme synthesis

    DEFF Research Database (Denmark)

    Post, David A.; Switzer, Robert L.; Hove-Jensen, Bjarne

    1996-01-01

    at 25 degrees C. The mutant enzyme had nearly normal heat stability, as long as it was synthesized at 25 degrees C. In contrast, there was hardly any PRPP synthase activity or anti-PRPP synthase antibody cross-reactive material present in cells harbouring the glycine to serine alteration following...... synthase activity as a strain harbouring a plasmid-borne wild-type prs allele. In cells harbouring both mutations, the C -> T mutation appeared to compensate for the G -> A mutation by increasing the amount of a partially defective enzyme at the permissive temperature....

  14. Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate.

    Science.gov (United States)

    Schilmiller, Anthony L; Schauvinhold, Ines; Larson, Matthew; Xu, Richard; Charbonneau, Amanda L; Schmidt, Adam; Wilkerson, Curtis; Last, Robert L; Pichersky, Eran

    2009-06-30

    We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

  15. Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme.

    Science.gov (United States)

    Tittmann, Kai; Schröder, Kathrin; Golbik, Ralph; McCourt, Jennifer; Kaplun, Alexander; Duggleby, Ronald G; Barak, Ze'ev; Chipman, David M; Hübner, Gerhard

    2004-07-13

    Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

  16. Caged Protein Prenyltransferase Substrates: Tools for Understanding Protein Prenylation

    Energy Technology Data Exchange (ETDEWEB)

    DeGraw, Amanda J.; Hast, Michael A.; Xu, Juhua; Mullen, Daniel; Beese, Lorena S.; Barany, George; Distefano, Mark D. (Duke); (UMM)

    2010-11-15

    Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase (Protein Farnesyltransferase) inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and X-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate and their photolysis products are benign. These properties highlight the utility of those analogs towards a variety of in vitro and in vivo applications.

  17. GGPPS, a new EGR-1 target gene, reactivates ERK 1/2 signaling through increasing Ras prenylation.

    Science.gov (United States)

    Shen, Ning; Shao, Yue; Lai, Shan-Shan; Qiao, Long; Yang, Run-Lin; Xue, Bin; Pan, Fei-Yan; Chen, Hua-Qun; Li, Chao-Jun

    2011-12-01

    Cigarette smoke activates the extracellular signal-regulated kinase (ERK) 1/2 mitogen activated-protein kinase pathway, which, in turn, is responsible for early growth response gene-1 (EGR-1) activation. Here we provide evidence that EGR-1 activation can also reactivate ERK 1/2 mitogen activated-protein kinase through a positive feedback loop through its target gene (geranylgeranyl diphosphate synthase) GGPPS. For the first time, the GGPPS gene is identified as a target of EGR-1, as EGR-1 can directly bind to the predicted consensus-binding site in the GGPPS promoter and regulate its transcription. Long-term observations show that there are two ERK 1/2 phosphorylation peaks after cigarette smoke extract stimulation in human lung epithelial Beas-2B cells. The first peak (at 10 minutes) is responsible for EGR-1 accumulation, and the second (at 4 hours) is diminished after the disruption of EGR-1 transcriptional activity. EGR-1 overexpression enhances Ras prenylation and membrane association in a GGPPS-dependent manner, and it augments ERK 1/2 activation. Likewise, a great reduction of the second peak of ERK 1/2 phosphorylation is observed during long-term cigarette smoke extract stimulation in cells where GGPPS is disrupted. Thus, we have uncovered an intricate positive feedback loop in which ERK 1/2-activated EGR-1 promotes ERK 1/2 reactivation through promoting GGPPS transcription, which might affect cigarette smoke-related lung pathological processes.

  18. Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare.

    Science.gov (United States)

    Zerbe, Philipp; Chiang, Angela; Dullat, Harpreet; O'Neil-Johnson, Mark; Starks, Courtney; Hamberger, Björn; Bohlmann, Jörg

    2014-09-01

    Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS-e/f clade with unusual βα-domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by nuclear magnetic resonance (NMR) analysis, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids.

  19. Prenylated flavones from Artocarpus altilis.

    Science.gov (United States)

    Shamaun, Shireen Shaharina; Rahmani, Mawardi; Hashim, Najihah Mohd; Ismail, Hazar Bebe Mohd; Sukari, Mohd Aspollah; Lian, Gwendoline Ee Cheng; Go, Rusea

    2010-10-01

    Six prenylated flavones, including one new compound, were isolated and identified from the stem bark extracts of Artocarpus altilis. The new prenylated flavone hydroxyartocarpin (1) was characterized as 3-(gamma,gamma-dimethylallyl)-6-isopentenyl-5,8,2',4'-tetrahydroxy-7-methoxyflavone and the known compounds were artocarpin (2), morusin (3), cycloartobiloxanthone (4), cycloartocarpin A (5) and artoindonesianin V (6). The structures of the compounds were determined by spectroscopic methods (IR, MS, (1)H-NMR and (13)C-NMR) and comparison with published data for the known compounds.

  20. Characterisation of a Recombinant Patchoulol Synthase Variant for Biocatalytic Production of Terpenes.

    Science.gov (United States)

    Frister, Thore; Hartwig, Steffen; Alemdar, Semra; Schnatz, Katharina; Thöns, Laura; Scheper, Thomas; Beutel, Sascha

    2015-08-01

    The patchoulol synthase (PTS) is a multi-product sesquiterpene synthases which is the central enzyme for biosynthesis of patchouli essential oil in the patchouli plant. Sesquiterpene synthases catalyse the formation of various complex carbon backbones difficult to approach by organic synthesis. Here, we report the characterisation of a recombinant patchoulol synthase complementary DNA (cDNA) variant (PTS var. 1), exhibiting significant amino acid exchanges compared to the native PTS. The product spectrum using the natural substrate E,E-farnesyl diphosphate (FDP) as well as terpenoid products resulting from conversions employing alternative substrates was analysed by GC-MS. In respect to a potential use as a biocatalyst, important enzymatic parameters such as the optimal reaction conditions, kinetic behaviour and the product selectivity were studied as well. Adjusting the reaction conditions, an increased patchoulol ratio in the recombinant essential oil was achieved. Nevertheless, the ratio remained lower than in plant-derived patchouli oil. As alternative substrates, several prenyl diposphates were accepted and converted in numerous compounds by the PTS var. 1, revealing its great biocatalytic potential.

  1. Analysis of ent-copalyl diphosphate aynthase and ent-kaurene synthase (CPS & KS) gene family in cotton genome databases%棉花基因组数据库中CPS & KS基因的查找与分析

    Institute of Scientific and Technical Information of China (English)

    赵亮; 狄佳春; 陈旭升

    2016-01-01

    The study focused on the distribution of ent⁃copalyl diphosphate aynthase and ent⁃kaurene synthase (CPS&KS), the key enzymes for biosynthesis of GA, in cotton genome. The CPS&KS gene family has two conservative struc⁃ture domains, Terpene_synth and Terpene_synth_C, based on the analysis of their amino acid sequences from Arabidopsis thaliana, and the seed files in Pfam database are PF01397 and PF03936, respectively. By using Hmmsearch of HMMER, 72 sequences were harvested in the genome database from Gossypium raimondii. Five genes exhibiting homologies with 10 genes in subgenomes At and Dt of genome TM⁃1 of tetraploid cotton were identified. Based on the alignment of transcriptome database of an ultra⁃dwarf mutant with GA sensitivity before and after GA treatment, two KS genes were found up⁃regulated.%为了探究赤霉素合成关键酶CPS&KS类基因在棉花中的分布情况,分析了已发表的拟南芥CPS&KS基因编码的蛋白质氨基酸序列,发现该类基因存在2个保守结构域,Terpene_synth和Terpene_synth_C,其在Pfam数据库中的种子文件分别为PF01397和PF03936。利用软件HMMER中的Hmmsearch程序在雷蒙德氏棉蛋白质数据库中调取72条序列,通过与拟南芥中的CPS&KS基因序列进行比对,最终在雷蒙德氏棉基因组中筛选到5个CPS&KS候选基因,这5个基因与四倍体棉花TM⁃1基因组At和Dt亚组的10个基因具有同源关系。通过对赤霉素敏感的棉花超矮杆突变体赤霉素处理前后转录组数据库分析比对,最终确定2个KS候选基因在转录组中表现为上调作用。

  2. A Versatile Photoactivatable Probe Designed to Label the Diphosphate Binding Site of Farnesyl Diphosphate Utilizing Enzymes

    Science.gov (United States)

    Henry, Olivier; Lopez-Gallego, Fernando; Agger, Sean A.; Schmidt-Dannert, Claudia; Sen, Stephanie; Shintani, David; Cornish, Katrina; Distefano, Mark D.

    2009-01-01

    Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit S. cerevisiae protein farnesyltransferase (ScPFTase) with IC50 values of 5.8 (meta isomer) and 3.0 µM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a KI of 0.46 µM. Radiolabeled forms of both analogues selectively labelled the β-subunit of ScPFTase. The para isomer was also shown to label E. coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes. PMID:19447628

  3. Multifunctional Prenylated Peptides for Live Cell Analysis

    Science.gov (United States)

    Wollack, James W.; Zeliadt, Nicholette A.; Mullen, Daniel G.; Amundson, Gregg; Geier, Suzanne; Falkum, Stacy; Wattenberg, Elizabeth V.; Barany, George; Distefano, Mark D.

    2009-01-01

    Protein prenylation is a common post-translational modification present in eukaryotic cells. Many key proteins involved in signal transduction pathways are prenylated and inhibition of prenylation can be useful as a therapeutic intervention. While significant progress has been made in understanding protein prenylation in vitro, we have been interested in studying this process in living cells, including the question of where prenylated molecules localize. Here, we describe the synthesis and live cell analysis of a series of fluorescently labeled multifunctional peptides, based on the C-terminus of the naturally prenylated protein CDC42. A synthetic route was developed that features a key Acm to Scm protecting group conversion. This strategy was compatible with acid-sensitive isoprenoid moieties, and allowed incorporation of an appropriate fluorophore as well as a cell-penetrating sequence (penetratin). These peptides are able to enter cells through different mechanisms, depending on the presence or absence of the penetratin vehicle and the nature of the prenyl group attached. Interestingly, prenylated peptides lacking penetratin are able to enter cells freely through an energy-independent process, and localize in a perinuclear fashion. This effect extends to a prenylated peptide that includes a full “CAAX box” sequence (specifically, CVLL). Hence, these peptides open the door for studies of protein prenylation in living cells, including enzymatic processing and intracellular peptide trafficking. Moreover, the synthetic strategy developed here should be useful for the assembly of other types of peptides that contain acid sensitive functionalities. PMID:19425596

  4. Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes.

    Science.gov (United States)

    Kim, Yeon-Bok; Kim, Sang-Min; Kang, Min-Kyoung; Kuzuyama, Tomohisa; Lee, Jong Kyu; Park, Seung-Chan; Shin, Sang-Chul; Kim, Soo-Un

    2009-05-01

    Pinus densiflora Siebold et Zucc. is the major green canopy species in the mountainous area of Korea. To assess the response of resin acid biosynthetic genes to mechanical and chemical stimuli, we cloned cDNAs of genes encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (1-deoxy-d-xylulose 5-phosphate synthase (PdDXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PdDXR) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (PdHDR)) by the rapid amplification of cDNA ends (RACE) technique. In addition, we cloned the gene encoding abietadiene synthase (PdABS) as a marker for the site of pine resin biosynthesis. PdHDR and PdDXS occurred as two gene families. In the phylogenetic trees, PdDXSs, PdDXR and PdHDRs each formed a separate clade from their respective angiosperm homologs. PdDXS2, PdHDR2 and PdDXR were most actively transcribed in stem wood, whereas PdABS was specifically transcribed. The abundance of PdDXS2 transcripts in wood in the resting state was generally 50-fold higher than the abundance of PdDXS1 transcripts, and PdHDR2 transcripts were more abundant by an order of magnitude in wood than in other tissues, with the ratio of PdHDR2 to PdHDR1 transcripts in wood being about 1. Application of 1 mM methyl jasmonate (MeJA) selectively enhanced the transcript levels of PdDXS2 and PdHDR2 in wood. The ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 reached 900 and 20, respectively, on the second day after MeJA treatment, whereas the transcript level of PdABS increased twofold by 3 days after MeJA treatment. Wounding of the stem differentially enhanced the transcript ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 to 300 and 70, respectively. The increase in the transcript levels of the MEP pathway genes in response to wounding was accompanied by two orders of magnitude increase in PdABS transcripts. These observations indicated that resin acid biosynthesis activity, represented by PdABS transcription, was correlated

  5. Manganese dipyridoxyl diphosphate:

    DEFF Research Database (Denmark)

    H, Brurok; Ardenkjær-Larsen, Jan Henrik; G, Hansson

    1999-01-01

    Manganese dipyridoxyl diphosphate (MnDPDP) is a contrast agent for magnetic resonance imaging (MRI) of the liver. Aims of the study were to examine if MnDPDP possesses superoxide dismutase (SOD) mimetic activity in vitro, and if antioxidant protection can be demonstrated in an ex vivo rat heart...

  6. Prenyldiphosphate synthases and gibberellin biosynthesis

    NARCIS (Netherlands)

    van Schie, C.C.N.; Haring, M.A.; Schuurink, R.C.; Bach, T.J.; Rohmer, M.

    2013-01-01

    Gibberellins are derived from the diterpene precursor geranylgeranyl diphophosphate (GGPP). GGPP is converted to ent-kaurene, which contains the basic structure of gibberellins, in the plastids by the combined actions of copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Generally, ge

  7. Estrogenicity and metabolism of prenylated flavonoids and isoflavonoids

    NARCIS (Netherlands)

    Schans, van de M.G.M.

    2015-01-01

      Binding of (prenylated) flavonoids and isoflavonoids to the human estrogen receptors (hERs) might result in beneficial health effects in vivo. To understand structure-activity relationships of prenylated (iso)flavonoids towards the hERs, prenylated (iso)flavonoids were purified from extracts

  8. A new prenylated aurone from Artocarpus altilis.

    Science.gov (United States)

    Huong, Tran Thu; Cuong, Nguyen Xuan; Tram, Le Huyen; Quang, Tran Thuong; Duong, Le Van; Nam, Nguyen Hoai; Dat, Nguyen Tien; Huong, Phan Thi Thanh; Diep, Chau Ngoc; Kiem, Phan Van; Minh, Chau Van

    2012-01-01

    Phytochemical study of the methanol extract of Artocarpus altilis resulted in the isolation of a new prenylated aurone, artocarpaurone (1), together with eight known compounds including two prenylated chalcones (2 and 3), three prenylated flavanones (4-6), and three triterpenes (7-9). The structure of 1 was elucidated as 6-hydroxy-2-[8-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-ylmethylene]-3(2H)-benzofuranone by spectroscopic methods including 1D and 2D NMR spectra and FT-ICR-MS. Compound 1 showed moderate nitric oxide radical scavenging activity, whereas 2 and 3 had moderate 2,2-diphenyl-1-picrylhydrazyl radical scavenging effect, compared with the positive control (+)-catechin.

  9. Regiospecificities and Prenylation Mode Specificities of the Fungal Indole Diterpene Prenyltransferases AtmD and PaxD

    Science.gov (United States)

    Liu, Chengwei; Minami, Atsushi; Noike, Motoyoshi; Toshima, Hiroaki; Oikawa, Hideaki

    2013-01-01

    We recently reported the function of paxD, which is involved in the paxilline (compound 1) biosynthetic gene cluster in Penicillium paxilli. Recombinant PaxD catalyzed a stepwise regular-type diprenylation at the 21 and 22 positions of compound 1 with dimethylallyl diphosphate (DMAPP) as the prenyl donor. In this study, atmD, which is located in the aflatrem (compound 2) biosynthetic gene cluster in Aspergillus flavus and encodes an enzyme with 32% amino acid identity to PaxD, was characterized using recombinant enzyme. When compound 1 and DMAPP were used as substrates, two major products and a trace of minor product were formed. The structures of the two major products were determined to be reversely monoprenylated compound 1 at either the 20 or 21 position. Because compound 2 and β-aflatrem (compound 3), both of which are compound 1-related compounds produced by A. flavus, have the same prenyl moiety at the 20 and 21 position, respectively, AtmD should catalyze the prenylation in compound 2 and 3 biosynthesis. More importantly and surprisingly, AtmD accepted paspaline (compound 4), which is an intermediate of compound 1 biosynthesis that has a structure similar to that of compound 1, and catalyzed a regular monoprenylation of compound 4 at either the 21 or 22 position, though the reverse prenylation was observed with compound 1. This suggests that fungal indole diterpene prenyltransferases have the potential to alter their position and regular/reverse specificities for prenylation and could be applicable for the synthesis of industrially useful compounds. PMID:24038699

  10. Rare prenylated flavonoids from Tephrosia purpurea.

    Science.gov (United States)

    Hegazy, Mohamed-Elamir F; Abd el-Razek, Mohamed H; Nagashima, Fumihiro; Asakawa, Yoshinori; Paré, Paul W

    2009-01-01

    Chemical investigations of aerial parts of Tephrosia purpurea yielded the rare prenylated flavonoids, tephropurpulin A (1) and isoglabratephrin (2), in addition to a previously identified flavonoid, glabratephrin (3). Structures were established by 1D and 2D NMR spectroscopy, as well as by HR-MS analysis; for compounds 2 and 3, structures were confirmed by X-ray analysis.

  11. Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Andersen, Kasper R; Kilstrup, Mogens;

    2017-01-01

    is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP......Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds...... synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number...

  12. Bis(4-methoxybenzylammonium dihydrogen diphosphate

    Directory of Open Access Journals (Sweden)

    Adel Elboulali

    2013-02-01

    Full Text Available In the title compound, 2C8H12NO+·H2P2O72−, the linked PO4 groups of the diphosphate anion are almost eclipsed and the P—O—P angle is 134.45 (7°. In the crystal, infinite ribbons of H2P2O72− anions propagate in [100], being linked by strong O—H...O hydrogen bonds. The 4-methoxybenzylammonium cations bond to the diphosphate chains by N—H...O and C—H...O links, and are themselves linked by C—H...π interactions.

  13. Prenylated Rab acceptor protein is a receptor for prenylated small GTPases.

    Science.gov (United States)

    Figueroa, C; Taylor, J; Vojtek, A B

    2001-07-27

    Localization of Ras and Ras-like proteins to the correct subcellular compartment is essential for these proteins to mediate their biological effects. Many members of the Ras superfamily (Ha-Ras, N-Ras, TC21, and RhoA) are prenylated in the cytoplasm and then transit through the endomembrane system on their way to the plasma membrane. The proteins that aid in the trafficking of the small GTPases have not been well characterized. We report here that prenylated Rab acceptor protein (PRA1), which others previously identified as a prenylation-dependent receptor for Rab proteins, also interacts with Ha-Ras, RhoA, TC21, and Rap1a. The interaction of these small GTPases with PRA1 requires their post-translational modification by prenylation. The prenylation-dependent association of PRA1 with multiple GTPases is conserved in evolution; the yeast PRA1 protein associates with both Ha-Ras and RhoA. Earlier studies reported the presence of PRA1 in the Golgi, and we show here that PRA1 co-localizes with Ha-Ras and RhoA in the Golgi compartment. We suggest that PRA1 acts as an escort protein for small GTPases by binding to the hydrophobic isoprenoid moieties of the small GTPases and facilitates their trafficking through the endomembrane system.

  14. Mechanistic insights into Mg2+-independent prenylation by CloQ from classical molecular mechanics and hybrid quantum mechanics/molecular mechanics molecular dynamics simulations.

    Science.gov (United States)

    Bayse, Craig A; Merz, Kenneth M

    2014-08-05

    Understanding the mechanism of prenyltransferases is important to the design of engineered proteins capable of synthesizing derivatives of naturally occurring therapeutic agents. CloQ is a Mg(2+)-independent aromatic prenyltransferase (APTase) that transfers a dimethylallyl group to 4-hydroxyphenylpyruvate in the biosynthetic pathway for clorobiocin. APTases consist of a common ABBA fold that defines a β-barrel containing the reaction cavity. Positively charged basic residues line the inside of the β-barrel of CloQ to activate the pyrophosphate leaving group to replace the function of the Mg(2+) cofactor in other APTases. Classical molecular dynamics simulations of CloQ, its E281G and F68S mutants, and the related NovQ were used to explore the binding of the 4-hydroxyphenylpyruvate (4HPP) and dimethylallyl diphosphate substrates in the reactive cavity and the role of various conserved residues. Hybrid quantum mechanics/molecular mechanics potential of mean force (PMF) calculations show that the effect of the replacement of the Mg(2+) cofactor with basic residues yields a similar activation barrier for prenylation to Mg(2+)-dependent APTases like NphB. The topology of the binding pocket for 4HPP is important for selective prenylation at the ortho position of the ring. Methylation at this position alters the conformation of the substrate for O-prenylation at the phenol group. Further, a two-dimensional PMF scan shows that a "reverse" prenylation product may be a possible target for protein engineering.

  15. A single residue change leads to a hydroxylated product from the class II diterpene cyclization catalyzed by abietadiene synthase

    Science.gov (United States)

    Criswell, Jared; Potter, Kevin; Shephard, Freya; Beale, Michael H.; Peters, Reuben J.

    2012-01-01

    Class II diterpene cyclases catalyze bicyclization of geranylgeranyl diphosphate. While this reaction typically is terminated via methyl deprotonation to yield copalyl diphosphate, in rare cases hydroxylated bicycles are produced instead. Abietadiene synthase is a bifunctional diterpene cyclase that usually produces a copalyl diphosphate intermediate. Here it is shown that substitution of aspartate for a conserved histidine in the class II active site of abietadiene synthase leads to selective production of 8α-hydroxy-CPP instead, demonstrating striking plasticity. PMID:23167845

  16. Prenylated flavonoids from maclura tinctoria fruits

    Energy Technology Data Exchange (ETDEWEB)

    Oyama, Sayuri de Oliveira; Souza, Luiz Antonio de [Departamento de Biologia, Universidade Estadual de Maringa, PR (Brazil); Baldoqui, Debora Cristina; Sarragiotto, Maria Helena [Departamento de Quimica, Universidade Estadual de Maringa, PR (Brazil); Silva, Adriano Antonio, E-mail: aasilva@ufac.br [Departamento de Ciencias da Natureza, Universidade Federal do Acre, AC (Brazil)

    2013-09-01

    A phytochemical investigation of Maclura tinctoria fruits yielded five flavonoids, including one prenylated flavonol (licoflavonol) and four prenylated isoflavones (wighteone, derrone, alpinum isoflavone, and 6-(2,2-dimethyl-2H-chromen-6-yl)-2-(1-hydroxy-1-methylethyl) -2,3-dihydro-5H-furo[3,2-g]chromen-5-one). The structures of the isolates were established by analyzing their spectroscopic data. Compound 6-(2,2-dimethyl-2H-chromen-6-yl) -2-(1-hydroxy-1-methylethyl)-2,3-ihydro-5H-furo[3,2-g]chromen-5-one is new and the other isolates are described for the first time in this species. (author)

  17. Five new prenylated chalcones from Desmodium renifolium.

    Science.gov (United States)

    Li, Yan-Ping; Yang, Yu-Chun; Li, Yin-Ke; Jiang, Zhi-Yong; Huang, Xiang-Zhong; Wang, Wei-Guang; Gao, Xue-Mei; Hu, Qiu-Fen

    2014-06-01

    Five unusual new prenylated chalcones, renifolins D-H (1-5), were isolated from whole Desmodium renifolium plants. All of their structures were determined by spectroscopic methods including 1D and 2D NMR. All of the isolates were evaluated for cytotoxicity using five tumor cell lines. Compounds 2 and 3 exhibited cytotoxicity against A549 cells, with IC50 values of 2.8 and 2.2 μM, respectively.

  18. Anti-inflammatory Natural Prenylated Phenolic Compounds - Potential Lead Substances.

    Science.gov (United States)

    Brezáni, Viliam; Šmejkal, Karel; Hošek, Jan; Tomášová, Veronika

    2017-08-10

    Natural phenolics are secondary plant metabolites, which can be divided into several categories with the common structural feature of phenolic hydroxyl. The biological activity of phenolics is often modified and enhanced by prenylation by prenyl and geranyl; higher terpenoid chains are rare. The type of prenyl connection and modification affects their biological activity. This review summarizes information about prenylated phenols and some of their potential sources, and provides an overview of their anti-inflammatory potential in vitro and in vivo. The literature search was performed using Scifinder and keywords prenyl, phenol, and inflammation. For individual compounds, an additional search was performed to find information about further activities and mechanisms of effects. We summarized the effects of prenylated phenolics in vitro in cellular or biochemical systems on the production and release of inflammation-related cytokines; their effects on inhibition of cyclooxygenases and lipoxygenases; the effects on production of nitric oxide, antiradical and antioxidant activity; and the effect on the inhibition of the release of enzymes and mediators from neutrophils, mast cells and macrophages. The information about the antiphlogistic potential of prenylated phenolics is further supported by a review of their action in animal models. Almost 400 prenylated phenols were reviewed to overview their anti-inflammatory effect. The bioactivity of several prenylated phenols was confirmed also using in vivo assays. A pool of natural prenylated phenols represents a source of inspiration for synthesis, and prenylated phenols as components of various medicinal plants used to combat inflammation could be their active principles. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Prenyl sulfates as alkylating reagents for mercapto amino acids.

    Science.gov (United States)

    Maltsev, Sergey; Sizova, Olga; Utkina, Natalia; Shibaev, Vladimir; Chojnacki, Tadeusz; Jankowski, Wieslaw; Swiezewska, Ewa

    2008-01-01

    A new methodology for prenylation of thiol compounds has been developed. The approach is based on the use of prenyl sulfates as new reagents for S-prenylation of benzenethiol and cysteamine in aqueous systems. The C(10)-prenols geraniol and nerol that differ in the configuration (E or Z, correspondingly) of the alpha-isoprene unit were efficiently O-sulfated in the presence of a pyridine-SO(3') complex. The obtained geranyl and neryl sulfates were tested as alkylating agents. These compounds were chosen to reveal the influence of the alpha-isoprene unit configuration on their alkylation (prenylation) ability. S-Geranyl cysteine was prepared to demonstrate the applicability of this method for prenylation of peptides containing mercapto amino acids.

  20. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    Science.gov (United States)

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.

  1. Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine Uridine Diphosphate Donors: Chain Terminators in Glycosaminoglycan Synthesis.

    Science.gov (United States)

    Schultz, Victor L; Zhang, Xing; Linkens, Kathryn; Rimel, Jenna; Green, Dixy E; DeAngelis, Paul L; Linhardt, Robert J

    2017-02-17

    Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). The resulting unnatural UDP-sugar donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various synthases. UDP-4FGlcNAc was transferred onto an acceptor by Pastuerella multocida heparosan synthase 1 and subsequently served as a chain terminator.

  2. Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance.

    Science.gov (United States)

    Hove-Jensen, Bjarne; Andersen, Kasper R; Kilstrup, Mogens; Martinussen, Jan; Switzer, Robert L; Willemoës, Martin

    2017-03-01

    Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

  3. Activation of thiamin diphosphate in enzymes.

    Science.gov (United States)

    Hübner, G; Tittmann, K; Killenberg-Jabs, M; Schäffner, J; Spinka, M; Neef, H; Kern, D; Kern, G; Schneider, G; Wikner, C; Ghisla, S

    1998-06-29

    Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

  4. Prenyl transfer to aromatic substrates: genetics and enzymology.

    Science.gov (United States)

    Heide, Lutz

    2009-04-01

    Aromatic prenyltransferases catalyze the transfer of prenyl moieties to aromatic acceptor molecules and give rise to an astounding diversity of primary and secondary metabolites in plants, fungi and bacteria. Significant progress has been made in the biochemistry and genetics of this heterogeneous group of enzymes in the past years. After 30 years of extensive research on plant prenylflavonoid biosynthesis, finally the first aromatic prenyltransferases involved in the formation of these compounds have been cloned. In bacteria, investigations of the newly discovered family of ABBA prenyltransferases revealed a novel type of protein fold, the PT barrel. In fungi, a group of closely related indole prenyltransferase was found to carry out aromatic prenylations with different substrate specificity and regiospecificity, and to catalyze both regular and reverse prenylations.

  5. Rapid membrane permeabilization of Listeria monocytogenes and Escherichia coli induced by antibacterial prenylated phenolic compounds from legumes

    NARCIS (Netherlands)

    Araya-Cloutier, Carla; Vincken, Jean Paul; Ederen, van Roan; Besten, den Heidy M.W.; Gruppen, Harry

    2018-01-01

    Prenylated phenolics from the Fabaceae are promising lead compounds for new antibacterials. Pools enriched in prenylated phenolics were made from lupine, peanut and soybean seedlings. One pool was rich in chain prenylated isoflavones (cIsf), one in chain prenylated stilbenoids (cSti), one in chain

  6. Prenylated Dihydrochalcones from Artocarpus altilis as Antiausterity Agents.

    Science.gov (United States)

    Nguyen, Mai Thanh Thi; Nguyen, Nhan Trung; Awale, Suresh

    2015-01-01

    Human pancreatic cancer cell lines have remarkable tolerance to nutrition starvation, which enables them to survive under a tumor microenvironment. A novel antiausterity strategy in anticancer drug discovery led to the discovery of agents that preferentially inhibit the survival of cancer cells under low nutrient conditions. Artocarpus altilis (Family: Moraceae) is commonly referred to as breadfruit, traditionally for the treatment of many diseases. Many prenylated flavonoid and prenylated chalocones together with their cancer cell cytotoxicity were reported from this plant. This chapter briefly summarizes the constituents, biosynthesis, cytotoxicity, and antiausterity activity on PANC-1 human pancreatic cancer cell line of A. altilis. © 2015 Elsevier Inc. All rights reserved.

  7. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named

  8. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPIN

  9. Ammonium ytterbium(III diphosphate(V

    Directory of Open Access Journals (Sweden)

    Michal Dušek

    2008-12-01

    Full Text Available The title compound, NH4YbP2O7, crystallizes in the KAlP2O7 structure type and consists of distorted YbO6 octahedra and bent P2O74− diphosphate units forming together a three-dimensional network. There are channels in the structure running along the c axis, where the NH4+ cations are located. They are connected via N—H...O hydrogen bonds to the terminal O atoms of the diphosphate anions.

  10. Induction of prenylated isoflavonoids and stilbenoids in legumes

    NARCIS (Netherlands)

    Aisyah, S.

    2015-01-01

    The germination of legume seeds in the presence or absence of stress factors was studied with respect to compositional changes in prenylated isoflavonoids and stilbenoids. Different strategies were applied using (i) different types of legume seed, (ii) different stress factors i.e. biotic, abiotic

  11. Induction of prenylated isoflavonoids and stilbenoids in legumes

    NARCIS (Netherlands)

    Aisyah, S.

    2015-01-01

    The germination of legume seeds in the presence or absence of stress factors was studied with respect to compositional changes in prenylated isoflavonoids and stilbenoids. Different strategies were applied using (i) different types of legume seed, (ii) different stress factors i.e. biotic, abiotic a

  12. A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

    Directory of Open Access Journals (Sweden)

    Vogel Constantin

    2012-11-01

    Full Text Available Abstract Background Standard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD is still missing. Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult. Results We developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8 was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the ScPDC mutant E477Q, the cofactors ThDP and Mg2+ as well as the substrate analogue (2S-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED and was used to systematically analyze functionally and structurally relevant

  13. Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

    2016-05-01

    Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes.

  14. A photoactive isoprenoid diphosphate analogue containing a stable phosphonate linkage: synthesis and biochemical studies with prenyltransferases

    Science.gov (United States)

    DeGraw, Amanda J.; Zhao, Zongbao; Strickland, Corey L.; Taban, A. Huma; Hsieh, John; Michael, Jefferies; Xie, Wenshuang; Shintani, David; McMahan, Colleen; Cornish, Katrina; Distefano, Mark D.

    2008-01-01

    A number of biochemical processes rely on isoprenoids, including the post-translational modification of signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltransferases. While these initial analogues proved to be excellent structural analogues with good cross linking capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl diphosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics, photolabeling experiments, as well as x-ray crystallographic analysis with a protein prenyltransferase are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new analogue was explored by using it to photoaffinity label crude protein extracts obtained from Hevea brasiliensis latex. Those experiments suggest that a small protein, Rubber Elongation Factor, interacts directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a substrate. PMID:17477573

  15. A New Prenylated Xanthone from Root Barks of Cudrania cochinchinensis

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-ping; WEI Wan-xing; ZHOU Min; GAN Chun-fang; LIU Sheng

    2013-01-01

    Objective To study the chemical constituents from the root barks of Cudrania cochinchinensis.Methods The chemical constituents were isolated and purified by silica gel column chromatography.The structures of the compounds were identified on the basis of spectral data (MS,1H-NMR,13C-NMR,and 2D NMR) and by the comparison of spectroscopic data with the reported values in the literatures.Results A new xanthone,1,6,7-trihydroxy-4-(1,1-dimethylallyl)-3-methoxyxanthone (1) and a known prenylated xanthone 1,5,6-trihydroxy-4-(1,l-dimethylallyl)-3-methoxyxanthone (isocudraniaxanthone B,2) were isolated from the root barks of C.cochinchinensis.Conclusion Compound 1 is a new prenylated xanthone.Isomers 1 and 2 are obtained from this plantfor the first time.

  16. Synthesis and DPPH Radical Scavenging Activity of Prenylated Phenol Derivatives

    Directory of Open Access Journals (Sweden)

    Héctor Carrasco

    2012-01-01

    Full Text Available The synthesis of twenty six prenylated phenols derivatives is reported. These compounds were obtained under mild conditions via Electrophilic Aromatic Substitution (EAS coupling reactions between phenol derivatives containing electron-donor subtituents and 3-methyl-2-buten-1-ol using BF3×OEt2. Dialkylations were also produced with this method. The formation of a chroman ring by intramolecular cyclization between a sp2 carbon from the prenyl group with the hydroxyl substituent in the ortho position occurred with some phenols. All the synthesized compounds were evaluated as antioxidants according to a DPPH radical scavenging activity assay. IC50 values of five synthesized compounds indicated they were as good antioxidants as Trolox™.

  17. Unusual prenylated phenols with antioxidant activities from Ganoderma cochlear.

    Science.gov (United States)

    Peng, XingRong; Liu, JieQing; Wang, CuiFang; Han, ZhongHui; Shu, Yi; Li, XuYang; Zhou, Lin; Qiu, MingHua

    2015-03-15

    Seven new prenylated phenols, five novel phenols (1-5) with polycyclic skeleton and two new phenols (6 and 7) with a carbon chain, along with one known compound (8) were isolated from the fruiting bodies of Ganoderma cochlear. The structures of new compounds were elucidated by the spectroscopic technologies, X-ray crystallography analysis and chiral HPLC chromatography. All compounds showed antioxidant effect in radical scavenging assays and a plausible biosynthetic pathway for 1-8 was proposed.

  18. Prenylated xanthone glucosides from Ural's lichen Umbilicaria proboscidea.

    Science.gov (United States)

    Rezanka, Tomás; Jáchymová, Jitka; Dembitsky, Valery M

    2003-02-01

    Two new compounds isolated from an extract of a Central Asian lichen [Umbilicaria proboscidea (L.) Schrader=Syn.: Gyrophora proboscidea (L.) Ach.] are glucosides with mono- and di-prenylated xanthones as the aglycones and a saccharide moiety from two glucoses linked at C-7. The structures were elucidated on the basis of extensive spectroscopic analysis (1D and 2D NMR, MS, IR and UV) and by hydrolysis.

  19. Motesanib diphosphate in progressive differentiated thyroid cancer

    DEFF Research Database (Denmark)

    Sherman, Steven I; Wirth, Lori J; Droz, Jean-Pierre

    2008-01-01

    BACKGROUND: The expression of vascular endothelial growth factor (VEGF) is characteristic of differentiated thyroid cancer and is associated with aggressive tumor behavior and a poor clinical outcome. Motesanib diphosphate (AMG 706) is a novel oral inhibitor of VEGF receptors, platelet......-derived growth-factor receptor, and KIT. METHODS: In an open-label, single-group, phase 2 study, we treated 93 patients who had progressive, locally advanced or metastatic, radioiodine-resistant differentiated thyroid cancer with 125 mg of motesanib diphosphate, administered orally once daily. The primary end...... point was an objective response as assessed by an independent radiographic review. Additional end points included the duration of the response, progression-free survival, safety, and changes in serum thyroglobulin concentration. RESULTS: Of the 93 patients, 57 (61%) had papillary thyroid carcinoma...

  20. How thiamine diphosphate is activated in enzymes.

    Science.gov (United States)

    Kern, D; Kern, G; Neef, H; Tittmann, K; Killenberg-Jabs, M; Wikner, C; Schneider, G; Hübner, G

    1997-01-03

    The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

  1. (3R)-Linalool synthase from Artemisia annua L.: cDNA isolation, characterization, and wound induction.

    Science.gov (United States)

    Jia, J W; Crock, J; Lu, S; Croteau, R; Chen, X Y

    1999-12-01

    Artemisia annua is an annual herb used in traditional Chinese medicine. A cDNA library was constructed from leaves of A. annua seedlings and target sequences were amplified by PCR using degenerate primers derived from a consensus sequence of angiosperm terpene synthases. Two clones, QH1 and QH5, with high sequence similarity to plant monoterpene synthases were ultimately obtained and expressed in Escherichia coli. These cDNAs encode peptides of 567 aa (65.7 kDa) and 583 aa (67.4 kDa), respectively, and display 88% identity with each other and 42% identity with Mentha spicata limonene synthase. The two recombinant enzymes yielded no detectable activity with isopentenyl diphosphate, dimethylallyl diphosphate, chrysanthemyl diphosphate, farnesyl diphosphate, (+)-copalyl diphosphate, or geranylgeranyl diphosphate, but were active with geranyl diphosphate in yielding (3R)-linalool as the sole product in the presence of divalent metal cation cofactors. QH1-linalool synthase displays a K(m) value of 64 microM for geranyl diphosphate, which is considerably higher than other known monoterpene synthases, and a K(m) value of 4.6 mM for Mg(+2). Transcripts of QH1 and QH5 could be detected by RT-PCR in the leaves and inflorescence of A. annua, but not in the stem stele or roots; transcripts of QH5 could also be detected in stem epidermis. Linalool could not be detected by GC-MS in the essential oil of A. annua, nor in acid or base hydrolysates of aqueous extracts of leaves. RT-PCR demonstrated a wound-inducible increase in QH1 and QH5 transcript abundance in both leaves and stems over a 3-day time course. Copyright 1999 Academic Press.

  2. Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris.

    Science.gov (United States)

    Rudolph, Kristin; Parthier, Christoph; Egerer-Sieber, Claudia; Geiger, Daniel; Muller, Yves A; Kreis, Wolfgang; Müller-Uri, Frieder

    2016-01-01

    The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata.

  3. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  4. Identification of prenylated pterocarpans and other isoflavonoids in Rhizopus spp. elicited soya bean seedlings by electrospray ionisation mass spectrometry.

    Science.gov (United States)

    Simons, Rudy; Vincken, Jean-Paul; Bohin, Maxime C; Kuijpers, Tomas F M; Verbruggen, Marian A; Gruppen, Harry

    2011-01-15

    Phytoalexins from soya are mainly characterised as prenylated pterocarpans, the glyceollins. Extracts of non-soaked and soaked soya beans, as well as that of soya seedlings, grown in the presence of Rhizopus microsporus var. oryzae, were screened for the presence of prenylated flavonoids with a liquid chromatography/mass spectrometry (LC/MS)-based screening method. The glyceollins I-III and glyceollidins I-II, belonging to the isoflavonoid subclass of the pterocarpans, were tentatively assigned. The formation of these prenylated pterocarpans was accompanied by that of other prenylated isoflavonoids of the subclasses of the isoflavones and the coumestans. It was estimated that approx. 40% of the total isoflavonoid content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans, whereas 7% comprised prenylated isoflavones and prenylated coumestans. The site of prenylation (A-ring or B-ring) of the prenylated isoflavones was tentatively annotated using positive-ion mode MS by comparing the (1,3) A(+) retro-Diels-Alder (RDA) fragments of prenylated and non-prenylated isoflavones. Furthermore, the fragmentation pathways of the five pterocarpans in negative-ion (NI) mode were proposed, which involved the cleavage of the C-ring and/or D-ring. The absence of the ring-closed prenyl (pyran or furan) gave exclusively -H(2) O(x,y) RDA fragments, whereas its presence gave predominantly the common RDA fragments. Copyright © 2010 John Wiley & Sons, Ltd.

  5. Prenylated flavonoids from Desmodium caudatum and evaluation of their anti-MRSA activity.

    Science.gov (United States)

    Sasaki, Hisako; Kashiwada, Yoshiki; Shibata, Hirofumi; Takaishi, Yoshihisa

    2012-10-01

    Seven prenylated flavonoids and a prenylated chromanochroman derivative, together with eight known flavonoids, were isolated from roots of Desmodium caudatum. The 15 structures were elucidated by extensive spectroscopic analyses. The antibacterial activity of many of other compounds was evaluated against methicillin-resistant Staphylococcus aureus (MRSA: COL and 5) by a disc diffusion method, and the minimum inhibitory concentrations (MICs) to MRSA were determined.

  6. Amyrisins A - C, O-Prenylated Flavonoids from Amyris madrensis⊥

    Science.gov (United States)

    Peng, Jiangnan; Hartley, Rachel M.; Fest, Gary A.; Mooberry, Susan L.

    2012-01-01

    Three new O-prenylated flavonoids, amyrisins A – C (1 –2), were isolated from the leaves and twigs of Amyris madrensis, along with the known compound, polygamain (4). The structures of 1 – 3 were elucidated based on the analysis of spectroscopic data. Amyrisins B (2) and C (3) showed moderate cytotoxicity against PC-3 and DU 145 prostate cancer cells with IC50 values of 17.5 and 23 μM, respectively while amyrisin A (1) did not show any cytotoxicity at the highest concentration tested, 50 μM. Polygamain (4) exhibited potent antiproliferative and microtubule depolymerizing activities. PMID:22260294

  7. Taichunamides: Prenylated Indole Alkaloids from Aspergillus taichungensis (IBT 19404)

    DEFF Research Database (Denmark)

    Kagiyama, Ippei; Kato, Hikaru; Nehira, Tatsuo

    2016-01-01

    Seven new prenylated indole alkaloids, taichunamides A–G, were isolated from the fungus Aspergillus taichungensis (IBT 19404). Taichunamides A and B contained an azetidine and 4‐pyridone units, respectively, and are likely biosynthesized from notoamide S via (+)‐6‐epi‐stephacidin A. Taichunamides C...... and D contain endoperoxide and methylsulfonyl units, respectively. This fungus produced indole alkaloids containing an anti‐bicyclo[2.2.2]diazaoctane core, whereas A. protuberus and A. amoenus produced congeners with a syn‐bicyclo[2.2.2]diazaoctane core. Plausible biosynthetic pathways to access...

  8. A New Prenylated Indole Diketopiperazine Alkaloid from Eurotium cristatum

    Directory of Open Access Journals (Sweden)

    Xianwei Zou

    2014-11-01

    Full Text Available A new prenylated indole diketopiperazine alkaloid, cristatumin F (1, and four known metabolites, echinulin (2, dehydroechinulin (3, neoechinulin A (4 and variecolorin O (5, were isolated from the crude extract of the fungus Eurotium cristatum. The structure of 1 was elucidated primarily by NMR and MS methods. The absolute configuration of 1 was assigned using Marfey’s method applied to its acid hydrolyzate. Cristatumin F (1 showed modest radical scavenging activity against DPPH radicals, and exhibited marginal attenuation of 3T3L1 pre-adipocytes.

  9. A new prenylated indole diketopiperazine alkaloid from Eurotium cristatum.

    Science.gov (United States)

    Zou, Xianwei; Li, Ying; Zhang, Xiaona; Li, Qian; Liu, Xuan; Huang, Yun; Tang, Tao; Zheng, Saijing; Wang, Weimiao; Tang, Jintian

    2014-11-03

    A new prenylated indole diketopiperazine alkaloid, cristatumin F (1), and four known metabolites, echinulin (2), dehydroechinulin (3), neoechinulin A (4) and variecolorin O (5), were isolated from the crude extract of the fungus Eurotium cristatum. The structure of 1 was elucidated primarily by NMR and MS methods. The absolute configuration of 1 was assigned using Marfey's method applied to its acid hydrolyzate. Cristatumin F (1) showed modest radical scavenging activity against DPPH radicals, and exhibited marginal attenuation of 3T3L1 pre-adipocytes.

  10. First total synthesis of two nematicidal prenylated flavanones

    Institute of Scientific and Technical Information of China (English)

    Jin Hui Yang; Shi Zhi Jiang; Yan Min Zhao; Yun Feng Li; Cong Bin Ji; Wan Yi Liu

    2009-01-01

    The total synthesis of(±)-8-(3-methylbut-2-enyl)-2-phenyl-2,3-dihydrochromen-4-one and(±)-2-(4-hydroxyphenyl)-8-(3-methylbut-2-enyl)-2,3-dihydrochromen-4-one was first achieved through C-prenylation,protection of phenolic hydroxyl group,aldol condensation,cyclization and deprotection starting from cheap benzaldehyde,4-hydroxybenzaldehyde and 2-hydroxyacetophenone,with total yield of 20 and 16.3%.All structures of new compounds were confirmed by IR,1H NMR and MS.

  11. Pseudouridine synthases.

    Science.gov (United States)

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  12. Studies on thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Leeper, F J; Hawksley, D; Mann, S; Perez Melero, C; Wood, M D H

    2005-08-01

    The 3-deaza analogue of TPP (thiamine diphosphate), a close mimic of the ylid intermediate, has been synthesized and is an extremely potent inhibitor of a variety of TPP-dependent enzymes, binding much more tightly than TPP itself. Results using deazaTPP complexed with the E1 subunit of PDH (pyruvate dehydrogenase) have led to a novel proposal about the mechanism of this enzyme. The 2-substituted forms of deazaTPP, which mimic other intermediates in the catalytic mechanism, can also be synthesized and 2-(1-hydroxyethyl)deazaTPP is also an extremely potent inhibitor of PDC (pyruvate decarboxylase). Attachment of such 2-substituents is expected to be a way to introduce selectivity in the inhibition of various TPP-dependent enzymes.

  13. Pentalenene Synthase: Analysis of Active Site Residues by Site-Directed Mutagenesis

    NARCIS (Netherlands)

    Seemann, M.; Zhai, G.; Kraker, de J.W.; Paschall, C.M.; Christianson, D.W.; Cane, D.E.

    2002-01-01

    Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions of (+)-germacrene A (7) with relatively minor changes in kcat and kcat/Km. By contrast, single H309 mu

  14. The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts.

    Science.gov (United States)

    Kowalska, Ewa; Kozik, Andrzej

    2008-01-01

    Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)-4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge.

  15. Biochemical characterization and homology modeling of methylbutenol synthase and implications for understanding hemiterpene synthase evolution in plants.

    Science.gov (United States)

    Gray, Dennis W; Breneman, Steven R; Topper, Lauren A; Sharkey, Thomas D

    2011-06-10

    2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ~90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K(+), whereas isoprene production is inhibited by K(+) such that, at physiologically relevant [K(+)], little or no isoprene emission should be detected from MBO-emitting trees. The K(m) of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site.

  16. A new bioactive prenylated dihydroflavanoid from Dolichos tenuicaulis (Baker) Craib

    Institute of Scientific and Technical Information of China (English)

    Jin Yong Peng; Hai Yan Liu; Yan Qi; Xu Han; You Wei Xu; Li Na Xu; Qi Wei Xu

    2007-01-01

    A new prenylated dihydroflavonoid was obtained from the root of Dolichos tenuicaulis (Baker) Craib. The structure was elucidated as (2S)-5,2',6'-trihydroxy-3''', 8-di (γ, γ-dimethyl-allyl)-2''', 2'''-dimethylpyrano-[5''',6''':6,7]-2''''',4'''''-cyclohexadiene-1'''''-one-[2''''',3''''': 3',4']-flavanone, named dolichnin A, by spectroscopic methods including UV, IR, HR-EI-MS, 1D NMR and 2D NMR techniques, and subsequently, the anticancer activity of this compound to inhibit human cancer cells' growth including A549, BEL-7402, Hep-3B, SMMC7721, HT-29, MCF-7, SGC-7902, K562, A498 and PC3 cell lines by MTT method was evaluated in vitro.

  17. A New Prenylated Xanthone from Latex of Garcinia cowa Roxb.

    Directory of Open Access Journals (Sweden)

    Zhi Na

    2013-05-01

    Full Text Available A new prenylated xanthone, 1,6-dihydroxy-3,7-dimethoxy-2-(3,7-dimethyloct-2,6-dienyl xanthone (3-O-methylcowaxanthone (1, together with four known xanthones, cowaxanthone (2, 7-O-methylgarcinone (3, α-mangostin (4 and γ-mangostin (5 were isolated from the latex of Garcinia cowa. The structure of compound 1 was elucidated on the basis of spectroscopic data interpretation, including 1D and 2D NMR and HREIMS. The cytotoxic activitiy of 1 against five human cancer cell lines, HL-60, SMMC-7721, A-549, MCF-7 and SW480, was evaluated, but it was inactive (IC 50>40μM.

  18. PRENYLATED BENZOIC ACID DERIVATIVES FROM PIPER SPECIES AS SOURCE OF ANTI-INFECTIVE AGENTS

    OpenAIRE

    2012-01-01

    A number of prenylated benzoic acids derivatives with interesting biological activities have been previously isolated and characterized from different species of piperaceae family. Several Piper species contained structurally similar compounds with diverse biological activities such as anti-bacterial, anti-fungal, insecticidal as well as anti-parasitic all of which produces compounds that can be classified as prenylated benzoic acid derivatives. Piper sp has proven to serve as a source of pot...

  19. Strategies for the Preparation of Differentially Protected ortho-Prenylated Phenols.

    Science.gov (United States)

    Hoarau, Christophe; Pettus, Thomas R R

    2003-01-01

    A new process for ortho-prenylation of phenols is presented within the context of known methods. All of the processes are briefly assessed with regards to the substitution patterns and accompanying functional groups tolerated by each strategy. The conclusion reached is that a new procedure using ortho-quinone methides, for which an experimental protocol is provided, offers the greatest generality and flexibility in the preparation of ortho-prenylated phenol derivatives.

  20. Modification of Prenylated Stilbenoids in Peanut (Arachis hypogaea) Seedlings by the Same Fungi That Elicited Them: The Fungus Strikes Back.

    Science.gov (United States)

    Aisyah, Siti; Gruppen, Harry; Slager, Mathijs; Helmink, Bianca; Vincken, Jean-Paul

    2015-10-28

    Aspergillus oryzae and Rhizopus oryzae were compared for inducing the production of prenylated stilbenoids in peanut seedlings. The fungus was applied at two different time points: directly after soaking (day 1) or after 2 days of germination (day 3). Aspergillus- and Rhizopus-elicited peanut seedlings accumulated an array of prenylated stilbenoids, with overlap in compounds induced, but also with compounds specific to the fungal treatment. The differences were confirmed to be due to modification of prenylated stilbenoids by the fungus itself. Each fungus appeared to deploy different strategies for modification. The content of prenylated stilbenoids modified by fungi accounted for around 8% to 49% (w/w) of total stilbenoids. The contents of modified prenylated stilbenoids were higher when the fungus was applied on day 1 instead of day 3. Altogether, type of fungus and time point of inoculation appeared to be crucial parameters for optimizing accumulation of prenylated stilbenoids in peanut seedlings.

  1. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  2. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase.

    Science.gov (United States)

    Bohlmann, J; Steele, C L; Croteau, R

    1997-08-29

    Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA

  3. Radioimmunoassay for guanosin-5'-diphosphate-3'-diphosphate and adenosine-5'-triphosphate-3'-diphosphate

    Energy Technology Data Exchange (ETDEWEB)

    Hamagishi, Y.; Oki, T.; Tone, H.; Inui, T. (Sanraku-Ocean Co. Ltd., Fujisawa, Kanagawa (Japan). Central Research Lab.)

    1980-12-01

    A radioimmunoassay for guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and adenosine-5'-triphosphate-3'-diphosphate (pppApp) has been developed. The assay method is based on competition of an unlabeled highly phosphorylated nucleotide with /sup 3/H-labeled highly phosphorylated nucleotide for binding sites on a specific antibody. Antibodies to ppGpp and pppApp were obtained by immunizing rabbits with the antigen prepared by conjugating ppGpp with human serum albumin using 1-ethyl-3-(3-dimethylaminoprophyl)carbodiimide, and with the antigen prepared by conjugating 8-(6-aminohexyl)amino-adenosine-5'-triphosphate-3'-diphosphate with human serum albumin using glutaraldehyde, respectively. Antibody-bound /sup 3/H-labeled highly phosphorylated nucleotides were separated from the free /sup 3/H-labeled highly phosphorylated nucleotides by selective adsorption on dextran-coated charcoal. Displacement plots were linear over a concentration range of 5 - 1,000 pmol/assay tube in a log-probit percentage plot. Application of this method to biological systems offers improved accuracy and convenience compared with the previous /sup 32/PO/sub 4/-labeling technique.

  4. Antiherbivore prenylated benzoic acid derivatives from Piper kelleyi.

    Science.gov (United States)

    Jeffrey, Christopher S; Leonard, Michael D; Glassmire, Andrea E; Dodson, Craig D; Richards, Lora A; Kato, Massuo J; Dyer, Lee A

    2014-01-24

    The known prenylated benzoic acid derivative 3-geranyl-4-hydroxy-5-(3″,3″-dimethylallyl)benzoic acid (1) and two new chromane natural products were isolated from the methanolic extract of the leaves of Piper kelleyi Tepe (Piperaceae), a midcanopy tropical shrub that grows in lower montane rain forests in Ecuador and Peru. Structure determination using 1D and 2D NMR analysis led to the structure of the chromene 2 and to the reassignment of the structure of cumanensic acid as 4, an isomeric chromene previously isolated from Piper gaudichaudianum. The structure and relative configuration of new chromane 3 was determined using 1D and 2D NMR spectroscopic analysis and was found to be racemic by ECD spectropolarimetry. The biological activity of 1-3 was evaluated against a lab colony of the generalist caterpillar Spodoptera exigua (Noctuidae), and low concentrations of 2 and 3 were found to significantly reduce fitness. Further consideration of the biosynthetic relationship of the three compounds led to the proposal that 1 is converted to 2 via an oxidative process, whereas 3 is produced through hetero-[4+2] dimerization of a quinone methide derived from the chromene 2.

  5. Biosynthesis of the diterpenoid lycosantalonol via nerylneryl diphosphate in Solanum lycopersicum.

    Science.gov (United States)

    Matsuba, Yuki; Zi, Jiachen; Jones, A Daniel; Peters, Reuben J; Pichersky, Eran

    2015-01-01

    We recently reported that three genes involved in the biosynthesis of monoterpenes in trichomes, a cis-prenyltransferase named neryl diphosphate synthase 1 (NDPS1) and two terpene synthases (TPS19 and TPS20), are present in close proximity to each other at the tip of chromosome 8 in the genome of the cultivated tomato (Solanum lycopersicum). This terpene gene "cluster" also contains a second cis-prenyltransferase gene (CPT2), three other TPS genes, including TPS21, and the cytochrome P450-oxidoreductase gene CYP71BN1. CPT2 encodes a neryneryl diphosphate synthase. Co-expression in E. coli of CPT2 and TPS21 led to the formation of the diterpene lycosantalene, and co-expression in E. coli of CPT2, TPS21 and CYP71BN1 led to the formation of lycosantalonol, an oxidation product of lycosantalene. Here we show that maximal expression of all three genes occurs in the petiolule part of the leaf, but little expression of these genes occurs in the trichomes present on the petiolules. While lycosantalene or lycosantalonol cannot be detected in the petiolules of wild-type plants (or anywhere else in the plant), lycosantalene and lycosantalonol are detected in petiolules of transgenic tomato plants expressing CPT2 under the control of the 35S CaMV promoter. These results suggest that lycosantalene and lycosantalonol are produced in the petiolules and perhaps in other tissues of wild-type plants, but that low rate of synthesis, controlled by the rate-limiting enzyme CPT2, results in product levels that are too low for detection under our current methodology. It is also possible that these compounds are further modified in the plant. The involvement of CPT2, TPS21 and CYP71BN1 in a diterpenoid biosynthetic pathway outside the trichomes, together with the involvement of other genes in the cluster in the synthesis of monoterpenes in trichomes, indicates that this cluster is further evolving into "sub-clusters" with unique biochemical, and likely physiological, roles.

  6. Evaluation of a cell penetrating prenylated peptide lacking an intrinsic fluorophore via in situ click reaction.

    Science.gov (United States)

    Ochocki, Joshua D; Mullen, Daniel G; Wattenberg, Elizabeth V; Distefano, Mark D

    2011-09-01

    Protein prenylation involves the addition of either a farnesyl (C(15)) or geranylgeranyl (C(20)) isoprenoid moiety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the plasma membrane of the cell. A recently discovered application of prenylated peptides is that they have inherent cell-penetrating ability, and are hence termed cell penetrating prenylated peptides. These peptides are able to efficiently cross the cell membrane in an ATP independent, non-endocytotic manner and it was found that the sequence of the peptide does not affect uptake, so long as the geranylgeranyl group is still present [Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.; Geier, S.; Falkum, S.; Wattenberg, E. V.; Barany, G.; Distefano, M. D. Multifunctional Prenylated Peptides for Live Cell Analysis. J. Am. Chem. Soc.2009, 131, 7293-7303]. The present study investigates the effect of removing the fluorophore from the peptides and investigating the uptake by confocal microscopy and flow cytometry. Our results show that the fluorophore is not necessary for uptake of these peptides. This information is significant because it indicates that the prenyl group is the major determinant in allowing these peptides to enter cells; the hydrophobic fluorophore has little effect. Moreover, these studies demonstrate the utility of the Cu-catalyzed click reaction for monitoring the entry of nonfluorescent peptides into cells.

  7. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

    Full Text Available Abstract Background Isopentenyl diphosphate (IPP, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  8. Differential effects of prenylation and s-acylation on type I and II ROPS membrane interaction and function.

    Science.gov (United States)

    Sorek, Nadav; Gutman, Orit; Bar, Einat; Abu-Abied, Mohamad; Feng, Xuehui; Running, Mark P; Lewinsohn, Efraim; Ori, Naomi; Sadot, Einat; Henis, Yoav I; Yalovsky, Shaul

    2011-02-01

    Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gγ proteins, while type II ROPs are attached to the plasma membrane by S-acylation. Yet, it is not known how prenylation affects ROP membrane interaction dynamics and what are the functional redundancy and specificity of type I and type II ROPs. Here, we have used the expression of ROPs in mammalian cells together with geranylgeranylation and CaaX prenylation-deficient mutants to answer these questions. Our results show that the mechanism of type II ROP S-acylation and membrane attachment is unique to plants and likely responsible for the viability of plants in the absence of CaaX prenylation activity. The prenylation of ROPs determines their steady-state distribution between the plasma membrane and the cytosol but has little effect on membrane interaction dynamics. In addition, the prenyl group type has only minor effects on ROP function. Phenotypic analysis of the CaaX prenylation-deficient pluripetala mutant epidermal cells revealed that type I ROPs affect cell structure primarily on the adaxial side, while type II ROPs are functional and induce a novel cell division phenotype in this genetic background. Taken together, our studies show how prenyl and S-acyl lipid modifications affect ROP subcellular distribution, membrane interaction dynamics, and function.

  9. Neuroprotective effects of xanthohumol, a prenylated flavonoid from hops (Humulus lupulus), in ischemic stroke of rats.

    Science.gov (United States)

    Yen, Ting-Lin; Hsu, Chung-King; Lu, Wan-Jung; Hsieh, Cheng-Ying; Hsiao, George; Chou, Duen-Suey; Wu, Gong-Jhe; Sheu, Joen-Rong

    2012-02-29

    Xanthohumol is the principal prenylated flavonoid in hops (Humulus lupulus L.), an ingredient of beer. Xanthohumol was found to be a potent chemopreventive agent; however, no data are available concerning its neuroprotective effects. In the present study, the neuroprotective activity and mechanisms of xanthohumol in rats with middle cerebral artery occlusion (MCAO)-induced cerebral ischemia were examined. Treatment with xanthohumol (0.2 and 0.4 mg/kg; intraperitoneally) 10 min before MCAO dose-dependently attenuated focal cerebral ischemia and improved neurobehavioral deficits in cerebral ischemic rats. Xanthohumol treatment produced a marked reduction in infarct size compared to that in control rats. MCAO-induced focal cerebral ischemia was associated with increases in hypoxia-inducible factor (HIF)-1α, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), and active caspase-3 protein expressions in ischemic regions. These expressions were obviously inhibited by treatment with xanthohumol. In addition, xanthohumol (3-70 μM) concentration-dependently inhibited platelet aggregation stimulated by collagen (1 μg/mL) in human platelet-rich plasma. An electron spin resonance (ESR) method was used to examine the scavenging activity of xanthohumol on free radicals which had formed. Xanthohumol (1.5 and 3 μM) markedly reduced the ESR signal intensity of hydroxyl radical (OH•) formation in the H₂O₂/NaOH/DMSO system. In conclusion, this study demonstrates for the first time that in addition to its originally being considered an agent preventing tumor growth, xanthohumol possesses potent neuroprotective activity. This activity is mediated, at least in part, by inhibition of inflammatory responses (i.e., HIF-1α, iNOS expression, and free radical formation), apoptosis (i.e., TNF-α, active caspase-3), and platelet activation, resulting in a reduction of infarct volume and improvement in neurobehavior in rats with cerebral ischemia. Therefore, this

  10. PRENYLATED BENZOIC ACID DERIVATIVES FROM PIPER SPECIES AS SOURCE OF ANTI-INFECTIVE AGENTS

    Directory of Open Access Journals (Sweden)

    Ibrahim Malami

    2012-06-01

    Full Text Available A number of prenylated benzoic acids derivatives with interesting biological activities have been previously isolated and characterized from different species of piperaceae family. Several Piper species contained structurally similar compounds with diverse biological activities such as anti-bacterial, anti-fungal, insecticidal as well as anti-parasitic all of which produces compounds that can be classified as prenylated benzoic acid derivatives. Piper sp has proven to serve as a source of potential anti-infective agents by developing a biosynthetic route to the synthesis of prenylated benzoic acids derivative natural products as their chemical defense system. Despite the frequent isolation of these interesting compounds with strong biological activity, yet none have been taking into consideration for development into therapeutic agents.

  11. Statin treatment increases lifespan and improves cardiac health in Drosophila by decreasing specific protein prenylation.

    Directory of Open Access Journals (Sweden)

    Stephen R Spindler

    Full Text Available Statins such as simvastatin are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase inhibitors and standard therapy for the prevention and treatment of cardiovascular diseases in mammals. Here we show that simvastatin significantly increased the mean and maximum lifespan of Drosophila melanogaster (Drosophila and enhanced cardiac function in aging flies by significantly reducing heart arrhythmias and increasing the contraction proportion of the contraction/relaxation cycle. These results appeared independent of internal changes in ubiquinone or juvenile hormone levels. Rather, they appeared to involve decreased protein prenylation. Simvastatin decreased the membrane association (prenylation of specific small Ras GTPases in mice. Both farnesyl (L744832 and type 1 geranylgeranyl transferase (GGTI-298 inhibitors increased Drosophila lifespan. These data are the most direct evidence to date that decreased protein prenylation can increase cardiac health and lifespan in any metazoan species, and may explain the pleiotropic (non-cholesterol related health effects of statins.

  12. A new prenylated flavanonol from Seseli annuum roots showing protective effect on human lymphocytes DNA.

    Science.gov (United States)

    Vucković, Ivan; Vajs, Vlatka; Stanković, Miroslava; Tesević, Vele; Milosavljević, Slobodan

    2010-03-01

    A new prenylated flavanonol named seselinonol (1) was isolated from the roots of Seseli annuum, together with the well-known biologically active polyacetylenes falcarinol (2) and falcarindiol (3), and the prenylated furanocoumarin phellopterin (4). Its structure was elucidated by extensive spectroscopic analysis, including HR-ESI-MS, 1D- and 2D-NMR. Seselinonol and phellopterin were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B blocked micronucleus (CBMN) assay. The new compound exerted a beneficial effect by decreasing DNA damage of human lymphocytes.

  13. Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP ...

  14. Antiparasitic activity of prenylated benzoic acid derivatives from Piper species.

    Science.gov (United States)

    Flores, Ninoska; Jiménez, Ignacio A; Giménez, Alberto; Ruiz, Grace; Gutiérrez, David; Bourdy, Genevieve; Bazzocchi, Isabel L

    2009-03-01

    Fractionation of dichloromethane extracts from the leaves of Piper heterophyllum and P. aduncum afforded three prenylated hydroxybenzoic acids, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl-2,6,10,14-hexadecatetraenyl)-4,5-dihydroxybenzoic acid, 3-[(2E,6E,10E)-11-carboxy-13-hydroxy-3,7,15-trimethyl-2,6,10,14-hexadecatetraenyl]-4,5-dihydroxybenzoic acid and 3-[(2E,6E,10E)-11-carboxy-14-hydroxy-3,7,15-trimethyl-2,6,10,15-hexadecatetraenyl]-4,5-dihydroxybenzoic acid, along with the known compounds, 4,5-dihydroxy-3-(E,E,E-11-formyl-3,7,15-trimethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid (arieianal), 3,4-dihydroxy-5-(E,E,E-3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid, 4-hydroxy-3-(E,E,E-3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid, 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxy-benzoic acid, 4-hydroxy-3-(3,7-dimethyl-2,6-octadienyl)benzoic acid and 4-hydroxy-3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid. Their structures were elucidated on the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC) and comparison with data reported in the literature. Riguera ester reactions and optical rotation measurements established the compounds as racemates. The antiparasitic activity of the compounds were tested against three strains of Leishmania spp., Trypanosoma cruzi and Plasmodium falciparum. The results showed that 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxy-benzoic acid exhibited potent and selective activity against L. braziliensis (IC(50) 6.5 microg/ml), higher that pentamidine used as control. Moreover, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl- 2,6,10,14-hexadecatetraenyl)-4,5-dihydroxybenzoic acid and 4-hydroxy-3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid showed moderate antiplasmodial (IC(50) 3.2 microg/ml) and trypanocidal (16.5 microg/ml) activities, respectively.

  15. Prenylated isoflavonoids from soya and licorice : analysis, induction and in vitro estrogenicity

    NARCIS (Netherlands)

    Simons, R.

    2011-01-01


    Prenylatedisoflavonoids are found in large amountsin soya bean (Glycine max) germinated under stress and in licorice (Glycyrrhiza glabra). Prenylation of isoflavonoids has been associated with modification of theirestrogenic activity.
    The aims of this thesis were (1) to provide a struct

  16. Prenylated isoflavonoids from soya and licorice : analysis, induction and in vitro estrogenicity

    NARCIS (Netherlands)

    Simons, R.

    2011-01-01


    Prenylatedisoflavonoids are found in large amountsin soya bean (Glycine max) germinated under stress and in licorice (Glycyrrhiza glabra). Prenylation of isoflavonoids has been associated with modification of theirestrogenic activity.
    The aims of this thesis were (1) to provide a

  17. How prenylation and S-acylation regulate subcellular targeting and function of ROP GTPases.

    Science.gov (United States)

    Sorek, Nadav; Henis, Yoav I; Yalovsky, Shaul

    2011-07-01

    Rho of Plants (ROP) small G proteins function at discrete domains of the plasma and possibly endo membranes. ROPs are synthesized as soluble proteins and their attachment to membranes and partitioning in membrane microdomains are facilitated by the posttranslational lipid modifications prenylation and/or S-acylation. Based on their amino acid sequences, ROPs can be classified into two major subgroups: type-I ROPs terminate with a canonical CaaX box motif and are prenylated primarily by geranylgeranyltransferase-I (GGT-I) and to a lesser extent by farnesyltransferase (FT). Type-II ROPs terminate with a plant specific GC-CG box domain and are attached to the plasma membrane by stable S-acylation. In addition, type-I and possibly also type-II ROPs undergo activation dependent transient S-acylation in the G-domain and consequent partitioning into lipid rafts. Surprisingly, although geranylgeranylation is required for the membrane attachment of type-I ROPs and the γ subunits of heterotrimeric G proteins, Arabidopsis mutants lacking GGT-I function have a mild phenotype compared to wild type plants. The mild phenotype of the ggt-I mutants suggested that farnesylation by FT may compensate for the loss of GGT-I function and that possibly the prenylated type-I and S-acylated type-II ROPS have some overlapping functions. In a paper recently published in Plant Physiology we examined the role of the prenyl group type in type-I ROP function and membrane interaction dynamics and the functional redundancy between type-I and type-II ROPs. This study complements a second paper in which we examined the role of G-domain transient S-acylation in the membrane interaction dynamics and signaling by type-I ROPs. Together these two studies provide a framework for realizing the role of prenylation and S-acylation in subcellular targeting, membrane interaction dynamics and signaling by ROP GTPases.

  18. Molecular cloning and functional analysis of a 10-epi-junenol synthase from Inula hupehensis.

    Science.gov (United States)

    Gou, Jun-Bo; Li, Zhen-Qiu; Li, Chang-Fu; Chen, Fang-Fang; Lv, Shi-You; Zhang, Yan-Sheng

    2016-09-01

    Junenol based-eudesmanolides have been detected in many compositae plant species and were reported to exhibit various pharmacological activities. So far, the gene encoding junenol synthase has never been isolated. Here we report the molecular cloning and functional analysis of a 10-epi-junenol synthase from Inula hupehensis (designated IhsTPS1). IhsTPS1 converts the substrate farnesyl diphosphate into multiple sesquiterpenes with the product 10-epi-junenol being predominant. The transcript levels of IhsTPS1 correlate well with the accumulation pattern of 10-epi-junenol in I. hupehensis organs, supporting its biochemical roles in vivo.

  19. Synthesis, Properties and Applications of Diazotrifluropropanoyl-Containing Photoactive Analogues of Farnesyl Diphosphate Containing Modified Linkages for Enhanced Stability

    Science.gov (United States)

    Hovlid, Marisa L.; Edelstein, Rebecca L.; Henry, Olivier; Ochocki, Joshua; DeGraw, Amanda; Lenevich, Stepan; Talbot, Trista; Young, Victor G.; Hruza, Alan W.; Lopez-Gallego, Fernando; Labello, Nicholas P.; Strickland, Corey L.; Schmidt-Dannert, Claudia; Distefano, Mark D.

    2009-01-01

    Photoactive analogues of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogues that contain diazotrifluoropropionyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analogue (3) was synthesized in 32P-labeled form from geraniol in 7 steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [32P]3 demonstrate that this compound labels the β-subunits of both farnesyl- and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analogue 4 was prepared in 6 steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to PFTase in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase and a 32P-labeled form selectively photocrosslinks the β-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [32P]4 manifests significant selectivity and should hence be useful for identifying novel FPP utilizing enzymes in crude protein preparations. PMID:19954434

  20. New function of the amino group of thiamine diphosphate in thiamine catalysis.

    Science.gov (United States)

    Meshalkina, L E; Kochetov, G A; Hübner, G; Tittmann, K; Golbik, R

    2009-03-01

    In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4'-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase--4'-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4'-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH2-methylated ThDP exhibits dihydroxyethyl-4'-methylamino-ThDP-synthase activity. Thus, the 4'-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.

  1. Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin.

    Science.gov (United States)

    Hartmann, M A; Wentzinger, L; Hemmerlin, A; Bach, T J

    2000-12-01

    Plant isoprenoids represent a large group of compounds with a wide range of physiological functions. In the cytosol, isoprenoids are synthesized via the classical acetate/mevalonate pathway. In this pathway, farnesyl diphosphate (FPP) occupies a central position, from which isoprene units are dispatched to the different classes of isoprenoids, with sterols as the major end products. The present work deals with effects of squalestatin (SQ) on the metabolism of FPP in proliferating and synchronized cultured tobacco cv. Bright Yellow-2 cells. SQ is a potent inhibitor of squalene synthase (SQS), the first committed enzyme in the sterol pathway. At nanomolar concentrations, SQ severely impaired cell growth and sterol biosynthesis, as attested by the rapid decrease in SQS activity. At the same time, it triggered a several-fold increase in both the enzymic activity and mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase. When SQ was added to cells synchronized by aphidicolin treatment, it was found to block the cell cycle at the end of G(1) phase, but no cell death was induced. Tobacco cells were also fed exogenous tritiated trans-trans farnesol, the allylic alcohol derived from FPP, in the presence and absence of SQ. Evidence is presented that this compound was incorporated into sterols and ubiquinone Q(10). In the presence of SQ, the sterol pathway was inhibited, but no increase in the radioactivity of ubiquinone was observed, suggesting that this metabolic channel was already saturated under normal conditions.

  2. The Metabolism of 2-Caroboxy-4-Ketopentitol Diphosphate

    Energy Technology Data Exchange (ETDEWEB)

    Moses, V.; Calvin, M.

    1958-07-15

    2-Carboxy-4-ketopentitol is converted enzymatically by a cell-free preparation from spinach leaves into a substance undergoing acid-lactone interconversion. This substance has no phosphate or letone group and is probably a dicarboxylic, six-carbon sugar acid or the saccharic or saccharinic acid type. The significance of these findings with regard to the metabolic role of 2-carboxy-4-ketopentitol diphosphate is discussed.

  3. Activation of thiamin diphosphate and FAD in the phosphatedependent pyruvate oxidase from Lactobacillus plantarum

    OpenAIRE

    Tittmann, Kai; Proske, Daniela; Spinka, Michael; Ghisla, Sandro; Rudolph, Rainer; Hübner, Gerhard; Kern, Gunther

    1998-01-01

    The phosphate- and oxygen-dependent pyruvate oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold...

  4. Xanthohumol, a prenylated chalcone from Humulus lupulus L., inhibits cholesteryl ester transfer protein.

    Science.gov (United States)

    Hirata, Hiroshi; Takazumi, Koji; Segawa, Shuichi; Okada, Yukio; Kobayashi, Naoyuki; Shigyo, Tatsuro; Chiba, Hitoshi

    2012-10-01

    High density lipoprotein (HDL)-cholesterol levels are correlated with a low risk of atherosclerosis. The inhibition of cholesteryl ester transfer protein (CETP), which catalyses cholesterol transfer between lipoproteins, leads to an increase in HDL-cholesterol and is expected to be the next anti-atherogenic target. This study revealed that xanthohumol, a prenylated chalcone, showed the highest inhibition against CETP from screening of natural products in various plants. We investigated the inhibitory activity of some chalcones and flavanones. Naringenin chalcone showed weak CETP inhibition compared with xanthohumol. In addition, isoxanthohumol and naringenin drastically decreased the inhibitory activity. These results suggest that the prenyl group and chalcone structure of xanthohumol were responsible for the CETP inhibitory activity.

  5. Synthesis of the naturally occurring prenylated coumarins balsamiferone and cedrelopsin by domino reactions

    Digital Repository Service at National Institute of Oceanography (India)

    Patre, R.E.; Parameswaran, P.S.; Tilve, S.G.

    = Prenyl 6c, R = Benzyl 8, R = Allyl 2, R = Prenyl 9, R = Benzyl Scheme 1 OH OO R O O COOEt R (3,3) (3,3) O O COOEt R H 7 11 12 Scheme 2 Kapil et al. 12 have synthesized balsamiferone 2 beginning from umbelliferone-3-carboxylic acid in 13....04, 165.33, 188.39; 1 H NMR (300 MHz, CDCl 3 ) δ 1.76 (3H, s, CH 3 ), 1.77 (3H, s, CH 3 ), 1.82 (6H, s, 2 X CH 3 ), 4.59 (4H, m, CH 2 ), 5.50 (2H, m, CH), 6.48 (1H, s, H-3), 6.54 (1H, d, J = 8.7 Hz, H-5), 7.80 (1H, d, J = 8.7 Hz, H-6), 10.32 (1H, s...

  6. Prenylated flavonoids from the roots of Desmodium caudatum and evaluation of their antifungal activity.

    Science.gov (United States)

    Sasaki, Hisako; Kashiwada, Yoshiki; Shibatav, Hirofumi; Takaishi, Yoshihisa

    2012-11-01

    Two new prenylated flavonoids (1, 4) and two new prenylated C-methyl-flavonoids (6, 7), together with four known flavonoids (2, 3, 5, 8), were isolated from the roots of Desmodium caudatum. The structures of the new compounds were elucidated by extensive spectroscopic analyses including 1D-, 2D-NMR and MS. The antifungal activities of five compounds (1, 2, 4, 6, 8) as well as nine flavonoids (9-17) previously isolated from this plant against Aspergillus niger, Penicillium sp., Rhizopus sp., and Trichophyton sp. were evaluated. Compound 6 showed potent antifungal activity against Trichophyton sp. with a minimum inhibitory concentration (MIC) value of 1.95 µg/mL.

  7. Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation

    DEFF Research Database (Denmark)

    López-Posadas, Rocío; Becker, Christoph; Günther, Claudia

    2016-01-01

    the transcriptome of IECs from IBD patients using a genome-wide approach. We observed disease-specific alterations in IECs with markedly impaired Rho-A signaling in active IBD patients. Localization of epithelial Rho-A was shifted to the cytosol in IBDs, and inflammation was associated with suppressed Rho......-A activation due to reduced expression of the Rho-A prenylation enzyme geranylgeranyltransferase-I (GGTase-I). Functionally, we found that mice with conditional loss of Rhoa or the gene encoding GGTase-I, Pggt1b, in IECs exhibit spontaneous chronic intestinal inflammation with accumulation of granulocytes...... and CD4+ T cells. This phenotype was associated with cytoskeleton rearrangement and aberrant cell shedding, ultimately leading to loss of epithelial integrity and subsequent inflammation. These findings uncover deficient prenylation of Rho-A as a key player in the pathogenesis of IBDs. As therapeutic...

  8. Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus), Prevents Platelet Activation in Human Platelets

    OpenAIRE

    Ye-Ming Lee; Kuo-Hsien Hsieh; Wan-Jung Lu; Hsiu-Chu Chou; Duen-Suey Chou; Li-Ming Lien; Joen-Rong Sheu; Kuan-Hung Lin

    2012-01-01

    Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.). Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation...

  9. Cytotoxic and leishmanicidal properties of garcinielliptone FC, a prenylated benzophenone from Platonia insignis.

    Science.gov (United States)

    Costa Júnior, Joaquim S; de Almeida, Antonia Amanda Cardoso; de Barros Falcão Ferraz, Alexandre; Rossatto, Raíssa Rebés; Silva, Teresinha G; Silva, Paulo B N; Militão, Gardenia C G; das Graças Lopes Citó, Antonia Maria; Santana, Lorena Citó Lopes Resende; de Amorim Carvalho, Fernando Aécio; Freitas, Rivelilson M

    2013-03-01

    Garcinielliptone FC (GFC), a natural prenylated benzophenone, was extracted from Platonia insignis Mart. (Clusiaceae), a native plant commonly known as bacuri and used in traditional Brazilian medicine for the treatment of skin diseases. The aim of this study was to evaluate the cytotoxic and leishmanicidal effects of GFC using in vitro models. The experimental data demonstrated that the polyisoprenylated benzophenone GFC possesses cytotoxic and leishmanicidal activities.

  10. Prenylated flavanones and flavanonols as chemical markers in Glycosmis species (Rutaceae).

    Science.gov (United States)

    Lukaseder, Brigitte; Vajrodaya, Srunya; Hehenberger, Tina; Seger, Christoph; Nagl, Michael; Lutz-Kutschera, Gerda; Robien, Wolfgang; Greger, Harald; Hofer, Otmar

    2009-05-01

    Fifteen prenylated or geranylated flavanones and flavanonols were isolated from the leaf extracts of different Glycosmis species collected in Thailand and Malaysia. All structures were elucidated by spectroscopic methods, especially 1D and 2D NMR. Six compounds were described for the first time and two were only known so far as synthetic products. The chemotaxonomic significance of flavanoid accumulation within the genus Glycosmis is highlighted.

  11. Increased RhoA prenylation in the loechrig (loe mutant leads to progressive neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Mandy Cook

    Full Text Available The Drosophila mutant loechrig (loe shows age-dependent degeneration of the nervous system and is caused by the loss of a neuronal isoform of the AMP-activated protein kinase (AMPK γ-subunit (also known as SNF4Aγ. The trimeric AMPK complex is activated by low energy levels and metabolic insults and regulates multiple important signal pathways that control cell metabolism. A well-known downstream target of AMPK is hydroxyl-methylglutaryl-CoA reductase (HMGR, a key enzyme in isoprenoid synthesis, and we have previously shown that HMGR genetically interacts with loe and affects the severity of the degenerative phenotype. Prenylation of proteins like small G-proteins is an important posttranslational modification providing lipid moieties that allow the association of these proteins with membranes, thereby facilitating their subsequent activation. Rho proteins have been extensively studied in neuronal outgrowth, however, much less is known about their function in neuronal maintenance. Here we show that the loe mutation interferes with isoprenoid synthesis, leading to increased prenylation of the small GTPase Rho1, the fly orthologue of vertebrate RhoA. We also demonstrate that increased prenylation and Rho1 activity causes neurodegeneration and aggravates the behavioral and degenerative phenotypes of loe. Because we cannot detect defects in the development of the central nervous system in loe, this suggests that loe only interferes with the function of the RhoA pathway in maintaining neuronal integrity during adulthood. In addition, our results show that alterations in isoprenoids can result in progressive neurodegeneration, supporting findings in vertebrates that prenylation may play a role in neurodegenerative diseases like Alzheimer's Disease.

  12. Unusual cyclic terpenoids with terminal pendant prenyl moieties: from occurrence to synthesis.

    Science.gov (United States)

    Kulcitki, Veaceslav; Harghel, Petru; Ungur, Nicon

    2014-12-01

    The paper reviews the known examples of cyclic terpenoids produced from open chain polyenic precursors by an "unusual" biosynthetic pathway, involving selective electrophilic attack on an internal double bond followed by cyclization. The resulting compounds possess cyclic backbones with pendant terminal prenyl groups. Synthetic approaches applied for the synthesis of such specifically functionalized compounds are also discussed, as well as biological activity of reported representatives.

  13. Promoting effects of isobavachin on neurogenesis of mouse embryonic stem cells were associated with protein prenylation

    Institute of Scientific and Technical Information of China (English)

    Dan-yin WANG; Yu-zhe HU; Si-si KONG; Yong-ping YU; Dan-yan ZHU; Yi-dia LOU

    2011-01-01

    Aim:Some small molecules can induce mouse embryonic stem(ES)cells to differentiate into neuronal cells.Here.we explored the effect of isobavachin(IBA),a compound with a prenyl group at position 8 of ring A.on promoting neuronal differentiation and the potential role of its protein prenylation.Methods:The hanging drop method was employed for embryonic body(EB)formation to mimic embryo development in vivo.The EBs were treated with IBA at a final concentration of 10-7mol/L from EB stage(d 4)to d 8+10.Geranylgeranyltransferase l inhibitor GGTI298 was subsequently used to disrupt protein prenylation.Neuronal subtypes.including neurons and astrocytes, were observed by fluorescence microscopy.Gene and protein expression levels were detected using RT-PCR and Western blot analysis, respectively.Results:With IBA treatment,nestin was highly expressed in the neural progenitors generated from EBs(d 4,d 8+O).EBs then further differentiated into neurons(marked by 13-tubulin III)and astrocytes(marked by GF_AP), which were both Up-regulated in a timedependent manner on d 8;+5 and d 8+10.Co-treatment with GGTI-298 selectively abolished the IBA-induced neuronal differentiatjon.Moreover,in the MAPK pathway.p38 and JNK phosphorylation were down-regulated,while ERK phosphorylation was up-regulated after IBA treatment at different neuronaI differentiation passages.Conclusion:IBA can facilitate mouse ES cells differentiating into neuronal cells.The mechanism involved protein prenylation and,Subseauently,phos-ERK activation and the phos-p38 off pathway.

  14. Studies of the ADP/ATP carrier of mitochondria with fluorescent ADP analogue formycin diphosphate.

    Science.gov (United States)

    Graue, C; Klingenberg, M

    1979-06-05

    The ADP/ATP carrier was studied by a fluorescent substrate, formycin diphosphate which is the only fluorescent ADP analogue to bind. Its low quantum yield, short decay time and spectral overlap with tryptophan has as yet prevented its wider use. By incorporating fluorescent acceptors of formycin diphosphate fluorescence, anthracene-maleimide and vinylanthracene, into the membrane, these difficulties were circumvented. Only bound formycin diphosphate transfers energy to the probes so that the secondary emission of these probes is a measure for membrane-bound formycin diphosphate. The fluorescent transfer is inhibited by ADP, bongkrekate and carboxyatractylate whether added before or after incubation of formycin diphosphate showing that only binding to the adenine nucleotide carrier is measured. It also shows directly that the earlier demonstrated ADP fixation by bongkrekate is indeed a displacement into the matrix. The fluorescence decay time of the bound formycin diphosphate is measured as 1.95 ns compared to 0.95 ns of the free formycin diphosphate, indicating that formycin diphosphate is bound at the carrier in a non-polar environment. The depolarization decay time was found to be larger than 15 ns, indicating that carrier-bound formycin diphosphate is immobile within this time period.

  15. Role of Intestinal Hydrolase in the Absorption of Prenylated Flavonoids Present in Yinyanghuo

    Directory of Open Access Journals (Sweden)

    Ming Hu

    2011-02-01

    Full Text Available Purpose: Yinyanghuo (Herba Epimdii is a traditional Chinese herb containing prenylated flavonoids as its active constituents. The aim of this study was to examine the significance of the intestinal hydrolysis of prenylated flavonoids by lactase phlorizin hydrolase (LPH, an enzyme at the brush border membrane of intestinal cells. Methods: A four-site perfused rat intestinal model was used. The concentration of the flavonoids of interest and their metabolites in different intestinal segements were analyzed by HPLC, and the apparent permeabilities were calculated. A lactase phlorizin hydrolase inhibitor (gluconolactone was employed to investigate the mechanism of the intestinal absorption, and the metabolites of the four flavonoids were identified using LC/MS/MS. Results: Diglycosides (icariin or triglycosides (epimedin A, epimedin B, and epimedin C were hydrolyzed rapidly in duodenum and jejunum producing one or two metabolites, while a monoglycoside (baohuoside I was absorbed directly. When co-perfused with glucono-lactone, both the hydrolysis of diglycosides and triglycosides were significantly inhibited, with inhibition rates for icariin (62%, 50%, 40%, 46%, epimedin A, (55%, 26%, 21%, 14%; epimedin B (42%, 40%, 74%, 22%, and epimedin C (42%, 40%, 52%, 35% in duodenum, jejunum, ileum, and colon, respectively. Also the metabolites of icariin, epimedin A, epimedin B, and epimedin C were identified as baohuoside I (one of two, sagittatoside A, sagittatoside B, and 2"-O-rhamnosylicariside II, respectively. Conclusions: The results showed that lactase phlorizin hydrolase was a major determinant of the intestinal absorption of prenylated flavonoids present in Yinyanghuo.

  16. Geosmin biosynthesis in Streptomyces avermitilis. Molecular cloning, expression, and mechanistic study of the germacradienol/geosmin synthase.

    Science.gov (United States)

    Cane, David E; He, Xiaofei; Kobayashi, Seiji; Omura, Satoshi; Ikeda, Haruo

    2006-08-01

    Geosmin (1) is responsible for the characteristic odor of moist soil. The Gram-positive soil bacterium Streptomyces avermitilis produces geosmin (1) as well as its precursor germacradienol (3). The S. avermitilis gene SAV2163 (geoA) is extremely similar to the S. coelicolor A3(2) SCO6073 gene that encodes a germacradienol/geosmin synthase. S. avermitilis mutants with a deleted geoA were unable to produce either germacradienol (3) or geosmin (1). Biosynthesis of both compounds was restored by introducing an intact geoA gene into the mutants. Incubation of recombinant GeoA, encoded by the SAV2163 gene of S. avermitilis, with farnesyl diphosphate (2) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (66%), (7S)-germacrene D (4) (24%), geosmin (1) (8%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (2%). Incubation of this germacradienol/geosmin synthase with [1,1-(2)H2] FPP (2a) gave geosmin-d1 (1a), as predicted. When recombinant GeoA from either S. avermitilis or S. coelicolor A3(2) was incubated with nerolidyl diphosphate (8), only the acyclic elimination products beta3-farnesene (10), (Z)-alpha-farnesene (11), and (E)-alpha-farnesene (12) were formed, thereby ruling out nerolidyl diphosphate as an intermediate in the conversion of farnesyl diphosphate to geosmin, germacradienol, and germacrene D.

  17. Biochemistry: Acetohydroxyacid Synthase

    Directory of Open Access Journals (Sweden)

    Pham Ngoc Chien

    2010-02-01

    Full Text Available Acetohydroxyacid synthase (AHAS, EC 2.2.1.6; formerly known as acetolactate synthase, ALS is a thiamin-and FAD-dependent enzyme which catalyses the first common step in the biosynthesis of the branched-chain amino acids (BCAA isoleucine, leucine and valine. The enzyme is inhibited by several commercial herbicides and has been studied over the last 20 to 30 years. A short introductory note about acetohydroxyacid synthase has been provided.

  18. Cloning and characterization of squalene synthase gene from Poria cocos and its up-regulation by methyl jasmonate.

    Science.gov (United States)

    Wang, Jian-Rong; Lin, Jun-Fang; Guo, Li-Qiong; You, Lin-Feng; Zeng, Xian-Lu; Wen, Jia-Ming

    2014-02-01

    Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate to give presqualene diphosphate and the subsequent rearrangement to form squalene. The gene encoding squalene synthase was cloned from Poria cocos by degenerate PCR and inverse PCR. The open reading frame of the gene is 1,497 bp, which encodes 499 amino acid residues. A phylogenetic analysis revealed that P. cocos SQS belonged to the fungus group, and was more closely related to the SQS of Ganoderma lucidum than other fungi. The treatment of P. cocos with methyl jasmonate (MeJA) significantly enhanced the transcriptional level of P. cocos sqs gene and the content of squalene in P. cocos. The transcriptional level of sqs gene was approximately fourfold higher than the control sample and the squalene content reached 128.62 μg/g, when the concentration of MeJA was 300 μM after 72 h induction.

  19. Nuclear magnetic resonance-based quantification of organic diphosphates.

    Science.gov (United States)

    Lenevich, Stepan; Distefano, Mark D

    2011-01-15

    Phosphorylated compounds are ubiquitous in life. Given their central role, many such substrates and analogs have been prepared for subsequent evaluation. Prior to biological experiments, it is typically necessary to determine the concentration of the target molecule in solution. Here we describe a method where concentrations of stock solutions of organic diphosphates and bisphosphonates are quantified using (31)P nuclear magnetic resonance (NMR) spectroscopy with standard instrumentation using a capillary tube with a secondary standard. The method is specific and is applicable down to a concentration of 200 μM. The capillary tube provides the reference peak for quantification and deuterated solvent for locking.

  20. Domain relationships in thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Duggleby, Ronald G

    2006-08-01

    Three-dimensional structures have been determined for 13 different enzymes that use thiamine diphosphate (ThDP) as a cofactor. These enzymes fall into five families, where members within a family have similar structures. In different families, there are similarities between some domains that clearly point to a common ancestor for all of these enzymes. Where the enzyme structures differ, evolutionary relationships between families can be discerned. Here, I present an analysis of these families and propose an evolutionary pathway to explain the diversity of structures that are now known.

  1. Molecular Characterization of Soybean Pterocarpan 2-Dimethylallyltransferase in Glyceollin Biosynthesis: Local Gene and Whole-Genome Duplications of Prenyltransferase Genes Led to the Structural Diversity of Soybean Prenylated Isoflavonoids.

    Science.gov (United States)

    Yoneyama, Keisuke; Akashi, Tomoyoshi; Aoki, Toshio

    2016-12-01

    Soybean (Glycine max) accumulates several prenylated isoflavonoid phytoalexins, collectively referred to as glyceollins. Glyceollins (I, II, III, IV and V) possess modified pterocarpan skeletons with C5 moieties from dimethylallyl diphosphate, and they are commonly produced from (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(-)-glycinol]. The metabolic fate of (-)-glycinol is determined by the enzymatic introduction of a dimethylallyl group into C-4 or C-2, which is reportedly catalyzed by regiospecific prenyltransferases (PTs). 4-Dimethylallyl (-)-glycinol and 2-dimethylallyl (-)-glycinol are precursors of glyceollin I and other glyceollins, respectively. Although multiple genes encoding (-)-glycinol biosynthetic enzymes have been identified, those involved in the later steps of glyceollin formation mostly remain unidentified, except for (-)-glycinol 4-dimethylallyltransferase (G4DT), which is involved in glyceollin I biosynthesis. In this study, we identified four genes that encode isoflavonoid PTs, including (-)-glycinol 2-dimethylallyltransferase (G2DT), using homology-based in silico screening and biochemical characterization in yeast expression systems. Transcript analyses illustrated that changes in G2DT gene expression were correlated with the induction of glyceollins II, III, IV and V in elicitor-treated soybean cells and leaves, suggesting its involvement in glyceollin biosynthesis. Moreover, the genomic signatures of these PT genes revealed that G4DT and G2DT are paralogs derived from whole-genome duplications of the soybean genome, whereas other PT genes [isoflavone dimethylallyltransferase 1 (IDT1) and IDT2] were derived via local gene duplication on soybean chromosome 11.

  2. A procedure for the preparation and isolation of nucleoside-5’-diphosphates

    Directory of Open Access Journals (Sweden)

    Heidi J. Korhonen

    2015-04-01

    Full Text Available Tris[bis(triphenylphosphoranylideneammonium] pyrophosphate (PPN pyrophosphate was used in the SN2 displacements of the tosylate ion from 5’-tosylnucleosides to afford nucleoside-5’-diphosphates. Selective precipitation permitted the direct isolation of nucleoside-5’-diphosphates from crude reaction mixtures.

  3. Enlarging the scope of cell penetrating prenylated peptides to include farnesylated “CAAX” box sequences and diverse cell types

    Science.gov (United States)

    Ochocki, Joshua D.; Igbavboa, Urule; Wood, W. Gibson; Wattenberg, Elizabeth V.; Distefano, Mark D.

    2010-01-01

    Protein prenylation is a post-translational modification that is present in a large number of proteins; it has been proposed to be responsible for membrane association and protein-protein interactions which contribute to its role in signal transduction pathways. Research has been aimed at inhibiting prenylation with farnesyltransferase inhibitors (FTIs) based on the finding that the farnesylated protein Ras is implicated in 30% of human cancers. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation as it occurs in living cells. Here we describe the preparation of a series of farnesylated peptides that contain sequences recognized by protein farnesyltransferase. Using a combination of flow cytometry and confocal microscopy, we show that these peptides enter a variety of different cell types. A related peptide where the farnesyl group has been replaced by a disulfide-linked decyl group is also shown to be able to efficiently enter cells. These results highlight the applicability of these peptides as a platform for further study of protein prenylation and subsequent processing in live cells. PMID:20584014

  4. 1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants.

    Science.gov (United States)

    Estévez, J M; Cantero, A; Reindl, A; Reichler, S; León, P

    2001-06-22

    The initial step of the plastidic 2C-methyl-D-erythritol 4-phosphate (MEP) pathway that produces isopentenyl diphosphate is catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase. To investigate whether or not 1-deoxy-d-xylulose-5-phosphate synthase catalyzes a limiting step in the MEP pathway in plants, we produced transgenic Arabidopsis plants that over- or underexpress this enzyme. Compared with non-transgenic wild-type plants, the transgenic plants accumulate different levels of various isoprenoids such as chlorophylls, tocopherols, carotenoids, abscisic acid, and gibberellins. Phenotypically, the transgenic plants had slight alterations in growth and germination rates. Because the levels of several plastidic isoprenoids correlate with changes in 1-deoxy-D-xylulose-5-phosphate synthase levels, we conclude that this enzyme catalyzes one of the rate-limiting steps of the MEP biosynthetic pathway. Furthermore, since the product of the MEP pathway is isopentenyl diphosphate, our results suggest that in plastids the pool of isopentenyl diphosphate is limiting to isprenoid production.

  5. [Interaction of pyruvate dehydrogenase complex from the heart muscle with thiamine diphosphate and its derivatives].

    Science.gov (United States)

    Strumilo, S A; Kiselevskiĭ, Iu V; Taranda, N I; Zabrodskaia, S V; Oparin, D A

    1989-01-01

    Inhibitory effects of 23 thiamin derivatives on the bovine heart pyruvate dehydrogenase complex (PDC) were studied. Oxythiamin diphosphate and tetrahydroxythiamin diphosphate exhibited the most pronounced effect on the PDC activity, affecting the complex by a competitive type of inhibition for thiamin diphosphate (TDP). The apparent affinity of TDP and the anticoenzyme derivatives for apo PDC depended on presence of phosphate and divalent metal ions. Phosphate considerably increased the Km values for TDP (up to 0.17 microM) and the Ki values for oxythiamin diphosphate (0.40 microM) as well as for tetrahydroxythiamin diphosphate (0.23 microM). In presence of Mn2+, Km value for TDP was 3.5-fold lower as compared with Mg2+ containing medium.

  6. Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution.

    OpenAIRE

    Lindqvist, Y; Schneider, G; Ermler, U.; Sundström, M

    1992-01-01

    The crystal structure of Saccharomyces cerevisiae transketolase, a thiamine diphosphate dependent enzyme, has been determined to 2.5 A resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits. The cofactor, vitamin B1 derived thiamine diphosphate, is bound at the interface between the two subunits. The enzyme subunit is built up of three domains of the alpha/beta type. The diphosphate moiety of thiamine diphosphate is bound to the enz...

  7. A rapid screening method for prenylated flavonoids with ultra-high-performance liquid chromatography/electrospray ionisation mass spectrometry in licorice root extracts

    NARCIS (Netherlands)

    Simons, R.; Vincken, J.P.; Bakx, E.J.; Verbruggen, M.A.; Gruppen, H.

    2009-01-01

    Due to their substitution with an isoprenoid group, prenylated flavonoids have an increased affinity for biological membranes and target proteins, enhancing their potential bioactivity. Although many prenylated flavonoids have been described, there are no methods that specifically screen for their p

  8. Differential Effects of Prenylation and S-Acylation on Type I and II ROPS Membrane Interaction and Function1[W][OA

    Science.gov (United States)

    Sorek, Nadav; Gutman, Orit; Bar, Einat; Abu-Abied, Mohamad; Feng, Xuehui; Running, Mark P.; Lewinsohn, Efraim; Ori, Naomi; Sadot, Einat; Henis, Yoav I.; Yalovsky, Shaul

    2011-01-01

    Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gγ proteins, while type II ROPs are attached to the plasma membrane by S-acylation. Yet, it is not known how prenylation affects ROP membrane interaction dynamics and what are the functional redundancy and specificity of type I and type II ROPs. Here, we have used the expression of ROPs in mammalian cells together with geranylgeranylation and CaaX prenylation-deficient mutants to answer these questions. Our results show that the mechanism of type II ROP S-acylation and membrane attachment is unique to plants and likely responsible for the viability of plants in the absence of CaaX prenylation activity. The prenylation of ROPs determines their steady-state distribution between the plasma membrane and the cytosol but has little effect on membrane interaction dynamics. In addition, the prenyl group type has only minor effects on ROP function. Phenotypic analysis of the CaaX prenylation-deficient pluripetala mutant epidermal cells revealed that type I ROPs affect cell structure primarily on the adaxial side, while type II ROPs are functional and induce a novel cell division phenotype in this genetic background. Taken together, our studies show how prenyl and S-acyl lipid modifications affect ROP subcellular distribution, membrane interaction dynamics, and function. PMID:21139084

  9. Observation of an Acryloyl–Thiamin Diphosphate Adduct in the First Step of Clavulanic Acid Biosynthesis

    Science.gov (United States)

    Merski, Matthew

    2011-01-01

    The first committed biosynthetic step toward clavulanic acid, the clinically-important β-lactamase inhibitor, is catalyzed by the thiamin diphosphate (ThDP)-dependent enzyme N2-(2-carboxyethyl)arginine synthase (CEAS). This protein carries out a unique reaction among ThDP-dependent processes in which a C–N bond is formed, and an electrophilic acryloyl–thiazolium intermediate of ThDP is proposed to be involved, unlike the nucleophilic enamine species typically generated by this class of enzymes. Here we present evidence for the existence of the putative acryloyl adduct, and report the unexpected observation of a long-wavelength chromophore (λ = 433 nm), which we attribute to this enzyme bound species. Chemical models were synthesized that both confirm its expected absorption (λ = 310–320 nm), and exclude self-condensation and intramolecular imine formation with the cofactor as its cause. Circular dichroism experiments and others discount charge transfer as a likely explanation for the ~120 nm red shift of the chromophore (~25 kcal). Examples are well-known of charged molecules that exhibit significantly red-shifted UV-visible spectra compared to their neutral forms as, for example, polyene cations and dyes such as indigo and the cyanines. Rhodopsin is the classic biochemical example where the protein (opsin)-bound protonated Schiff base of retinal displays a remarkable range of red-shifted absorptions modulated by the protein environment. Similar tuning of the chromophoric behavior of the enzyme-bound CEAS acryloyl•ThDP species may be occurring. PMID:18052280

  10. Transcriptomic insight into terpenoid and carbazole alkaloid biosynthesis, and functional characterization of two terpene synthases in curry tree (Murraya koenigii)

    Science.gov (United States)

    Meena, Seema; Rajeev Kumar, Sarma; Dwivedi, Varun; Kumar Singh, Anup; Chanotiya, Chandan S.; Akhtar, Md. Qussen; Kumar, Krishna; Kumar Shasany, Ajit; Nagegowda, Dinesh A.

    2017-01-01

    Curry tree (Murraya koenigii L.) is a rich source of aromatic terpenes and pharmacologically important carbazole alkaloids. Here, M. koenigii leaf transcriptome was generated to gain insight into terpenoid and alkaloid biosynthesis. Analysis of de novo assembled contigs yielded genes for terpene backbone biosynthesis and terpene synthases. Also, gene families possibly involved in carbazole alkaloid formation were identified that included polyketide synthases, prenyltransferases, methyltransferases and cytochrome P450s. Further, two genes encoding terpene synthases (MkTPS1 and MkTPS2) with highest in silico transcript abundance were cloned and functionally characterized to determine their involvement in leaf volatile formation. Subcellular localization using GFP fusions revealed the plastidial and cytosolic localization of MkTPS1 and MkTPS2, respectively. Enzymatic characterization demonstrated the monoterpene synthase activity of recombinant MkTPS1, which produced primarily (−)-sabinene from geranyl diphosphate (GPP). Recombinant MkTPS2 exhibited sesquiterpene synthase activity and formed (E,E)-α-farnesene as the major product from farnesyl diphosphate (FPP). Moreover, mRNA expression and leaf volatile analyses indicated that MkTPS1 accounts for (−)-sabinene emitted by M. koenigii leaves. Overall, the transcriptome data generated in this study will be a great resource and the start point for characterizing genes involved in the biosynthetic pathway of medicinally important carbazole alkaloids. PMID:28272514

  11. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    赵勇[1; 余龙[2; 高洁[3; 付强[4; 华益民[5; 张宏来[6; 赵寿元[7

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas

  12. Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Andersen, Kasper R; Kilstrup, Mogens

    2017-01-01

    is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP...... synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number...... phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic...

  13. Evaluation of anti-inflammatory activity of prenylated substances isolated from Morus alba and Morus nigra.

    Science.gov (United States)

    Zelová, Hana; Hanáková, Zuzana; Čermáková, Zuzana; Šmejkal, Karel; Dalĺ Acqua, Stefano; Babula, Petr; Cvačka, Josef; Hošek, Jan

    2014-06-27

    Chromatographic separation of root extracts of Morus alba and M. nigra led to the identification of the 2-arylbenzofurans moracin C (1), mulberrofuran Y (2), and mulberrofuran H (3), and the prenylated flavonoids kuwanon E (4), kuwanon C (5), sanggenon H (6), cudraflavone B (7), and morusinol (8), and the Diels-Alder adducts soroceal (9), and sanggenon E (10). The cytotoxicity and their antiphlogistic activity, determined as the attenuation of the secretion of TNF-α and IL-1β and the inhibition of NF-κB nuclear translocation in LPS-stimulated macrophages, were evaluated for compounds 1-10.

  14. Prenylated Coumarins from Heracleum stenopterum, Peucedanum praeruptorum, Clausena lansium, and Murraya paniculata

    Directory of Open Access Journals (Sweden)

    Xiang-Mei Li

    2016-09-01

    Full Text Available Abstract Four hitherto unknown prenylated coumarins, namely 6″-O-β-d-apiofuranosylapterin (1, 4′-O-isobutyroylpeguangxienin (2, 6-(3-methyl-2-oxobutyroyl-7-methoxycoumarin (3, and 6-hydroxycoumurrayin (4, were isolated from the ethanol extract of Heracleum stenopterum, Peucedanum praeruptorum, Clausena lansium, and Murraya paniculata, respectively. Their chemical structures were established on the basis of extensive spectroscopic analysis. Compound 2 exhibited in vitro cytotoxic activity against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW-480 with IC50 values ranging from 15.9 to 23.2 μM. Graphical Abstract

  15. Two New Prenylated Stilbenes with an Irregular Sesquiterpenyl Side Chain from Propolis from Fiji Islands

    Directory of Open Access Journals (Sweden)

    Boryana Trusheva

    2016-01-01

    Full Text Available Two new prenylated stilbenes with an irregular sesquiterpenyl side chain, solomonin B (1 and solomonin C (2, together with four known compounds, glyasperin A (3, isolated for the first time from propolis, kumatakenin (4, macarangin (5 and mangiferolic acid (6 were isolated from ethanol extract of propolis from Fiji islands. The compounds structures were determined based on their spectral data analysis (1D- and 2D NMR, UV and HREIMS and comparison with literature data. The chemical composition of propolis from Fiji islands was determined for the first time.

  16. Biotransformation of Resveratrol: New Prenylated trans-Resveratrol Synthesized by Aspergillus sp. SCSIOW2

    Directory of Open Access Journals (Sweden)

    Liyan Wang

    2016-07-01

    Full Text Available Arahypin-16 (1, a new prenylated resveratrol with a unique dihydrobenzofuran ring, has been isolated as a microbial metabolite of resveratrol (2 from whole-cell fermentation of Aspergillus sp. SCSIOW2. The stereochemistry of 1 was determined by ECD calculations. 1 showed about half of the extracellular radical scavenging effect (IC50 = 161.4 μM compared with resveratrol (IC50 = 80.5 μM, while on biomembranes it exhibited the same range of protection effects against free radicals generated from AAPH (IC50 = 78.6 μM and 87.9 μM.

  17. Use of synthetic isoprenoids to target protein prenylation and Rho GTPases in breast cancer invasion.

    Directory of Open Access Journals (Sweden)

    Min Chen

    Full Text Available Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase and geranylgeranyltransferases (GGTase for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs, which are farnesol or geranyl-geraniol analogs that act as alternate substrates for FTase or GGTase. Here, we test the ability of our lead PFIs, anilinogeraniol (AGOH and anilinofarnesol (AFOH, to block the invasion of breast cancer cells. We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D culture model at as little as 20 µM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase. To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA and RhoC activation and invasive growth. Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells. Collectively, this study demonstrates that AGOH and AFOH dramatically inhibit breast cancer invasion, at least in part by blocking Rho function, thus, suggesting that targeting

  18. Amyrisins A-C, O-prenylated flavonoids from Amyris madrensis.

    Science.gov (United States)

    Peng, Jiangnan; Hartley, Rachel M; Fest, Gary A; Mooberry, Susan L

    2012-03-23

    Three new O-prenylated flavonoids, amyrisins A-C (1-3), were isolated from the leaves and twigs of Amyris madrensis, along with the known compound polygamain (4). The structures of 1-3 were elucidated on the basis of the analysis of spectroscopic data interpretation. Amyrisins B (2) and C (3) showed moderate cytotoxicity against PC-3 and DU 145 prostate cancer cells with IC(50) values of 17.5 and 23 μM, respectively, while amyrisin A (1) did not show any cytotoxicity at the highest concentration tested, 50 μM. Polygamain (4) exhibited potent antiproliferative and microtubule-depolymerizing activities.

  19. Prenylated Coumarins from Heracleum stenopterum, Peucedanum praeruptorum, Clausena lansium, and Murraya paniculata.

    Science.gov (United States)

    Li, Xiang-Mei; Jiang, Xian-Jun; Yang, Ku; Wang, Li-Xia; Wen, Shi-Zhen; Wang, Fei

    2016-10-01

    Four hitherto unknown prenylated coumarins, namely 6″-O-β-D-apiofuranosylapterin (1), 4'-O-isobutyroylpeguangxienin (2), 6-(3-methyl-2-oxobutyroyl)-7-methoxycoumarin (3), and 6-hydroxycoumurrayin (4), were isolated from the ethanol extract of Heracleum stenopterum, Peucedanum praeruptorum, Clausena lansium, and Murraya paniculata, respectively. Their chemical structures were established on the basis of extensive spectroscopic analysis. Compound 2 exhibited in vitro cytotoxic activity against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW-480) with IC50 values ranging from 15.9 to 23.2 μM.

  20. Cloning and enzymology analysis of farnesyl pyrophosphate synthase gene from a superior strain of Artemisia annua L

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A cDNA(af1) encoding farnesyl pyrophosphate synthase AaFPS1 (FPS, EC2.5.1.1/EC2.5.1.10) from a high yield Artemisia annua strain 025 has been cloned from its cDNA library. Sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with molecular weight of 39 kD. Deduced aa sequence of the cDNA was similar to FPS from other plants, yeast and mammals, containing 5 conserved domains found in both prenyl transferase and polyprenyl synthase. The expression of the cDNA in Escherichia coli showed measurable specific activity of FPS in vitro. The enzyme was purified by ion exchange chromatography and its kinetics was measured. These results would further promote the molecular regulation of artemisinin biosynthesis.

  1. Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation.

    Science.gov (United States)

    DeGraw, Amanda J; Palsuledesai, Charuta; Ochocki, Joshua D; Dozier, Jonathan K; Lenevich, Stepan; Rashidian, Mohammad; Distefano, Mark D

    2010-12-01

    Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here, we investigate the utility of alkyne-containing isoprenoid analogs for chemical proteomics experiments by showing that these compounds readily penetrate mammalian cells in culture and become incorporated into proteins that are normally prenylated. Derivatization via Cu(I) catalyzed click reaction with a fluorescent azide reagent allows the proteins to be visualized and their relative levels to be analyzed. Simultaneous treatment of cells with these probes and inhibitors of prenylation reveals decreases in the levels of some but not all of the labeled proteins. Two-dimensional electrophoretic separation of these labeled proteins followed by mass spectrometric analysis allowed several labeled proteins to be unambiguously identified. Docking experiments and density functional theory calculations suggest that the substrate specificity of protein farnesyl transferase may vary depending on whether azide- or alkyne-based isoprenoid analogs is employed. These results demonstrate the utility of alkyne-containing analogs for chemical proteomic applications.

  2. Discovery of prenylated flavonoids with dual activity against influenza virus and Streptococcus pneumoniae.

    Science.gov (United States)

    Grienke, Ulrike; Richter, Martina; Walther, Elisabeth; Hoffmann, Anja; Kirchmair, Johannes; Makarov, Vadim; Nietzsche, Sandor; Schmidtke, Michaela; Rollinger, Judith M

    2016-06-03

    Influenza virus neuraminidase (NA) is the primary target for influenza therapeutics. Severe complications are often related to secondary pneumonia caused by Streptococcus pneumoniae (pneumococci), which also express NAs. Recently, a NA-mediated lethal synergism between influenza A viruses and pneumococci was described. Therefore, dual inhibitors of both viral and bacterial NAs are expected to be advantageous for the treatment of influenza. We investigated the traditional Chinese herbal drug sāng bái pí (mulberry root bark) as source for anti-infectives. Two prenylated flavonoid derivatives, sanggenon G (4) and sanggenol A (5) inhibited influenza A viral and pneumococcal NAs and, in contrast to the approved NA inhibitor oseltamivir, also planktonic growth and biofilm formation of pneumococci. Evaluation of 27 congeners of 5 revealed a correlation between the degree of prenylation and bioactivity. Abyssinone-V 4'-methyl ether (27) inhibited pneumococcal NA with IC50 = 2.18 μM, pneumococcal growth with MIC = 5.63 μM, and biofilm formation with MBIC = 4.21 μM, without harming lung epithelial cells. Compounds 5 and 27 also disrupt the synergism between influenza A virus and pneumococcal NA in vitro, hence functioning as dual-acting anti-infectives. The results warrant further studies on whether the observed disruption of this synergism is transferable to in vivo systems.

  3. Solid-phase synthesis of C-terminal peptide libraries for studying the specificity of enzymatic protein prenylation.

    Science.gov (United States)

    Wang, Yen-Chih; Distefano, Mark D

    2012-08-25

    Prenylation is an essential post-translational modification in all eukaryotes. Here we describe the synthesis of a 340-member library of peptides containing free C-termini on cellulose membranes. The resulting library was then used to probe the specificity of protein farnesyltransferase from S. cerevisiae.

  4. Prenylation and Backbone Structure of Flavonoids and Isoflavonoids from Licorice and Hop Influence Their Phase i and II Metabolism

    NARCIS (Netherlands)

    De Schans, Van Milou G.M.; Bovee, Toine F.H.; Stoopen, Geert M.; Lorist, Marlies; Gruppen, Harry; Vincken, Jean Paul

    2015-01-01

    In vitro liver metabolism of 11 prenylated flavonoids and isoflavonoids was investigated by determining their phase I glucuronyl and sulfate metabolites using pork liver preparations. One hundred metabolites were annotated using RP-UHPLC-ESI-MSn. A mass spectrometry-based data interpre

  5. The Primary Diterpene Synthase Products of Picea abies Levopimaradiene/Abietadiene Synthase (PaLAS) Are Epimers of a Thermally Unstable Diterpenol*

    Science.gov (United States)

    Keeling, Christopher I.; Madilao, Lina L.; Zerbe, Philipp; Dullat, Harpreet K.; Bohlmann, Jörg

    2011-01-01

    The levopimaradiene/abietadiene synthase from Norway spruce (Picea abies; PaLAS) has previously been reported to produce a mixture of four diterpene hydrocarbons when incubated with geranylgeranyl diphosphate as the substrate: levopimaradiene, abietadiene, neoabietadiene, and palustradiene. However, variability in the assay products observed by GC-MS of this and orthologous conifer diterpene synthases over the past 15 years suggested that these diterpenes may not be the initial enzyme assay products but are rather the products of dehydration of an unstable alcohol. We have identified epimers of the thermally unstable allylic tertiary alcohol 13-hydroxy-8(14)-abietene as the products of PaLAS. The identification of these compounds, not previously described in conifers, as the initial products of PaLAS has considerable implications for our understanding of the complexity of the biosynthetic pathway of the structurally diverse diterpene resin acids of conifer defense. PMID:21518766

  6. Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit.

    Science.gov (United States)

    Bravdo, B A; Palgi, A; Lurie, S

    1977-08-01

    Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening.

  7. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase

    OpenAIRE

    Jeudy, Sandra; Coutard, Bruno; Lebrun, Régine; Abergel, Chantal

    2005-01-01

    A. polyphaga mimivirus, the largest known double-stranded DNA virus, is the first virus to exhibit a nucleoside diphosphate kinase gene. The expression and crystallization of the viral NDK are reported.

  8. Two residues determine the product profile of the class II diterpene synthases TPS14 and TPS21 of Tripterygium wilfordii

    DEFF Research Database (Denmark)

    Hansen, Nikolaj Lervad; Nissen, Jakob N.; Hamberger, Björn Robert

    2017-01-01

    The medicinal plant Tripterygium wilfordii (Celastraceae) contains a pair of class II diterpene synthases (diTPS) of specialized labdane-type metabolism that, despite remarkably close homology, form strikingly different products. TwTPS21 catalyzes bicyclization of the linear C20 precursor...... residue gave mixed product profiles. Two mutants, TwTPS14:Y265H and TwTPS21:A325V, also produced ent-copalyl diphosphate, highlighting the evolutionary potential of enzymes of this family to drive rapid diversification of plant diterpene biosynthesis through neo-functionalization. Our study contributes...... to the understanding of structure-function relation in plant class II diTPSs and complements previous mutational studies of Arabidopsis ent-copalyl diphosphate synthase with additional examples from the specialized metabolism of T. wilfordii....

  9. Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases1[W][OA

    Science.gov (United States)

    Hall, Dawn E.; Zerbe, Philipp; Jancsik, Sharon; Quesada, Alfonso Lara; Dullat, Harpreet; Madilao, Lina L.; Yuen, Macaire; Bohlmann, Jörg

    2013-01-01

    Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs. PMID:23370714

  10. Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Kasparyan, Elena; Richter, Michael; Dresen, Carola; Walter, Lydia S; Fuchs, Georg; Leeper, Finian J; Wacker, Tobias; Andrade, Susana L A; Kolter, Geraldine; Pohl, Martina; Müller, Michael

    2014-12-01

    The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

  11. Ribulose diphosphate carboxylase of the cyanobacterium Spirulina platensis

    Energy Technology Data Exchange (ETDEWEB)

    Terekhova, I.V.; Chernyad' ev, I.I.; Doman, N.G.

    1986-11-20

    The ribulose diphosphate (RDP) carboxylase activity of the cyanobacterium Spirulina platensis is represented by two peaks when a cell homogenate is centrifuged in a sucrose density gradient. In the case of differential centrifugation (40,000 g, 1 h), the activity of the enzyme was distributed between the supernatant liquid (soluble form) and the precipitate (carboxysomal form). From the soluble fraction, in which 80-95% of the total activity of the enzyme is concentrated, electrophoretically homogeneous RDP carboxylase was isolated by precipitation with ammonium sulfate and centrifugation in a sucrose density gradient. The purified enzyme possessed greater electrophoretic mobility in comparison with the RDP carboxylase of beans Vicia faba. The molecular weight of the enzyme, determined by gel filtration, was 450,000. The enzyme consists of monotypic subunits with a molecular weight of 53,000. The small subunits were not detected in electrophoresis in polyacrylamide gel in the presence of SDS after fixation and staining of the gels by various methods.

  12. Crystal structures of phosphoketolase: thiamine diphosphate-dependent dehydration mechanism.

    Science.gov (United States)

    Suzuki, Ryuichiro; Katayama, Takane; Kim, Byung-Jun; Wakagi, Takayoshi; Shoun, Hirofumi; Ashida, Hisashi; Yamamoto, Kenji; Fushinobu, Shinya

    2010-10-29

    Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,β-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.

  13. Propioin synthesis using thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Mikolajek, Renaud J; Spiess, Antje C; Pohl, Martina; Büchs, Jochen

    2009-01-01

    Benzaldehyde lyase (BAL, EC 4.1.2.38) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD, EC 4.1.1.7) from Pseudomonas putida are thiamine diphosphate-dependent enzymes. These enzymes share a common tetrameric structure and catalyze various C--C-bond forming and breaking reactions. Here we describe a detailed study of the asymmetric synthesis of propioin from propanal catalyzed by BAL or BFD in aqueous solution in a batch reactor. Both enzymes are deactivated in the presence of high concentration of propanal. Compared to BAL, BFD is more stable under reaction conditions as well as during storage. The kinetic studies showed a typical Michaelis-Menten kinetic for BAL with a maximal specific reaction rate of 26.2 U/mg and an unusually high K(M) of 415 mM, whereas the v/[S]-plot for BFD is almost linear in the concentration range (100-1500 mM) investigated. Both enzymes produce propioin with opposite enantiomeric excess: BAL produced the (S)-propioin (ee of 35%), whereas BFD yielded the (R)-enantiomer (ee of 67%).

  14. Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli

    Science.gov (United States)

    Moreno-Bruna, Beatriz; Baroja-Fernández, Edurne; Muñoz, Francisco José; Bastarrica-Berasategui, Ainara; Zandueta-Criado, Aitor; Rodríguez-López, Milagros; Lasa, Iñigo; Akazawa, Takashi; Pozueta-Romero, Javier

    2001-01-01

    An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli. PMID:11416161

  15. Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

    Science.gov (United States)

    Grausgruber-Gröger, Sabine; Schmiderer, Corinna; Steinborn, Ralf; Novak, Johannes

    2012-03-01

    Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and β-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/β-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively).

  16. Xanthohumol, a prenylated chalcone from beer hops, acts as an α-glucosidase inhibitor in vitro.

    Science.gov (United States)

    Liu, Ming; Yin, Hua; Liu, Ge; Dong, Jianjun; Qian, Zhonghua; Miao, Jinlai

    2014-06-18

    Xanthohumol (XN) is a unique prenylated flavonoid in hops (Humulus lupulus L.) and beer. XN alleviates hyperglycemia and has potential usage in the treatment of type 2 diabetes. In the present study, a series of in vitro experiments were performed to investigate whether XN was an effective inhibitor of α-glucosidase. The results showed that XN inhibited α-glucosidase in a reversible and noncompetitive manner, with an IC50 value of 8.8 μM and that XN inhibited the release of glucose from the maltose in the apical side of the Caco-2 cell monolayer. Fluorescence and circular dichroism spectra results indicated that XN directly bound to α-glucosidase and induced minor conformational changes of the enzyme. These results demonstrated that XN is a promising α-glucosidase inhibitor, which therefore could be used as functional food to alleviate postprandial hyperglycemia and as a potential candidate for the development of an antidiabetic agent.

  17. Antimicrobial prenylated benzoylphloroglucinol derivatives and xanthones from the leaves of Garcinia goudotiana.

    Science.gov (United States)

    Mahamodo, Sania; Rivière, Céline; Neut, Christel; Abedini, Amin; Ranarivelo, Heritiana; Duhal, Nathalie; Roumy, Vincent; Hennebelle, Thierry; Sahpaz, Sevser; Lemoine, Amélie; Razafimahefa, Dorothée; Razanamahefa, Bakonirina; Bailleul, François; Andriamihaja, Bakolinirina

    2014-06-01

    Bioassay-guided fractionation using antimicrobial assay of the crude acetonic extract of Garcinia goudotiana leaves and of its five partitions led to the isolation of two new prenylated benzoylphloroglucinol derivatives, goudotianone 1 (1) and goudotianone 2 (2), in addition to two known compounds including one xanthone, 1,3,7-trihydroxy-2-isoprenylxanthone (3), and one triterpenoid, friedelin (4). Their structures were elucidated on the basis of different spectroscopic methods, including extensive 1D- and 2D-NMR spectroscopy and mass spectrometry. The crude acetonic extract, the methylene chloride and ethyl acetate partitions, and some tested compounds isolated from this species (1-3) demonstrated selective significant antimicrobial activities against Gram-positive bacteria, in particular Staphylococcus lugdunensis, Enterococcus faecalis and Mycobacterium smegmatis. The potential cytotoxic activities of these extracts and compounds were evaluated against human colon carcinoma HT29 and human fetal lung fibroblast MRC5 cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Flemingin-Type Prenylated Chalcones from the Sarawak Rainforest Plant Desmodium congestum.

    Science.gov (United States)

    Rees, Karlee A; Bermudez, Cindy; Edwards, David J; Elliott, Alysha G; Ripen, Jovita E; Seta, Cynthia; Huang, Johnny X; Cooper, Matthew A; Fraser, James A; Yeo, Tiong Chia; Butler, Mark S

    2015-08-28

    In an ongoing program to identify new anti-infective leads, an extract derived from whole plant material of Desmodium congestum collected in the Sarawak rainforest was found to have anti-MRSA activity. Bioassay-guided isolation led to the isolation of two new prenylated chalcones, 5'-O-methyl-3-hydroxyflemingin A (1) and 5'-O-methylflemingin C (2), which were closely related to the flemingins previously isolated from various Flemingia species. Chalcones 1 and 2, which were determined to be 4:6 enantiomeric mixtures by chiral HPLC, exhibited moderate activity against a panel of Gram-positive bacteria and were also cytotoxic to the HEK293 human embryonic kidney cell line.

  19. Effects of prenyl pyrophosphates on the binding of PKCgamma with RACK1.

    Science.gov (United States)

    Chen, Yu-Hsun; Wang, Han-Chung; Lin, Ching-Yu; Chuang, Nin-Nin

    2003-01-01

    Receptors for activated C kinase (RACKs) are a group of PKC binding proteins that have been shown to mediate isoform-selective functions of PKC and to be crucial in the translocation and subsequent functioning of the PKC isoenzymes on activation. RACK1 cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. The hepatopancreas cDNA from this shrimp was found to encode a 318-residue polypeptide whose predicted amino acid sequence shared 91% homology with human G(beta2)-like proteins. Expression of the cDNA of shrimp RACK1 in vitro yielded a 45-kDa polypeptide with positive reactivity toward the monoclonal antibodies against RACK1 of mammals. The shrimp RACK1 was biotinylated and used to compare the effects of geranylgeranyl pyrophosphate and farnesyl pyrophosphate on its binding with PKCgamma in anti-biotin-IgG precipitates. PKCgammas were isolated from shrimp eyes and mouse brains. Both enzyme preparations were able to inhibit taxol-induced tubulin polymerization. Interestingly, when either geranylgeranyl pyrophosphate or farnesyl pyrophosphate was reduced to the submicrogram level, the recruitment activity of RACK1 with purified PKCgamma was found to increase dramatically. The activation is especially significant for RACK1 and PKCgamma from different species. The observation implies that the deprivation of prenyl pyrophosphate might function as a signal for RACK1 to switch the binding from the conventional isoenzymes of PKC (cPKC) to the novel isoenzymes of PKC (nPKC). A hydrophobic binding pocket for geranylgeranyl pyrophosphate in RACK1 is further revealed via prenylation with protein geranylgeranyl transferase I of shrimp P. japonicus.

  20. Prenylated Polyphenols from Clusiaceae and Calophyllaceae with Immunomodulatory Activity on Endothelial Cells.

    Science.gov (United States)

    Rouger, Caroline; Pagie, Sylvain; Derbré, Séverine; Le Ray, Anne-Marie; Richomme, Pascal; Charreau, Béatrice

    2016-01-01

    Endothelial cells (ECs) are key players in inflammation and immune responses involved in numerous pathologies. Although attempts were experimentally undertaken to prevent and control EC activation, drug leads and probes still remain necessary. Natural products (NPs) from Clusiaceous and Calophyllaceous plants were previously reported as potential candidates to prevent endothelial dysfunction. The present study aimed to identify more precisely the molecular scaffolds that could limit EC activation. Here, 13 polyphenols belonging to 5 different chemical types of secondary metabolites (i.e., mammea coumarins, a biflavonoid, a pyranochromanone acid, a polyprenylated polycyclic acylphloroglucinol (PPAP) and two xanthones) were tested on resting and cytokine-activated EC cultures. Quantitative and qualitative changes in the expression of both adhesion molecules (VCAM-1, ICAM-1, E-selectin) and major histocompatibility complex (MHC) molecules have been used to measure their pharmaceutical potential. As a result, we identified 3 mammea coumarins that efficiently reduce (up to >90% at 10 μM) both basal and cytokine-regulated levels of MHC class I, class II, MICA and HLA-E on EC surface. They also prevented VCAM-1 induction upon inflammation. From a structural point of view, our results associate the loss of the free prenyl group substituting mammea coumarins with a reduced cellular cytotoxicity but also an abrogation of their anti-inflammatory potential and a reduction of their immunosuppressive effects. A PPAP, guttiferone J, also triggers a strong immunomodulation but restricted to HLA-E and MHC class II molecules. In conclusion, mammea coumarins with a free prenyl group and the PPAP guttiferone J emerge as NPs able to drastically decrease both VCAM-1 and a set of MHC molecules and to potentially reduce the immunogenicity of the endothelium.

  1. Bis-Pyrano Prenyl Isoflavone Improves Glucose Homeostasis by Inhibiting Dipeptidyl Peptidase-4 in Hyperglycemic Rats.

    Science.gov (United States)

    Altenhofen, Delsi; da Luz, Gabrielle; Frederico, Marisa Jádna Silva; Venzke, Dalila; Brich, Mayara; Vigil, Silvana; Fröde, Tania Silvia; Linares, Carlos Eduardo Blanco; Pizzolatti, Moacir Geraldo; Silva, Fátima Regina Mena Barreto

    2017-01-01

    Isoflavones widely distributed in plants prevent diabetes. This study investigated the in vivo and in vitro effect of 3',4'-dihydroxy-6″,6″,6″',6″'-tetramethylbis(pyrano[2″,3″:5,6::2″',3″':7,8]isoflavone (bis-pyrano prenyl isoflavone) on glucose homeostasis in hyperglycemic rats. The ethyl acetate fraction from aerial parts of Polygala molluginifolia that contain isoflavones was assayed on glucose tolerance, on in vitro maltase activity and on protein glycation. The isoflavone bis-pyrano prenyl isolated from this fraction was investigated on glucose homeostasis. The in vivo action of the isoflavone exhibits an anti-hyperglycemic effect by improving glucose tolerance, augmenting the liver glycogen, inhibiting maltase activity, and stimulating glucagon-like peptide-1 (GLP-1) and insulin secretion. The in vitro isoflavone inhibits dipeptidyl peptidase-4 (DPP-4) activity since the glucose tolerance was improved in the presence of the isoflavone as much as sitagliptin, an inhibitor of DPP-4. However, the co-incubation with isoflavone and sitagliptin exhibited an additive anti-hyperglycemic action. The isoflavone increased the GLP-1 faster than the positive hyperglycemic group, which shows that the intestine is a potential target. Thus, to clarify the main site of action in which isoflavone improves glucose balance, the in vitro mechanism of action of this compound was tested in intestine using calcium influx as a trigger for the signal pathways for GLP-1 secretion. The isoflavone stimulates calcium influx in intestine and its mechanism involves voltage-dependent calcium channels, phospholipase C, protein kinase C, and stored calcium contributing for GLP-1 secretion. In conclusion, the isoflavone regulates glycaemia by acting mainly in a serum target, the DPP-4 inhibitor. Furthermore, the long-term effect of isoflavone prevents protein glycation. J. Cell. Biochem. 118: 92-103, 2017. © 2016 Wiley Periodicals, Inc.

  2. Prenylated Polyphenols from Clusiaceae and Calophyllaceae with Immunomodulatory Activity on Endothelial Cells

    Science.gov (United States)

    Rouger, Caroline; Pagie, Sylvain; Derbré, Séverine; Le Ray, Anne-Marie; Richomme, Pascal; Charreau, Béatrice

    2016-01-01

    Endothelial cells (ECs) are key players in inflammation and immune responses involved in numerous pathologies. Although attempts were experimentally undertaken to prevent and control EC activation, drug leads and probes still remain necessary. Natural products (NPs) from Clusiaceous and Calophyllaceous plants were previously reported as potential candidates to prevent endothelial dysfunction. The present study aimed to identify more precisely the molecular scaffolds that could limit EC activation. Here, 13 polyphenols belonging to 5 different chemical types of secondary metabolites (i.e., mammea coumarins, a biflavonoid, a pyranochromanone acid, a polyprenylated polycyclic acylphloroglucinol (PPAP) and two xanthones) were tested on resting and cytokine-activated EC cultures. Quantitative and qualitative changes in the expression of both adhesion molecules (VCAM-1, ICAM-1, E-selectin) and major histocompatibility complex (MHC) molecules have been used to measure their pharmaceutical potential. As a result, we identified 3 mammea coumarins that efficiently reduce (up to >90% at 10 μM) both basal and cytokine-regulated levels of MHC class I, class II, MICA and HLA-E on EC surface. They also prevented VCAM-1 induction upon inflammation. From a structural point of view, our results associate the loss of the free prenyl group substituting mammea coumarins with a reduced cellular cytotoxicity but also an abrogation of their anti-inflammatory potential and a reduction of their immunosuppressive effects. A PPAP, guttiferone J, also triggers a strong immunomodulation but restricted to HLA-E and MHC class II molecules. In conclusion, mammea coumarins with a free prenyl group and the PPAP guttiferone J emerge as NPs able to drastically decrease both VCAM-1 and a set of MHC molecules and to potentially reduce the immunogenicity of the endothelium. PMID:27907087

  3. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially......Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been...... in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...

  4. Bacterial diterpene synthases: new opportunities for mechanistic enzymology and engineered biosynthesis.

    Science.gov (United States)

    Smanski, Michael J; Peterson, Ryan M; Huang, Sheng-Xiong; Shen, Ben

    2012-04-01

    Diterpenoid biosynthesis has been extensively studied in plants and fungi, yet cloning and engineering diterpenoid pathways in these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial diterpene synthases are poised to make significant contributions to our understanding of terpenoid biosynthesis. Here we will first survey diterpenoid natural products of bacterial origin and briefly review their biosynthesis with emphasis on diterpene synthases (DTSs) that channel geranylgeranyl diphosphate to various diterpenoid scaffolds. We will then highlight differences of DTSs of bacterial and higher organism origins and discuss the challenges in discovering novel bacterial DTSs. We will conclude by discussing new opportunities for DTS mechanistic enzymology and applications of bacterial DTS in biocatalysis and metabolic pathway engineering.

  5. Bacterial Diterpene Synthases: New Opportunities for Mechanistic Enzymology and Engineered Biosynthesis

    Science.gov (United States)

    Smanski, Michael J.; Peterson, Ryan M.; Huang, Sheng-Xiong; Shen, Ben

    2012-01-01

    Diterpenoid biosynthesis has been extensively studied in plants and fungi, yet cloning and engineering diterpenoid pathways in these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial diterpene synthases are poised to make significant contributions to our understanding of terpenoid biosynthesis. Here we will first survey diterpenoid natural products of bacterial origin and briefly review their biosynthesis with emphasis on diterpene synthases (DTSs) that channel geranylgeranyl diphosphate to various diterpenoid scaffolds. We will then highlight differences of DTSs of bacterial and higher organism origins and discuss the challenges in discovering novel bacterial DTSs. We will conclude by discussing new opportunities for DTS mechanistic enzymology and applications of bacterial DTS in biocatalysis and metabolic pathway engineering. PMID:22445175

  6. Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne; Larsen, Sine

    2000-01-01

    saturation with ribose 5-phosphate leads to the binding of Mg2+ and substrates via a slow pathway where Pi binds to the enzyme last. The random mechanism for Pi binding was further supported by studies with competitive inhibitors of Mg2+, MgATP, and ribose 5-phosphate that all appeared noncompetitive when...... varying Pi at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing Pi concentrations. Results from ADP inhibition of Pi activation suggest that these effectors compete for binding to a common regulatory site....

  7. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture

    Science.gov (United States)

    2016-01-01

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D80DQFD and N218DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H218O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-2H2]FDP and (R)-[1-2H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were

  8. Functional characterization of the Xanthophyllomyces dendrorhous farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoding genes that are involved in the synthesis of isoprenoid precursors.

    Directory of Open Access Journals (Sweden)

    Jennifer Alcaíno

    Full Text Available The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20 in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5 and dimethylallyl pyrophosphate (DMAPP, C5 was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15 synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10 from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to

  9. Functional characterization of the Xanthophyllomyces dendrorhous farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoding genes that are involved in the synthesis of isoprenoid precursors.

    Science.gov (United States)

    Alcaíno, Jennifer; Romero, Ignacio; Niklitschek, Mauricio; Sepúlveda, Dionisia; Rojas, María Cecilia; Baeza, Marcelo; Cifuentes, Víctor

    2014-01-01

    The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor

  10. Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis.

    Science.gov (United States)

    Meshalkina, L; Nilsson, U; Wikner, C; Kostikowa, T; Schneider, G

    1997-03-01

    The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site-directed mutagenesis. Replacement of E162, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild-type enzyme. The two mutant enzymes show, however, significant increases in the K0.5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves. This suggests that the interaction of E162 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer. Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency. The 25-80-fold increase in K0.5 for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site. The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.

  11. The position of prenylation of isoflavonoids and stilbenoids from legumes (Fabaceae) modulates the antimicrobial activity against Gram positive pathogens.

    Science.gov (United States)

    Araya-Cloutier, Carla; den Besten, Heidy M W; Aisyah, Siti; Gruppen, Harry; Vincken, Jean-Paul

    2017-07-01

    The legume plant family (Fabaceae) is a potential source of antimicrobial phytochemicals. Molecular diversity in phytochemicals of legume extracts was enhanced by germination and fungal elicitation of seven legume species, as established by RP-UHPLC-UV-MS. The relationship between phytochemical composition, including different types of skeletons and substitutions, and antibacterial properties of extracts was investigated. Extracts rich in prenylated isoflavonoids and stilbenoids showed potent antibacterial activity against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus at concentrations between 0.05 and 0.1% (w/v). Prenylated phenolic compounds were significantly (plegume seedlings can serve multiple purposes, e.g. as phytoestrogens they can provide health benefits and as natural antimicrobials they offer preservation of foods.

  12. Anti-respiratory syncytial virus prenylated dihydroquinolone derivatives from the gorgonian-derived fungus Aspergillus sp. XS-20090B15.

    Science.gov (United States)

    Chen, Min; Shao, Chang-Lun; Meng, Hong; She, Zhi-Gang; Wang, Chang-Yun

    2014-12-26

    Two new prenylated dihydroquinolone derivatives, 22-O-(N-Me-l-valyl)aflaquinolone B (1) and 22-O-(N-Me-l-valyl)-21-epi-aflaquinolone B (2), and two known analogues, aflaquinolones A (3) and D (or a diastereomer of D, 4), were isolated from the mycelia of a gorgonian-derived Aspergillus sp. fungus. The structures of the new compounds were elucidated by spectroscopic methods, ECD spectra, Marfey's method, and chemical conversion. Compounds 1 and 2 display an unusual esterification of N-Me-l-Val to the side-chain prenyl group. Compound 2 exhibited outstanding anti-RSV activity with an IC50 value of 42 nM, approximately 500-fold stronger than that of the positive control ribavirin (IC50 = 20 μM), and showed a comparatively higher therapeutic ratio (TC50/IC50 = 520).

  13. Monoterpene synthases of loblolly pine (Pinus taeda) produce pinene isomers and enantiomers.

    Science.gov (United States)

    Phillips, M A; Savage, T J; Croteau, R

    1999-12-01

    The turpentine fraction of conifer oleoresin is a complex mixture of monoterpene olefins and plays important roles in defense and in the mediation of chemical communication between conifer hosts and insect predators. The stereochemistry of the turpentine monoterpenes is critical in these interactions, influencing host recognition, toxicity, and potency of derived pheromones, and the stereochemical composition of these compounds lends insight into their biogenetic origin, with implications for the numbers and types of enzymes responsible and their corresponding genes. Analysis of the oleoresin from several tissues of loblolly pine (Pinus taeda) showed the derived turpentine to consist mainly of (+)-(3R:5R)-alpha-pinene and (-)-(3S:5S)-beta-pinene. Cell-free extracts from xylem tissue yielded three monoterpene synthases which together account for the monoterpene isomer and enantiomer content of the turpentine of this tissue. The major products of these enzymes, produced from the universal precursor of monoterpenes, geranyl diphosphate, were shown to be (+)-alpha-pinene, (-)-alpha-pinene, and (-)-beta-pinene, respectively. In most properties (molecular mass of approximately 60 kDa, K(m) for geranyl diphosphate of 3 microM, requirement for monovalent and divalent cations), these enzymes resemble other monoterpene synthases from conifer species.

  14. Substrate geometry controls the cyclization cascade in multiproduct terpene synthases from Zea mays.

    Science.gov (United States)

    Vattekkatte, Abith; Gatto, Nathalie; Köllner, Tobias G; Degenhardt, Jörg; Gershenzon, Jonathan; Boland, Wilhelm

    2015-06-07

    Multiproduct terpene synthases TPS4-B73 and TPS5-Delprim from maize (Zea mays) catalyze the conversion of farnesyl diphosphate (FDP) and geranyl diphosphate (GDP) into a complex mixture of sesquiterpenes and monoterpenes, respectively. Various isotopic and geometric isomers of natural substrates like (2Z)-[2-(2)H]- and [2,4,4,9,9,9-(2)H6]-(GDP) and (2Z,6E)-[2-(2)H]- and [2,4,4,13,13,13-(2)H6]-(FDP) were synthesized analogous to presumptive reaction intermediates. On incubation with labeled (2Z) substrates, TPS4 and TPS5 showed much lower kinetic isotope effects than the labeled (2E) substrates. Interestingly, the products arising from the deuterated (2Z)-precursors revealed a distinct preference for cyclic products and exhibited an enhanced turnover on comparison with natural (2E)-substrates. This increase in the efficiency due to (2Z) configuration emphasizes the rate limiting effect of the initial (2E) → (2Z) isomerization step in the reaction cascade of the multiproduct terpene synthases. Apart from turnover advantages, these results suggest that substrate geometry can be used as a tool to optimize the biosynthetic reaction cascade towards valuable cyclic terpenoids.

  15. Characterization of a bifunctional enzyme with (p)ppGpp-hydrolase/synthase activity in Leptospira interrogans.

    Science.gov (United States)

    He, Ping; Deng, Cong; Liu, Boyu; Zeng, LingBing; Zhao, Wei; Zhang, Yan; Jiang, XuCheng; Guo, XiaoKui; Qin, JinHong

    2013-11-01

    Alarmone Guanosine 5'-diphosphate (or 5'-triphosphate) 3'-diphosphate [(p)ppGpp] is the key component that globally regulates stringent control in bacteria. There are two homologous enzymes, RelA and SpoT in Escherichia coli, which are responsible for fluctuations in (p)ppGpp concentration inside the cell, whereas there exists only a single RelA/SpoT enzyme in Gram-positive bacteria. We have identified a bifunctional enzyme with (p)ppGpp-hydrolase/synthase activity in Leptospira interrogans. We show that the relLin gene (LA_3085) encodes a protein that fully complements the relA/spoT double mutants in E. coli. The protein functions as a (p)ppGpp degradase as well as a (p)ppGpp synthase when the cells encounter amino acid stress and deprivation of carbon sources. N-terminus HD and RSD domains of relLin (relLinN ) were observed to restore growth of double mutants of E. coli. Finally, We demonstrate that purified RelLin and RelLinN show high (p)ppGpp synthesis activity in vitro. Taken together, our results suggest that L. interrogans contain a single Rel-like bifunctional protein, RelLin , which plays an important role in maintaining the basal level of (p)ppGpp in the cell potentially contributing to the regulation of bacterial stress response.

  16. Xanthohumol, a Prenylated Chalcone from Hops, Inhibits the Viability and Stemness of Doxorubicin-Resistant MCF-7/ADR Cells

    OpenAIRE

    Ming Liu; Hua Yin; Xiaokun Qian; Jianjun Dong; Zhonghua Qian; Jinlai Miao

    2016-01-01

    Xanthohumol is a unique prenylated flavonoid in hops (Humulus lupulus L.) and beer. Xanthohumol has been shown to possess a variety of pharmacological activities. There is little research on its effect on doxorubicin-resistant breast cancer cells (MCF-7/ADR) and the cancer stem-like cells exiting in this cell line. In the present study, we investigate the effect of xanthohumol on the viability and stemness of MCF-7/ADR cells. Xanthohumol inhibits viability, induces apoptosis, and arrests the ...

  17. 2-Methyl-3-buten-2-ol (MBO) synthase expression in Nostoc punctiforme leads to over production of phytols.

    Science.gov (United States)

    Gupta, Dinesh; Ip, Tina; Summers, Michael L; Basu, Chhandak

    2015-01-01

    Phytol is a diterpene alcohol of medicinal importance and it also has potential to be used as biofuel. We found over production of phytol in Nostoc punctiforme by expressing a 2-Methyl-3-buten-2-ol (MBO) synthase gene. MBO synthase catalyzes the conversion of dimethylallyl pyrophosphate (DMAPP) into MBO, a volatile hemiterpene alcohol, in Pinus sabiniana. The result of enhanced phytol production in N. punctiforme, instead of MBO, could be explained by one of the 2 models: either the presence of a native prenyltransferase enzyme with a broad substrate specificity, or appropriation of a MBO synthase metabolic intermediate by a native geranyl diphosphate (GDP) synthase. In this work, an expression vector with an indigenous petE promoter for gene expression in the cyanobacterium N. punctiforme was constructed and MBO synthase gene expression was successfully shown using reverse transcriptase (RT)-PCR and SDS-PAGE. Gas chromatography--mass spectrophotometry (GC-MS) was performed to confirm phytol production from the transgenic N. punctiforme strains. We conclude that the expression of MBO synthase in N. punctiforme leads to overproduction of an economically important compound, phytol. This study provides insights about metabolic channeling of isoprenoids in cyanobacteria and also illustrates the challenges of bioengineering non-native hosts to produce economically important compounds.

  18. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  19. 3-Hydroxymethyl coenzyme A reductase inhibition attenuates spontaneous smooth muscle tone via RhoA/ROCK pathway regulated by RhoA prenylation.

    Science.gov (United States)

    Rattan, Satish

    2010-06-01

    RhoA prenylation may play an important step in the translocation of RhoA in the basal internal anal sphincter (IAS) smooth muscle tone. Statins inhibit downstream posttranslational RhoA prenylation by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition (HMGCRI). The role of statins in relation to RhoA prenylation in the pathophysiology of the spontaneously tonic smooth muscle has not been investigated. In the present studies, we determined the effect of classical HMGCRI simvastatin on the basal IAS tone and RhoA prenylation and in the levels of RhoA/Rho kinase (ROCK) in the cytosolic vs. membrane fractions of the smooth muscle. Simvastatin produced concentration-dependent decrease in the IAS tone (via direct actions at the smooth muscle cells). The decrease in the IAS tone by simvastatin was associated with the decrease in the prenylation of RhoA, as well as RhoA/ROCK in the membrane fractions of the IAS, in the basal state. The inhibitory effects of the HMGCRI were completely reversible by geranylgeranyltransferase substrate geranylgeranyl pyrophosphate. Relaxation of the IAS smooth muscle via HMGCRI simvastatin is mediated via the downstream decrease in the levels of RhoA prenylation and ROCK activity. Studies support the concept that RhoA prenylation leading to RhoA/ROCK translocation followed by activation is important for the basal tone in the IAS. Data suggest that the role of HMG-CoA reductase may go beyond cholesterol biosynthesis, such as the regulation of the smooth muscle tone. The studies have important implications in the pathophysiological mechanisms and in the novel therapeutic approaches for anorectal motility disorders.

  20. Bioconversion of lactose/whey to fructose diphosphate with recombinant Saccharomyces cerevisiae cells

    Energy Technology Data Exchange (ETDEWEB)

    Compagno, C.; Tura, A.; Ranzi, B.M.; Martegani, E. (Univ. di Milano (Italy))

    1993-07-01

    Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli [beta]-galactosidase gene are able to bioconvert lactose or whey into fructose-1,6-diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. The authors showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate.

  1. Fruit color mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis

    Science.gov (United States)

    Isoprenoids are a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, IPP is synthesized in the cytoplasm from mevalonic acid via the “MVA pathway” a...

  2. Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare

    DEFF Research Database (Denmark)

    Zerbe, Philipp; Chiang, Angela; Dullat, Harpreet

    2014-01-01

    Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five...... (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce...

  3. Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus), Prevents Platelet Activation in Human Platelets.

    Science.gov (United States)

    Lee, Ye-Ming; Hsieh, Kuo-Hsien; Lu, Wan-Jung; Chou, Hsiu-Chu; Chou, Duen-Suey; Lien, Li-Ming; Sheu, Joen-Rong; Lin, Kuan-Hung

    2012-01-01

    Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.). Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca(2+)](i) mobilization, thromboxane A(2) formation, hydroxyl radical (OH(●)) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A(2) formation, thereby leading to inhibition of [Ca(2+)](i) and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

  4. Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus, Prevents Platelet Activation in Human Platelets

    Directory of Open Access Journals (Sweden)

    Ye-Ming Lee

    2012-01-01

    Full Text Available Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.. Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH● formation, and phospholipase C (PLCγ2, protein kinase C (PKC, mitogen-activated protein kinase (MAPK, and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

  5. Antiinvasive effect of xanthohumol, a prenylated chalcone present in hops (Humulus lupulus L.) and beer.

    Science.gov (United States)

    Vanhoecke, Barbara; Derycke, Lara; Van Marck, Veerle; Depypere, Herman; De Keukeleire, Denis; Bracke, Marc

    2005-12-20

    The female inflorescences of the hop plant (Humulus lupulus L.) are essential during brewing to add taste and flavor to beer and to stabilize beer foam. Xanthohumol, the main prenylated chalcone in hops, was investigated for its antiinvasive activity on human breast cancer cell lines (MCF-7 and T47-D) in vitro. Xanthohumol was able to inhibit the invasion of MCF-7/6 cells at 5 microM in the chick heart invasion assay and of T47-D cells in the collagen invasion assay. Xanthohumol inhibited growth of MCF-7/6 and T47-D cells, but not of chick heart cells. Moreover, it induced apoptosis of these tumor cells as demonstrated by the cleavage of nuclear PARP after 48 hr treatment. To probe the mechanism of the antiinvasive effect of xanthohumol, involvement of the E-cadherin/catenin invasion-suppressor complex was investigated. An aggregation assay demonstrated stimulation of aggregation of MCF-7/6 cells in the presence of 5 microM xanthohumol and this could be completely inhibited by an antibody against E-cadherin. Xanthohumol upregulates the function of the E-cadherin/catenin complex and inhibits invasion in vitro, indicating a possible role as an antiinvasive agent in vivo as well.

  6. O-prenylated acridone alkaloids from the stems of Balsamocitrus paniculata (Rutaceae).

    Science.gov (United States)

    Happi, Emmanuel Ngeufa; Waffo, Alain François; Wansi, Jean Duplex; Ngadjui, Bonaventure Tchaleu; Sewald, Norbert

    2011-06-01

    Two new O-prenylated acridone alkaloids, balsacridone A (1) and B (2), together with eighteen known compounds were isolated from the methanol extract from the stems of Balsamocitrus paniculata, a Cameroonian medicinal plant. The structures of all compounds were determined by comprehensive analyses of their 1D and 2D NMR, mass spectral (EI and ESI) data, and chemical reactions. N-methyl-6-methoxybenzoxazolinone (16) was isolated for the first time from a natural source while compounds 13, 14, and 15 for the first time from this genus. Pure compounds were tested for their activity against bacteria, fungi, and plant pathogen oomycetes, using the paper disk agar diffusion assay. The agar diffusion test delivered low to missing antimicrobial activities, corresponding to MICs > 1 mg/mL. However, compounds 1-15 exhibited a strong suppressive effect on phagocytosis response upon activation with serum opsonized zymosan in the range of IC50 = 0.5-7.2 μM, and the acridone alkaloids (1-5), N-trans-p-coumaroyltyramine (13), and N-trans-pcoumaroyloctopamine (14) displayed weak cytotoxic activity against the human Caucasian prostate adenocarcinoma cell line PC-3, with IC₅₀ values ranging from 69.8 to 99.0 μM.

  7. Prenylated flavonoids from Artocarpus altilis: antioxidant activities and inhibitory effects on melanin production.

    Science.gov (United States)

    Lan, Wen-Chun; Tzeng, Cheng-Wei; Lin, Chun-Ching; Yen, Feng-Lin; Ko, Horng-Huey

    2013-05-01

    Flavonoids, 10-oxoartogomezianone (1), 8-geranyl-3-(hydroxyprenyl)isoetin (2), hydroxyartoflavone A (3), isocycloartobiloxanthone (4), and furanocyclocommunin (5), together with 12 known compounds, were isolated from heartwood and cortex of Artocarpus altilis, and their structures were identified by comparing their spectra with those of similar compounds. To identify natural antioxidants and whitening agents, the ability of these prenylated flavonoids was assessed to scavenge the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(+·)) radical cation, and the superoxide anion (O2(-·)), and their abilities to inhibit tyrosinase and melanin production. It was found that compounds 3, 4, and artoflavone A (15) had moderate DPPH(·)-scavenging activity, whereas compound 4 exhibited significant ABTS(+·)-scavenging activity, and that norartocarpetin (7) and artogomezianone (8) exhibited moderate ABTS(+·)-scavenging activity, with compounds 2, 7, and artocarpin (6) displaying good superoxide anion-scavenging activity. In addition, compounds 7, 8, cudraflavone A (14), and artonin M (17), inhibited melanin production by strongly suppressing tyrosinase activity. Compound 6 reduced the melanin content without inhibiting tyrosinase activity. These results suggest that flavonoids isolated from A. altilis may be candidate antioxidants and/or skin-whitening agents. However, further investigations are required to determine their mechanisms of action. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Prenyl Ammonium Salts - New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model.

    Directory of Open Access Journals (Sweden)

    Emilia Grecka

    Full Text Available Prenyl ammonium iodides (Amino-Prenols, APs, semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents.AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells.All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation-considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2, introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination.Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.

  9. Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro

    DEFF Research Database (Denmark)

    Engel, M; Issinger, O G; Lascu, I;

    1994-01-01

    We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia...

  10. ACTIVATION OF G-PROTEINS BY RECEPTOR-STIMULATED NUCLEOSIDE DIPHOSPHATE KINASE IN DICTYOSTELIUM

    NARCIS (Netherlands)

    Bominaar, Anthony A.; Molijn, Anco C.; Pestel, Martine; Veron, Michel; Haastert, Peter J.M. van

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC 2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase

  11. The effect of ticlopidine administration to humans on the binding of adenosine diphosphate to blood platelets

    NARCIS (Netherlands)

    Lips, J.P.M.; Sixma, J.J.; Schiphorst, M.E.

    1980-01-01

    Administration of Ticlopidine to human volunteers resulted in a prolonged bleeding time and decreased or absent aggregation of platelets with collagen and epinephrine. Adenosine diphosphate (ADP) induced platelet aggregation was initiated by a normal shape change, but the rate of the first wave of a

  12. Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Georgescauld, Florian; Moynie, Lucile; Habersetzer, Johann;

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic acti...

  13. Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Arnvig, Kirsten; Hove-Jensen, Bjarne; Switzer, Robert L.

    1990-01-01

    Phosphoribosyl-diphosphate (PPRibP) synthetase from Bacillus subtiliis has been purified to near homogeneity from an Escherichia coli Δprs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthentase, on a plasmid. The Mr of the subunit (34,000) and its amino-terminal amino acid...

  14. METOVITAN PREVENTS THE ACCUMULATION OF THIAMINE DIPHOSPHATE OXIDIZED FORM IN RAT TISSUES UNDER IRRADIATION

    Directory of Open Access Journals (Sweden)

    Parkhomenko Yu. M.

    2015-08-01

    Full Text Available The aim of the research was to test the ability of the drug "Metovitan" to prevent the redox balance disturbance in the tissues and thiamine diphosphate irreversible oxidation upon exposure of ionizing radiation on the body. The rats were subjected to a single exposure of the X-ray therapeutic instrument RUM-17 to create a dose of 0.5, 1.0 and 5.0 Gray. Preparation "Metovitan" was administered at a dose of 25 mg per 1 kg body weight for 22–24 h before irradiation. Contents of thiamine diphosphate, reduced SH-groups and reactive oxygen species in blood and brain were determined using previously described methods. It has been shown that the active form of the thiamine diphosphate content in the blood was decreased depending on the doses (from 0.5 to 5.0 Gray. At the same time the content of thiamine diphosphate oxidized form was increased. Furthermore the critical changes occurred in metabolic processes redox state parameters, namely, the level of free SH-groups was reduced and the level of reactive oxygen species was increased. Similar changes were observed in the brain tissue. The Metovitan single administration to the animals the day before irradiation, at 25 mg per 1 kg of body weight dose, promotes the protection of the intracellular thiamine diphosphate and redox status in animal tissues (blood, brain tissue from the negative influence of irradiation (at 0.5; 1.0 Gray doses. These results give reason to recommend the drug for treating of the staff that is involved in the elimination of radioactive contamination. To provide the protection from the higher doses, other scheme of the treatment should be probably recommended.

  15. Characterization of an Isopentenyl Diphosphate Isomerase involved in the Juvenile Hormone pathway in Aedes aegypti

    Science.gov (United States)

    Diaz, Miguel; Mayoral, Jaime G.; Priestap, Horacio; Nouzova, Marcela; Rivera-Perez, Crisalejandra; Noriega, Fernando G.

    2012-01-01

    Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterwards IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg2+ or Mn2+ but not Zn2+ for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis (Li et al., 2003). This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect. PMID:22782071

  16. A sedge plant as the source of Kangaroo Island propolis rich in prenylated p-coumarate ester and stilbenes.

    Science.gov (United States)

    Duke, Colin C; Tran, Van H; Duke, Rujee K; Abu-Mellal, Abdallah; Plunkett, George T; King, Douglas I; Hamid, Kaiser; Wilson, Karen L; Barrett, Russell L; Bruhl, Jeremy J

    2017-02-01

    Propolis samples from Kangaroo Island, South Australia, were investigated for chemical constituents using high-field nuclear magnetic resonance spectral profiling. A type of propolis was found containing a high proportion of prenylated hydroxystilbenes. Subsequently, the botanical origin of this type of propolis was identified using a beehive propolis depletion method and analysis of flora. Ligurian honey bees, Apis mellifera ligustica Spinola, were found to produce propolis from resin exuded by the Australian native sedge plant Lepidosperma sp. Montebello (Cyperaceae). The plants, commonly known as sword sedge, were found to have resin that matched with the propolis samples identified as the most abundant propolis type on the island containing C- and O-prenylated tetrahydroxystilbenes (pTHOS) in addition to a small amount of prenylated p-coumarate. The isolation of five pTHOS not previously characterized are reported: (E)-4-(3-methyl-2-buten-1-yl)-3,4',5-trihydroxy-3'-methoxystilbene, (E)-2,4-bis(3-methyl-2-buten-1-yl)-3,3',4',5-tetrahydroxystilbene, (E)-2-(3-methyl-2-buten-1-yl)-3-(3-methyl-2-butenyloxy)-3',4',5-trihydroxystilbene, (E)-2,6-bis(3-methyl-2-buten-1-yl)-3,3',5,5'-tetrahydroxystilbene and (E)-2,6-bis(3-methyl-2-buten-1-yl)-3,4',5-trihydroxy-3'-methoxystilbene. A National Cancer Institute 60 human cell line anticancer screen of three of these compounds showed growth inhibitory activity. The large Australasian genus Lepidosperma is identified as a valuable resource for the isolation of substances with medicinal potential. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Prenylated Indolediketopiperazine Peroxides and Related Homologues from the Marine Sediment-Derived Fungus Penicillium brefeldianum SD-273

    Directory of Open Access Journals (Sweden)

    Chun-Yan An

    2014-01-01

    Full Text Available Three new indolediketopiperazine peroxides, namely, 24-hydroxyverruculogen (1, 26-hydroxyverruculogen (2, and 13-O-prenyl-26-hydroxyverruculogen (3, along with four known homologues (4–7, were isolated and identified from the culture extract of the marine sediment-derived fungus Penicillium brefeldianum SD-273. Their structures were determined based on the extensive spectroscopic analysis and compound 1 was confirmed by X-ray crystallographic analysis. The absolute configuration of compounds 1–3 was determined using chiral HPLC analysis of their acidic hydrolysates. Each of the isolated compounds was evaluated for antibacterial and cytotoxic activity as well as brine shrimp (Artemia salina lethality.

  18. Synthesis and evaluation of novel prenylated chalcone derivatives as anti-leishmanial and anti-trypanosomal compounds.

    Science.gov (United States)

    Passalacqua, Thais Gaban; Dutra, Luiz Antonio; de Almeida, Letícia; Velásquez, Angela Maria Arenas; Torres, Fabio Aurelio Esteves; Yamasaki, Paulo Renato; dos Santos, Mariana Bastos; Regasini, Luis Octavio; Michels, Paul A M; Bolzani, Vanderlan da Silva; Graminha, Marcia A S

    2015-08-15

    Chalcones form a class of compounds that belong to the flavonoid family and are widely distributed in plants. Their simple structure and the ease of preparation make chalcones attractive scaffolds for the synthesis of a large number of derivatives enabling the evaluation of the effects of different functional groups on biological activities. In this Letter, we report the successful synthesis of a series of novel prenylated chalcones via Claisen-Schmidt condensation and the evaluation of their effect on the viability of the Trypanosomatidae parasites Leishmania amazonensis, Leishmania infantum and Trypanosoma cruzi.

  19. Reactions of fac-[Re(CO)3(H2O)3]+ with nucleoside diphosphates and thiamine diphosphate in aqueous solution investigated by multinuclear NMR spectroscopy.

    Science.gov (United States)

    Adams, Kristie M; Marzilli, Patricia A; Marzilli, Luigi G

    2007-10-29

    Products formed between monoester diphosphates (MDPs) and fac-[Re(CO)3(H2O)3]OTf at pH 3.6 were examined. Such adducts of the fac-[Re(CO)3]+ moiety have an uncommon combination of properties for an "inert" metal center in that sharp NMR signals can be observed, yet the products are equilibrating at rates allowing NMR EXSY cross-peaks to be observed. Thiamine diphosphate (TDP) and uridine 5'-diphosphate (5'-UDP) form 1:1 bidentate {Palpha,Pbeta} chelates, in which the MDP binds Re(I) via Palpha and Pbeta phosphate groups. Asymmetric centers are created at Re(I) (RRe/SRe) and Palpha (Delta/Lambda), leading to four diastereomers. The two mirror pairs of diastereomers (RReDelta/SReLambda) and (RReLambda/SReDelta) for TDP (no ribose) and for all four diastereomers (RReDelta, RReLambda, SReDelta, SReLambda) for 5'-UDP (asymmetric ribose) gave two and four sets of NMR signals for the bound MDP, respectively. 31Palpha-31Palpha EXSY cross-peaks indicate that the fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- isomers interchange slowly on the NMR time scale, with an average k approximately equal to 0.8 s(-1) at 32 degrees C; the EXSY cross-peaks could arise from chirality changes at only Re(I) or at only Palpha. Guanosine 5'-diphosphate (5'-GDP), with a ribose moiety and a Re(I)-binding base, formed both possible diastereomers (RRe and SRe) of the fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- macrochelate, with one slightly more abundant diastereomer suggested to be RRe by Mn2+ ion 1H NMR signal line-broadening combined with distances from molecular models. Interchange of the diastereomers requires that the coordination site of either N7 or Pbeta move to the H2O site. 31Palpha-31Palpha EXSY cross-peaks indicate a k approximately equal to 0.5 s(-1) at 32 degrees C for RRe-to-SRe interchange. The similarity of the rate constants for interchange of fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- and fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- adducts suggest strongly that interchange of Pbeta and H2O coordination

  20. Taxadiene Synthase Structure and Evolution of Modular Architecture in Terpene Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    M Köksal; Y Jin; R Coates; R Croteau; D Christianson

    2011-12-31

    With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization and shows remarkable efficacy in cancer chemotherapy. The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia) is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene, which is catalysed by taxadiene synthase. The full-length form of this diterpene cyclase contains 862 residues, but a roughly 80-residue amino-terminal transit sequence is cleaved on maturation in plastids. We now report the X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N terminus, hereafter designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (1.82 {angstrom} resolution) and 2-fluorogeranylgeranyl diphosphate (2.25 {angstrom} resolution). The TXS structure reveals a modular assembly of three {alpha}-helical domains. The carboxy-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third 'insertion' domain together adopt the fold of a vestigial class II terpenoid cyclase. A class II cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TXS structure reveals a definitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis.

  1. New insight into the catalytic properties of rice sucrose synthase.

    Science.gov (United States)

    Huang, Yu-Chiao; Hsiang, Erh-Chieh; Yang, Chien-Chih; Wang, Ai-Yu

    2016-01-01

    Sucrose synthase (SuS), which catalyzes the reversible conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose, is a key enzyme in sucrose metabolism in higher plants. SuS belongs to family 4 of the glycosyltransferases (GT4) and contains an E-X7-E motif that is conserved in members of GT4 and two other GT families. To gain insight into the roles of this motif in rice sucrose synthase 3 (RSuS3), the two conserved glutamate residues (E678 and E686) in this motif and a phenylalanine residue (F680) that resides between the two glutamate residues were changed by site-directed mutagenesis. All mutant proteins maintained their tetrameric conformation. The mutants E686D and F680Y retained partial enzymatic activity and the mutants E678D, E678Q, F680S, and E686Q were inactive. Substrate binding assays indicated that UDP and fructose, respectively, were the leading substrates in the sucrose degradation and synthesis reactions of RSuS3. Mutations on E678, F680, and E686 affected the binding of fructose, but not of UDP. The results indicated that E678, F680, and E686 in the E-X7-E motif of RSuS3 are essential for the activity of the enzyme and the sequential binding of substrates. The sequential binding of the substrates implied that the reaction catalyzed by RSuS can be controlled by the availability of fructose and UDP, depending on the metabolic status of a tissue.

  2. SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides.

    Science.gov (United States)

    Hartwig, S; Frister, T; Alemdar, S; Li, Z; Scheper, T; Beutel, S

    2015-03-20

    An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L(-1) were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni(2+)-IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg(2+) containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC-MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis-Menten model, kinetic parameters of KM = 1.111 μM (±0.113), vmax = 0.3245 μM min(-1) (±0.0035), kcat = 2.95 min(-1), as well as a catalytic efficiency kcat/KM = 4.43 × 10(4) M(-1)s(-1) were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Influence of gibberellin and daminozide on the expression of terpene synthases and on monoterpenes in common sage (Salvia officinalis).

    Science.gov (United States)

    Schmiderer, Corinna; Grausgruber-Gröger, Sabine; Grassi, Paolo; Steinborn, Ralf; Novak, Johannes

    2010-07-01

    Common sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants, with antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, composed mainly of the monoterpenes 1,8-cineole, alpha-thujone, beta-thujone and camphor, is responsible for some of these effects. Gibberellins regulate diverse physiological processes in plants, such as seed germination, shoot elongation and cell division. In this study, we analyzed the effect of exogenously applied plant growth regulators, namely gibberellic acid (GA(3)) and daminozide, on leaf morphology and essential oil formation of two leaf stages during the period of leaf expansion. Essential oil content increased with increasing levels of gibberellins and decreased when gibberellin biosynthesis was blocked with daminozide. With increasing levels of gibberellins, 1,8-cineole and camphor contents increased. Daminozide blocked the accumulation of alpha- and beta-thujone. GA(3) at the highest level applied also led to a significant decrease of alpha- and beta-thujone. Monoterpene synthases are a class of enzymes responsible for the first step in monoterpene biosynthesis, competing for the same substrate geranylpyrophosphate. The levels of gene expression of the three most important monoterpene synthases in sage were investigated, 1,8-cineole synthase leading directly to 1,8-cineole, (+)-sabinene synthase responsible for the first step in the formation of alpha- and beta-thujone, and (+)-bornyl diphosphate synthase, the first step in camphor biosynthesis. The foliar application of GA(3) increased, while daminozide significantly decreased gene expression of the monoterpene synthases. The amounts of two of the end products, 1,8-cineole and camphor, were directly correlated with the levels of gene expression of the respective monoterpene synthases, indicating transcriptional control, while the formation of alpha- and beta

  4. Five new prenylated p-hydroxybenzoic acid derivatives with antimicrobial and molluscicidal activity from Piper aduncum leaves.

    Science.gov (United States)

    Orjala, J; Erdelmeier, C A; Wright, A D; Rali, T; Sticher, O

    1993-12-01

    Five new prenylated benzoic acid derivatives, methyl 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxybenzoate (1), 1-(1-methylethyl)-4-methyl-3-cyclohexenyl 3,5-bis(3-methyl-2-butenyl)-4-hydroxybenzoate (2), 1-(1-methylethyl)-4-methyl-3-cyclohexenyl 3,5-bis(3-methyl-2-butenyl)-4-methoxybenzoate (3), methyl 3,5-bis(3-methyl-2-butenyl)-4-methoxybenzoate (4), and 4-hydroxy-3-(3-methyl-2-butenyl)-5-(3-methyl-2-butenyl)-benzoic acid (5) were isolated from the dried leaves of Piper aduncum L. (Piperaceae). Together with the new metabolites, four known prenylated benzoic acid derivatives, 3,5-bis(3-methyl-2-butenyl)-4-methoxybenzoic acid (6), 4-hydroxy-3,5-bis(3-methyl-2-butenyl)-benzoic acid (nervogenic acid, 7), methyl 4-hydroxy-3,5-bis(3-methyl-2-butenyl)-benzoate (8), and methyl 4-hydroxy-3-(3-methyl-2-butenyl)-benzoate (9) as well as, dillapiol (10), myristicin, and the three sesquiterpenes humulene, caryophyllene epoxide, and humulene epoxide were isolated. Compounds 7, 8, and 9 are reported as natural products for the first time. The structures of the isolates were elucidated by spectroscopic methods, mainly 1D-and 2D-NMR spectroscopy. Isolates 4-7, 9, and 10 were molluscicidal while 2, 5-7, and 9 displayed significant antibacterial activities.

  5. Prenylated Chalcone 2 Acts as an Antimitotic Agent and Enhances the Chemosensitivity of Tumor Cells to Paclitaxel

    Directory of Open Access Journals (Sweden)

    Joana Fonseca

    2016-07-01

    Full Text Available We previously reported that prenylated chalcone 2 (PC2, the O-prenyl derivative (2 of 2′-hydroxy-3,4,4′,5,6′-pentamethoxychalcone (1, induced cytotoxicity of tumor cells via disruption of p53-MDM2 interaction. However, the cellular changes through which PC2 exerts its cytotoxic activity and its antitumor potential, remain to be addressed. In the present work, we aimed to (i characterize the effect of PC2 on mitotic progression and the underlying mechanism; and to (ii explore this information to evaluate its ability to sensitize tumor cells to paclitaxel in a combination regimen. PC2 was able to arrest breast adenocarcinoma MCF-7 and non-small cell lung cancer NCI-H460 cells in mitosis. All mitosis-arrested cells showed collapsed mitotic spindles with randomly distributed chromosomes, and activated spindle assembly checkpoint. Live-cell imaging revealed that the compound induced a prolonged delay (up to 14 h in mitosis, culminating in massive cell death by blebbing. Importantly, PC2 in combination with paclitaxel enhanced the effect on cell growth inhibition as determined by cell viability and proliferation assays. Our findings demonstrate that the cytotoxicity induced by PC2 is mediated through antimitotic activity as a result of mitotic spindle damage. The enhancement effects of PC2 on chemosensitivity of cancer cells to paclitaxel encourage further validation of the clinical potential of this combination.

  6. Bioactivity of natural O-prenylated phenylpropenes from Illicium anisatum leaves and their derivatives against spider mites and fungal pathogens.

    Science.gov (United States)

    Koeduka, T; Sugimoto, K; Watanabe, B; Someya, N; Kawanishi, D; Gotoh, T; Ozawa, R; Takabayashi, J; Matsui, K; Hiratake, J

    2014-03-01

    A variety of volatile phenylpropenes, C6-C3 compounds are widely distributed in the plant kingdom, whereas prenylated phenylpropenes are limited to a few plant species. In this study, we analysed the volatile profiles from Illicium anisatum leaves and identified two O-prenylated phenylpropenes, 4-allyl-2-methoxy-1-[(3-methylbut-2-en-1-yl)oxy]benzene [O-dimethylallyleugenol (9)] and 5-allyl-1,3-dimethoxy-2-(3-methylbut-2-en-1-yl)oxy]benzene [O-dimethylallyl-6-methoxyeugenol (11)] as major constituents. The structure-activity relationship of a series of eugenol derivatives showed that specific phenylpropenes, including eugenol (1), isoeugenol (2) and 6-methoxyeugenol (6), with a phenolic hydroxy group had antifungal activity for a fungal pathogen, whereas guaiacol, a simple phenolic compound, and allylbenzene had no such activity. The eugenol derivatives that exhibited antifungal activity, in turn, had no significant toxicant property for mite oviposition. Interestingly, O-dimethylallyleugenol (9) in which the phenolic oxygen was masked with a dimethylallyl group exhibited a specific, potent oviposition deterrent activity for mites. The sharp contrast in structural requirements of phenylpropenes suggested distinct mechanisms underlying the two biological activities and the importance of a phenolic hydroxy group and its dimethylallylation for the structure-based design of new functional properties of phenylpropenes.

  7. Prenylated Chalcone 2 Acts as an Antimitotic Agent and Enhances the Chemosensitivity of Tumor Cells to Paclitaxel.

    Science.gov (United States)

    Fonseca, Joana; Marques, Sandra; Silva, Patrícia M A; Brandão, Pedro; Cidade, Honorina; Pinto, Madalena M; Bousbaa, Hassan

    2016-07-29

    We previously reported that prenylated chalcone 2 (PC2), the O-prenyl derivative (2) of 2'-hydroxy-3,4,4',5,6'-pentamethoxychalcone (1), induced cytotoxicity of tumor cells via disruption of p53-MDM2 interaction. However, the cellular changes through which PC2 exerts its cytotoxic activity and its antitumor potential, remain to be addressed. In the present work, we aimed to (i) characterize the effect of PC2 on mitotic progression and the underlying mechanism; and to (ii) explore this information to evaluate its ability to sensitize tumor cells to paclitaxel in a combination regimen. PC2 was able to arrest breast adenocarcinoma MCF-7 and non-small cell lung cancer NCI-H460 cells in mitosis. All mitosis-arrested cells showed collapsed mitotic spindles with randomly distributed chromosomes, and activated spindle assembly checkpoint. Live-cell imaging revealed that the compound induced a prolonged delay (up to 14 h) in mitosis, culminating in massive cell death by blebbing. Importantly, PC2 in combination with paclitaxel enhanced the effect on cell growth inhibition as determined by cell viability and proliferation assays. Our findings demonstrate that the cytotoxicity induced by PC2 is mediated through antimitotic activity as a result of mitotic spindle damage. The enhancement effects of PC2 on chemosensitivity of cancer cells to paclitaxel encourage further validation of the clinical potential of this combination.

  8. Synthesis and anti-cancer activity evaluation of novel prenylated and geranylated chalcone natural products and their analogs.

    Science.gov (United States)

    Wang, Hao-Meng; Zhang, Li; Liu, Jiang; Yang, Zhao-Liang; Zhao, Hong-Ye; Yang, Yao; Shen, Di; Lu, Kui; Fan, Zhen-Chuan; Yao, Qing-Wei; Zhang, Yong-Min; Teng, Yu-Ou; Peng, Yu

    2015-03-06

    Four natural chalcones bearing prenyl or geranyl groups, i.e., bavachalcone (1a), xanthoangelol (1b), isobavachalcone (1c), and isoxanthoangelol (1d) were synthesized by using a regio-selective iodination and the Suzuki coupling reaction as key steps. The first total synthesis of isoxanthoangelol (1d) was achieved in 36% overall yield. A series of diprenylated and digeranylated chalcone analogs were also synthesized by alkylation, regio-selective iodination, aldol condensation, Suzuki coupling and [1,3]-sigmatropic rearrangement. The structures of the 11 new derivatives were confirmed by (1)H NMR, (13)C NMR and HRMS. The anticancer activity of these new chalcone derivatives against human tumor cell line K562 were evaluated by MTT assay in vitro. SAR studies suggested that the 5'-prenylation/geranylation of the chalcones significantly enhance their cytotoxic activity. Among them, Bavachalcone (1a) displayed the most potent cytotoxic activity against K562 with IC50 value of 2.7 μM. The morphology changes and annexin-V/PI staining studies suggested that those chalcone derivatives inhibited the proliferation of K562 cells by inducing apoptosis.

  9. A New Method of Crystallization of Octahydro Trisodium Salt of Fructose-1,6-diphosphate

    Institute of Scientific and Technical Information of China (English)

    应汉杰; 欧阳平凯

    2002-01-01

    In order to overcome the elementary heterogeneous nucleation while octahydro trisodium salt of fructose-1,6-diphosphate(FDPNaa.8 H2O) is crystallized with ethanol precipitation at low temperature, a new crystallizationmethod with alcohol precipitation combined with salt precipitation has been presented. The ethanol-sodium ac-etate system for crystallization of salt of fructose-1,6-diphosphate is based on the mechanism of crystallization ofFDPNa3.8 H2O in the ethanol-low temperature system. It is found that crystal size may be controlled by regulatingtemperature or pH value of solution in the crystallization process, and the crystal yield increases to 95% from 78%which obtained in the ethanol-low temperature system.

  10. Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase.

    Science.gov (United States)

    Versées, Wim; Spaepen, Stijn; Wood, Martin D H; Leeper, Finian J; Vanderleyden, Jos; Steyaert, Jan

    2007-11-30

    Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

  11. New Stetter reactions catalyzed by thiamine diphosphate dependent MenD from E. coli.

    Science.gov (United States)

    Beigi, Maryam; Waltzer, Simon; Zarei, Mostafa; Müller, Michael

    2014-12-10

    The intermolecular asymmetric Stetter reaction is a rarely found biocatalysts transformation. MenD, the second enzyme of the menaquinone biosynthetic pathway, catalyzes as a physiological reaction a Stetter-like addition of α-ketoglutarate to isochorismate. The substrate range of MenD for similar 1,4-additions is highly restricted. All other thiamine diphosphate dependent enzymes known to act as stetterases are members of the PigD enzyme subfamily, which accept aliphatic and aromatic α,β-unsaturated ketones and thioesters as Michael acceptor substrates. Here, we describe the unexpected activity of MenD with short-chain α,β-unsaturated acids and derivatives as substrates in Stetter reactions. MenD possesses a characteristic substrate range with respect to Michael acceptor substrates which is distinctly different from the classical stetterases. This provides biocatalytic access to new types of products which are not related to the products currently accessible by thiamine diphosphate dependent enzyme catalysis.

  12. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    Directory of Open Access Journals (Sweden)

    Juhi Sikarwar

    2013-01-01

    Full Text Available Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3. The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA chromatography.

  13. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    Science.gov (United States)

    Sikarwar, Juhi; Kaushik, Sanket; Sinha, Mau; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

    2013-01-01

    Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk) has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3). The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography. PMID:23662205

  14. Squalene Synthase As a Target for Chagas Disease Therapeutics

    Science.gov (United States)

    Chan, Hsiu-Chien; Li, Jikun; Zheng, Yingying; Huang, Chun-Hsiang; Ren, Feifei; Chen, Chun-Chi; Zhu, Zhen; Galizzi, Melina; Li, Zhu-Hong; Rodrigues-Poveda, Carlos A.; Gonzalez-Pacanowska, Dolores; Veiga-Santos, Phercyles; de Carvalho, Tecia Maria Ulisses; de Souza, Wanderley; Urbina, Julio A.; Wang, Andrew H.-J.; Docampo, Roberto; Li, Kai; Liu, Yi-Liang; Oldfield, Eric; Guo, Rey-Ting

    2014-01-01

    Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease. PMID:24789335

  15. Squalene synthase as a target for Chagas disease therapeutics.

    Directory of Open Access Journals (Sweden)

    Na Shang

    2014-05-01

    Full Text Available Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

  16. Products of the inactivation of ribonucleoside diphosphate reductase from Escherichia coli with 2'-azido-2'-deoxyuridine 5'-diphosphate

    Energy Technology Data Exchange (ETDEWEB)

    Salowe, S.P.; Ator, M.A.; Stubbe, J.A.

    1987-06-16

    Ribonucleoside diphosphate reductase (RDPR) from Escherichia coli was completely inactivated by 1 equiv of the mechanism-based inhibitor 2'-azido-2'-azido-2'-deoxyuridine 5'-diphosphate (N/sub 3/UDP). Incubation of RDPR with (3'-/sup 3/H)N/sub 3/UDP resulted in 0.2 mol of /sup 3/H released to solvent per mole of enzyme inactivated, indicating that cleavage of the 3' carbon-hydrogen bond occurred in the reaction. Incubation of RDPR with (..beta..-/sup 32/P)N/sub 3/UDP resulted in stoichiometric production of inorganic pyrophosphate. One equivalent of uracil was eliminated from N/sub 3/UDP, but no azide release was detected. Analysis of the reaction of RDPR with (/sup 15/N/sub 3/)N/sub 3/UDP by mass spectrometry revealed that the azide moiety was converted to 0.9 mol of nitrogen gas per mole of enzyme inactivated. The tyrosyl radical of the B2 subunit was destroyed during the inactivation by N/sub 3/UDP as reported previously, while the specific activity of the B1 subunit was reduced by half. Incubation of (5'-/sup 3/H)N/sub 3/UDP with RDPR resulted in stoichiometric covalent radiolabeling of the enzyme. Separation of the enzyme's subunits by chromatofocusing revealed that the modification was specific for the B1 subunit.

  17. Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms.

    Science.gov (United States)

    Jirgis, Ehab N M; Bashiri, Ghader; Bulloch, Esther M M; Johnston, Jodie M; Baker, Edward N

    2016-07-06

    Menaquinone (MQ) is an essential component of the respiratory chains of many pathogenic organisms, including Mycobacterium tuberculosis (Mtb). The first committed step in MQ biosynthesis is catalyzed by 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD), a thiamin diphosphate (ThDP)-dependent enzyme. Catalysis proceeds through two covalent intermediates as the substrates 2-oxoglutarate and isochorismate are successively added to the cofactor before final cleavage of the product. We have determined a series of crystal structures of Mtb-MenD that map the binding of both substrates, visualizing each step in the MenD catalytic cycle, including both intermediates. ThDP binding induces a marked asymmetry between the coupled active sites of each dimer, and possible mechanisms of communication can be identified. The crystal structures also reveal conformational features of the two intermediates that facilitate reaction but prevent premature product release. These data fully map chemical space to inform early-stage drug discovery targeting MenD.

  18. Inclusion of thiamine diphosphate and S-adenosylmethionine at their chemically active sites.

    Science.gov (United States)

    Schrader, Thomas; Fokkens, Michael; Klärner, Frank-Gerrit; Polkowska, Jolanta; Bastkowski, Frank

    2005-12-09

    [structure: see text] Molecular clips functionalized by phosphonate or phosphate groups bind thiamine diphosphate (TPP) and S-adenosylmethionine (SAM) with high affinity in water; both sulfur-based cofactors transfer organic groups to biomolecules. For TPP, various analytical tools point toward a simultaneous insertion of both heterocyclic rings into the electron-rich clip cavity. Similarly, SAM is also embedded with its sulfonium moiety inside the receptor cavity. This paves the way for enzyme models and direct interference with enzymatic processes.

  19. A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

    OpenAIRE

    Wei, Lei; Yin, Li; Hu,Xiaole; Xu, Ying; Chen,Fang

    2014-01-01

    Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene ...

  20. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. No obvious bands were detected in other examined tissues. The GGPPS gene was located on human chromosome 1q43 by Radiation Hybrid mapping method. It was proved that there was a putative predisposing gene for prostate cancer in this region, and that analogs of GGPP can inhibit the geranylgeranylation of p21rap protein in PC-3 prostate cancer cell lines. These facts suggest that GGPPS may be one of the candidate genes for prostate cancer.

  1. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a,3...... of substrates and products indicated a role of Mg2+ in preparing the active site of phosphoribosyl diphosphate synthetase for binding of the highly phosphorylated ligands MgATP and phosphoribosyl diphosphate, as evaluated by analysis of the effects of the inhibitors adenosine and ribose 1,5-bisphosphate....... Calcium ions, which inhibit the enzyme even in the presence of high concentrations of Mg2+, appeared to compete with free Mg2+ for binding to its activator site on the enzyme. Analysis of the inhibition of Mg2+ binding by MgADP indicated that MgADP binding to the allosteric site may occur in competition...

  2. A role for prenylated rab acceptor 1 in vertebrate photoreceptor development

    Directory of Open Access Journals (Sweden)

    Dickison Virginia M

    2012-12-01

    Full Text Available Abstract Background The rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day (P 10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration. Results Using a microarray approach, we performed gene profiling comparing rd1 and wild type (wt retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. Of the 143 identified differentially expressed genes, we focused on Rab acceptor 1 (Rabac1, which codes for the protein Prenylated rab acceptor 1 (PRA1 and plays an important role in vesicular trafficking. Quantitative RT-PCR analysis confirmed reduced expression of PRA1 in rd1 retina at all time points examined. Immunohistochemical observation showed that PRA1-like immunoreactivity (LIR co-localized with the cis-Golgi marker GM-130 in the photoreceptor as the Golgi translocated from the perikarya to the inner segment during photoreceptor differentiation in wt retinas. Diffuse PRA1-LIR, distinct from the Golgi marker, was seen in the distal inner segment of wt photoreceptors starting at P8. Both plexiform layers contained PRA1 positive punctae independent of GM-130 staining during postnatal development. In the inner retina, PRA1-LIR also colocalized with the Golgi marker in the perinuclear region of most cells. A similar pattern was seen in the rd1 mouse inner retina. However, punctate and significantly reduced PRA1-LIR was present throughout the developing rd1 inner segment, consistent with delayed photoreceptor development and abnormalities in Golgi sorting and vesicular trafficking. Conclusions We have identified genes that are differentially regulated in the rd1 retina at early

  3. Molecular Cloning and Functional Analysis of Squalene Synthase 2(SQS2 in Salvia miltiorrhiza Bunge

    Directory of Open Access Journals (Sweden)

    Qixian Rong

    2016-08-01

    Full Text Available Salvia miltiorrhiza Bunge,which is also known as a traditional Chinese herbal medicine,is widely studied for its ability to accumulate the diterpene quinone Tanshinones. In addition to producing a variety of diterpene quinone, S. miltiorrhiza Bunge also accumulates sterol, brassinosteroid and triterpenoids. During their biosynthesis, squalene synthase (SQS, EC 2.5.1.21 converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. In the present study, cloning and characterization of S. miltiorrhiza Bunge squalene synthase 2 (SmSQS2, Genbank Accession Number: KM408605 cDNA was investigated subsequently followed by its recombinant expression and preliminary enzyme activity. The full-length cDNA of SmSQS2 was 1 597 bp in length, with an open reading frame (ORF of 1 245 bp encoding 414 amino acids. The deduced amino acid sequence of SmSQS2 shared high similarity with those of SQSs from other plants. To obtain soluble recombinant enzymes, the truncated SmSQS2 in which 28 amino acids were deleted from the carboxy terminus was expressed as GST-Tag fusion protein in Escherichia coli BL21 (DE3 and confirmed by SDS-PAGE and Western Blot analysis, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. The gene expression level was analyzed through Quantitative real-time PCR, and was found to be higher in roots as compared to the leaves, and was up-regulated upon YE+ Ag+ treatment. These results could serve as an important to understand the function of the SQS family. In addition, the identification of SmSQS2 is important for further studies of terpenoid and sterol biosynthesis in S. miltiorrhiza Bunge.

  4. Structure of trihydrated rare-earth acid diphosphates LnHP 2O 7·3H 2O ( Ln=La, Er)

    Science.gov (United States)

    Ben Moussa, S.; Ventemillas, S.; Cabeza, A.; Gutierrez-Puebla, E.; Sanz, J.

    2004-06-01

    In trihydrated lanthanum acid-diphosphates LnHP 2O 7·3H 2O, prepared from acid LnCl 3 and Na 4P 2O 7 solutions (pH=1), two crystal forms were obtained. Layered structures of two representative members of this family have been determined by single-crystal X-ray diffraction (XRD) technique. In the case of orthorhombic LaHP 2O 7·3H 2O (type I), lanthanum cations are ninefold coordinated and diphosphate groups adopt a staggered (alternated) configuration. In the case of triclinic ErHP 2O 7·3H 2O (type II), erbium cations are eightfold coordinated and diphosphate groups adopt an eclipsed configuration. In agreement with Infrared (IR) spectroscopic data, a bended configuration for diphosphate groups has been deduced. In both structures, one-dimensional chains of edge-sharing rare-earth polyhedra are linked together by diphosphate groups to form the phosphate layers. In both diphosphates, PO 4 and HPO 4 environments have been identified by 31P MAS-NMR technique. In the two compounds, OH groups of HPO 4 tetrahedra point out of diphosphate planes interacting with adjacent layers. In La-diphosphate, the interaction between HPO 4 groups and water molecules of adjacent layers is favored; however, in Er-diphosphate, the interaction between phosphate acid groups of contiguous layers is produced. Based on structural information deduced, differences detected in IR and NMR spectra of two disphosphates are discussed.

  5. C2-alpha-lactylthiamin diphosphate is an intermediate on the pathway of thiamin diphosphate-dependent pyruvate decarboxylation. Evidence on enzymes and models.

    Science.gov (United States)

    Zhang, Sheng; Liu, Min; Yan, Yan; Zhang, Zhen; Jordan, Frank

    2004-12-24

    Thiamin diphosphate (ThDP)-dependent decarboxylations are usually assumed to proceed by a series of covalent intermediates, the first one being the C2-trimethylthiazolium adduct with pyruvate, C2-alpha-lactylthiamin diphosphate (LThDP). Herein is addressed whether such an intermediate is kinetically competent with the enzymatic turnover numbers. In model studies it is shown that the first-order rate constant for decarboxylation can indeed exceed 50 s(-1) in tetrahydrofuran as solvent, approximately 10(3) times faster than achieved in previous model systems. When racemic LThDP was exposed to the E91D yeast pyruvate decarboxylase variant, or to the E1 subunit of the pyruvate dehydrogenase complex (PDHc-E1) from Escherichia coli, it was partitioned between reversion to pyruvate and decarboxylation. Under steady-state conditions, the rate of these reactions is severely limited by the release of ThDP from the enzyme. Under pre-steady-state conditions, the rate constant for decarboxylation on exposure of LThDP to the E1 subunit of the pyruvate dehydrogenase complex was 0.4 s(-1), still more than a 100-fold slower than the turnover number. Because these experiments include binding, decarboxylation, and oxidation (for detection purposes), this is a lower limit on the rate constant for decarboxylation. The reasons for this slow reaction most likely include a slow conformational change of the free LThDP to the V conformation enforced by the enzyme. Between the results from model studies and those from the two enzymes, it is proposed that LThDP is indeed on the decarboxylation pathway of the two enzymes studied, and once LThDP is bound the protein needs to provide little assistance other than a low polarity environment.

  6. SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides

    Energy Technology Data Exchange (ETDEWEB)

    Hartwig, S.; Frister, T.; Alemdar, S.; Li, Z.; Scheper, T.; Beutel, S., E-mail: beutel@iftc.uni-hannover.de

    2015-03-20

    An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L{sup −1} were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni{sup 2+}-IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg{sup 2+} containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC–MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis–Menten model, kinetic parameters of K{sub M} = 1.111 μM (±0.113), v{sub max} = 0.3245 μM min{sup −1} (±0.0035), k{sub cat} = 2.95 min{sup −1}, as well as a catalytic efficiency k{sub cat}/K{sub M} = 4.43 × 10{sup 4} M{sup −1} s{sup −1} were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. - Highlights: • Uncharacterized (+)-zizaene synthase from C. zizanoides was cloned

  7. Impaired mechanical response of an EDMD mutation leads to motility phenotypes that are repaired by loss of prenylation.

    Science.gov (United States)

    Zuela, Noam; Zwerger, Monika; Levin, Tal; Medalia, Ohad; Gruenbaum, Yosef

    2016-05-01

    There are roughly 14 distinct heritable autosomal dominant diseases associated with mutations in lamins A/C, including Emery-Dreifuss muscular dystrophy (EDMD). The mechanical model proposes that the lamin mutations change the mechanical properties of muscle nuclei, leading to cell death and tissue deterioration. Here, we developed an experimental protocol that analyzes the effect of disease-linked lamin mutations on the response of nuclei to mechanical strain in living Caenorhabditis elegans We found that the EDMD mutation L535P disrupts the nuclear mechanical response specifically in muscle nuclei. Inhibiting lamin prenylation rescued the mechanical response of the EDMD nuclei, reversed the muscle phenotypes and led to normal motility. The LINC complex and emerin were also required to regulate the mechanical response of C. elegans nuclei. This study provides evidence to support the mechanical model and offers a potential future therapeutic approach towards curing EDMD.

  8. Xanthohumol, a prenylated chalcone derived from hops, inhibits proliferation, migration and interleukin-8 expression of hepatocellular carcinoma cells.

    Science.gov (United States)

    Dorn, Christoph; Weiss, Thomas S; Heilmann, Jörg; Hellerbrand, Claus

    2010-02-01

    Xanthohumol, the major prenylated chalcone found in hops, is well known to exert anti-cancer effects, but information regarding the impact on hepatocellular carcinoma (HCC) cells and potential adverse effects on non-tumorous hepatocytes is limited. Here, we show that xanthohumol at a concentration of 25 microM induced apoptosis in two HCC cell lines (HepG2 and Huh7). Furthermore, xanthohumol repressed proliferation and migration, as well as TNF induced NF-kappaB activity and interleukin-8 expression in both cell lines at even lower concentrations. In contrast, xanthohumol concentrations up to 100 microM did not affect viability of primary human hepatocytes in vitro. In summary, our data showed that xanthohumol can ameliorate different pro-tumorigenic mechanisms known to promote HCC progression, indicating its potential as promising therapeutic agent that selectively affects cancer cells.

  9. The inhibitory effects of xanthohumol, a prenylated chalcone derived from hops, on cell growth and tumorigenesis in human pancreatic cancer.

    Science.gov (United States)

    Jiang, Weiliang; Zhao, Senlin; Xu, Ling; Lu, Yingying; Lu, Zhanjun; Chen, Congying; Ni, Jianbo; Wan, Rong; Yang, Lijuan

    2015-07-01

    Pancreatic cancer (PC) is one of the most lethal human malignancies worldwide. Here, we demonstrated that xanthohumol (XN), the most abundant prenylated chalcone isolated from hops, inhibited the growth of cultured PC cells and their subcutaneous xenograft tumors. XN treatment was found to induce cell cycle arrest and apoptosis of PC cells (PANC-1, BxPC-3) by inhibiting phosphorylation of signal transducer and activator of transcription 3 (STAT3) and expression of its downstream targeted genes cyclinD1, survivin, and Bcl-xL at the messenger RNA level, which involved in regulation of apoptosis and the cell cycle. Overall, our results suggested that XN presents a promising candidate therapeutic agent against human PC and the STAT3 signaling pathway is its key molecular target.

  10. Prenylated flavone from roots of a hybrid between Artocarpus heterophyllus and Artocarpus integer and its biological activities

    Energy Technology Data Exchange (ETDEWEB)

    Panthong, Kanda, E-mail: kanda.p@psu.ac.th [Prince of Songkla University (Thailand). Natural Product Research Center of Excellence; Tohdee, Kanogwan [Prince of Songkla University (Thailand). Faculty of Science. Department of Chemistry; Hutadilok-Towatana, Nongporn [Prince of Songkla University (Thailand). Dept. of Biochemistry; Voravuthikunchai, Supayang P. [Prince of Songkla University (Thailand). Faculty of Science. Department of Microbiology; Chusri, Sasitorn [Prince of Songkla University (Thailand). Faculty of Traditional Thai Medicine

    2013-10-15

    One new prenylated flavone, 2,8-dihydroxy-3,10-dimethoxy-6-(2-methyl-1-propen-1-yl)- 6H,7H-[1]benzopyrano[4,3-b][1]-benzopyran-7-one, together with 24 known compounds were isolated from crude acetone extract from the roots of a hybrid between Artocarpus heterophyllus and Artocarpus integer. Their structures were determined by 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data. The antioxidant and antibacterial activities of the isolated compounds were evaluated. The new compound showed potent antioxidant activity against DPPH Bullet and superoxide with IC{sub 50} values of 0.033 and 0.125 mg mL{sup -1}, respectively. Significant antibacterial activity against Acinetobacter baumannii was observed with MIC value of 50 {mu}g mL{sup -1}. (author)

  11. Prenylated indole diketopiperazine alkaloids from a mangrove rhizosphere soil derived fungus Aspergillus effuses H1-1.

    Science.gov (United States)

    Gao, Huquan; Zhu, Tianjiao; Li, Dehai; Gu, Qianqun; Liu, Weizhong

    2013-08-01

    One new prenylated indole diketopiperazine alkaloid, named dihydroneochinulin B (1), one known spiro-polyketide-diketopiperazine hybrid cryptoechinuline D (2) and three related known metabolites didehydroechinulin B (3), neoechinulin B (4) and auroglaucin (5) were isolated from the mangrove rhizosphere soil derived fungus, Aspergillus effuses H1-1. The structures were assigned by detailed spectroscopic analysis. The enantiomers of cryptoechinuline D (2) were separated to be (+)-cryptoechinuline D (2a) and (-)-cryptoechinuline D (2b) by chiral HPLC, and their absolute configurations were determined by ECD analysis. The cytotoxic effects of the compounds were preliminarily evaluated on P388, HL-60, BEL-7402 and A-549 cell lines by SRB or MTT methods, and compounds 2, 2a and 3 showed significant activities.

  12. Identification of prenyl ethyl ether as a source of metallic, solvent-like off-flavor in hazelnut.

    Science.gov (United States)

    Amrein, Thomas M; Schwager, Hugo; Meier, Roberto; Frey, Peter; Gassenmeier, Klaus F

    2010-11-10

    In a large batch of ground hazelnuts, a metallic, solvent-like off-note was detected. In this investigation, the volatiles from the batch showing off-notes were compared to a batch without off-notes. On the basis of gas chromatography (GC) sniffing and instrumental analysis, a terpenoid compound, prenyl ethyl ether, was identified as a key contributor to the off-note. The compound was quantified, and its contribution to the metallic, solvent-like off-flavor was confirmed by spiking experiments and sensory evaluation. Analytical and sensory experiments found that the off-note was still present in hazelnut cakes. Fat oxidation did not contribute to the off-flavor. Analysis of market products demonstrated the correlation between the identified terpenoid and the off-flavor. It is assumed that fungi are involved in off-flavor formation.

  13. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr;

    2015-01-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichin......Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat...

  14. Biphenyl synthase, a novel type III polyketide synthase.

    Science.gov (United States)

    Liu, B; Raeth, T; Beuerle, T; Beerhues, L

    2007-05-01

    Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.

  15. Functional Analysis of the Isopentenyl Diphosphate Isomerase of Salvia miltiorrhiza via Color Complementation and RNA Interference

    Directory of Open Access Journals (Sweden)

    Xianan Zhang

    2015-11-01

    Full Text Available Isopentenyl diphosphate isomerase (IPI catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP and dimethylallyl diphosphate (DMAPP during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1 was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.

  16. Functional Analysis of the Isopentenyl Diphosphate Isomerase of Salvia miltiorrhiza via Color Complementation and RNA Interference.

    Science.gov (United States)

    Zhang, Xianan; Guan, Hongyu; Dai, Zhubo; Guo, Juan; Shen, Ye; Cui, Guanghong; Gao, Wei; Huang, Luqi

    2015-11-10

    Isopentenyl diphosphate isomerase (IPI) catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1) was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.

  17. Purification, crystallization and preliminary structural analysis of nucleoside diphosphate kinase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Misra, Gauri [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Aggarwal, Anita [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Mittal, Sonia [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Singh, Yogendra [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Ramachandran, Ravishankar, E-mail: r-ravishankar@cdri.res.in [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India)

    2007-12-01

    Nucleoside diphosphate kinase from B. anthracis has been crystallized. Preliminary crystallographic analysis shows that there is one monomer in the asymmetric unit of the crystal. Bacillus anthracis nucleoside diphosphate kinase (BaNdk) is an enzyme whose primary function is to maintain deoxynucleotide triphosphate (dNTP) pools by converting deoxynucleotide diphosphates to triphosphates using ATP as the major phosphate donor. Although the structures of Ndks from a variety of organisms have been elucidated, the enzyme from sporulating bacteria has not been structurally characterized to date. Crystals of the B. anthracis enzyme were grown using the vapour-diffusion method from a hanging drop consisting of 2 µl 10 mg ml{sup −1} protein in 50 mM Tris–HCl pH 8.0, 50 mM NaCl, 5 mM EDTA equilibrated against 500 µl reservoir solution consisting of 2.25 M ammonium formate and 0.1 M HEPES buffer pH 7.25. Diffraction data extending to 2.0 Å were collected at room temperature from a single crystal with unit-cell parameters a = b = 107.53, c = 52.3 Å. The crystals are hexagonal in shape and belong to space group P6{sub 3}22. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (V{sub M}) of 2.1 Å{sup 3} Da{sup −1} and a solvent content of about 36.9%.

  18. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2012-09-17

    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  19. Genetics Home Reference: GM3 synthase deficiency

    Science.gov (United States)

    ... Facebook Share on Twitter Your Guide to Understanding Genetic Conditions Search MENU Toggle navigation Home Page Search ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions GM3 synthase deficiency GM3 synthase ...

  20. Enzymatic production of fructose 1,6-diphosphate using crude cell extract of Bacillus stearothermophilus.

    Science.gov (United States)

    Widjaja, A; Yasuda, M; Ogino, H; Nakajima, H; Ishikawa, H

    1999-01-01

    The enzymatic production of fructose 1,6-diphosphate (FDP) from glucose was performed in a batch reactor and a semibatch reactor using the crude cell extract of Bacillus stearothermophilus which contains all four enzymes required for the synthesis. The experimental results of the yield and the time courses of FDP production obtained using various enzyme concentrations were in good agreement with the theoretical predictions calculated based on the differential equations including the rate equations of the four enzymes, which were determined using the purified enzymes of B. stearothermophilus.

  1. METOVITAN PREVENTS ACCUMULATION OF THIAMIN DIPHOSPHATE OXYGENIZED FORM IN RAT TISSUES UNDER IRRADIATION

    OpenAIRE

    Yu. M. Parkhomenko; G. V. Donchenko; L. I. Chehovskaya; S. P. Stepanenko; O. A. Mejenskaya; E. N. Gorban

    2015-01-01

    The aim of the research was to test the ability of the drug “Metovitan” to prevent the redox balance disturbance in the tissues and thiamine diphosphate irreversible oxidation upon exposure of ionizing radiation on the body. The rats were undergo to a single exposure of the X-ray therapeutic instrument RUM-17 to create a dose of 0.5, 1.0 and 5.0 Gy. Preparation “Metovitan” were administered at a dose of 25 mg per 1 kg body weight for 22–24 h before irradiation. Contents of thiamin...

  2. Influence of donor substrate on kinetic parameters of thiamine diphosphate binding to transketolase.

    Science.gov (United States)

    Ospanov, R V; Kochetov, G A; Kurganov, B I

    2007-01-01

    The two-step mechanism of interaction of thiamine diphosphate (ThDP) with transketolase (TK) has been studied: TK + ThDP TK...ThDP TK*-ThDP. The scheme involves the formation of inactive intermediate complex TK...ThDP followed by its transformation into catalytically active holoenzyme, TK*-ThDP. The dissociation and kinetic constants for individual stages of this process have been determined. The values of forward and backward rate constants change in the presence of the donor substrate hydroxypyruvate. This finally leads to an increase in the overall affinity of the coenzyme to TK.

  3. K channel activation by nucleotide diphosphates and its inhibition by glibenclamide in vascular smooth muscle cells.

    Science.gov (United States)

    Beech, D J; Zhang, H; Nakao, K; Bolton, T B

    1993-10-01

    1. Whole-cell and inside-out patch recordings were made from single smooth muscle cells that had been isolated enzymatically and mechanically from the rabbit portal vein. 2. In whole-cells the inclusion in the recording pipette solution of nucleotide diphosphates (NDPs), but not tri- or monophosphates, induced a K-current that developed gradually over 5 to 15 min. Intracellular 1 mM guanosine 5'-diphosphate (GDP) induced a slowly developing outward K-current at -37 mV that reached a maximum on average of 72 +/- 4 pA (n = 40). Half maximal effect was estimated to occur with about 0.2 mM GDP. Except for ADP, other NDPs had comparable effects. At 0.1 mM, ADP was equivalent to GDP but at higher concentration ADP was less effective. ADP induced its maximum effect at 1 mM but had almost no effect at 10 mM. 3. In 14% of inside-out patches exposed to 1 mM GDP at the intracellular surface, characteristic K channel activity was observed which showed long (> 1 s) bursts of openings separated by longer closed periods. The current-voltage relationship for the channel was linear in a 60 mM:130 mM K-gradient and the unitary conductance was 24 pS. 4. Glibenclamide applied via the extracellular solution was found to be a potent inhibitor of GDP-induced K-current (IK(GDP)) in the whole-cell. The Kd was 25 nM and the inhibition was fully reversible on wash-out. 5. IK(GDP) was not evoked if Mg ions were absent from the pipette solution. In contrast the omission of extracellular Mg ions had no effect on outward or inward IK(GDP). 6. Inclusion of 1 mM ATP in the recording pipette solution reduced IK(GDP) and also attenuated its decline during long (25 min) recordings. 7. When perforated-patch whole-cell recording was used, metabolic poisoning with cyanide and 2-deoxy-D-glucose induced a glibenclamide-sensitive K-current. This current was not observed when conventional whole-cell recording was used. Possible reasons for this difference are discussed. 8. These K channels appear similar to

  4. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    Science.gov (United States)

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

  5. Geranyllinalool Synthases in Solanaceae and Other Angiosperms Constitute an Ancient Branch of Diterpene Synthases Involved in the Synthesis of Defensive Compounds1[C][W][OPEN

    Science.gov (United States)

    Falara, Vasiliki; Alba, Juan M.; Kant, Merijn R.; Schuurink, Robert C.; Pichersky, Eran

    2014-01-01

    Many angiosperm plants, including basal dicots, eudicots, and monocots, emit (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, which is derived from geranyllinalool, in response to biotic challenge. An Arabidopsis (Arabidopsis thaliana) geranyllinalool synthase (GLS) belonging to the e/f clade of the terpene synthase (TPS) family and two Fabaceae GLSs that belong to the TPS-g clade have been reported, making it unclear which is the main route to geranyllinalool in plants. We characterized a tomato (Solanum lycopersicum) TPS-e/f gene, TPS46, encoding GLS (SlGLS) and its homolog (NaGLS) from Nicotiana attenuata. The Km value of SlGLS for geranylgeranyl diphosphate was 18.7 µm, with a turnover rate value of 6.85 s–1. In leaves and flowers of N. attenuata, which constitutively synthesize 17-hydroxygeranyllinalool glycosides, NaGLS is expressed constitutively, but the gene can be induced in leaves with methyl jasmonate. In tomato, SlGLS is not expressed in any tissue under normal growth but is induced in leaves by alamethicin and methyl jasmonate treatments. SlGLS, NaGLS, AtGLSs, and several other GLSs characterized only in vitro come from four different eudicot families and constitute a separate branch of the TPS-e/f clade that diverged from kaurene synthases, also in the TPS-e/f clade, before the gymnosperm-angiosperm split. The early divergence of this branch and the GLS activity of genes in this branch in diverse eudicot families suggest that GLS activity encoded by these genes predates the angiosperm-gymnosperm split. However, although a TPS sequence belonging to this GLS lineage was recently reported from a basal dicot, no representative sequences have yet been found in monocot or nonangiospermous plants. PMID:25052853

  6. Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-keto acid decarboxylase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Werther, Tobias; Spinka, Michael; Tittmann, Kai; Schütz, Anja; Golbik, Ralph; Mrestani-Klaus, Carmen; Hübner, Gerhard; König, Stephan

    2008-02-29

    The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

  7. Genome-based characterization of two prenylation steps in the assembly of the stephacidin and notoamide anticancer agents in a marine-derived Aspergillus sp.

    Science.gov (United States)

    Ding, Yousong; de Wet, Jeffrey R; Cavalcoli, James; Li, Shengying; Greshock, Thomas J; Miller, Kenneth A; Finefield, Jennifer M; Sunderhaus, James D; McAfoos, Timothy J; Tsukamoto, Sachiko; Williams, Robert M; Sherman, David H

    2010-09-15

    Stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a core bicyclo[2.2.2]diazaoctane ring system. These bioactive fungal secondary metabolites have a range of unusual structural and stereochemical features but their biosynthesis has remained uncharacterized. Herein, we report the first biosynthetic gene cluster for this class of fungal alkaloids based on whole genome sequencing of a marine-derived Aspergillus sp. Two central pathway enzymes catalyzing both normal and reverse prenyltransfer reactions were characterized in detail. Our results establish the early steps for creation of the prenylated indole alkaloid structure and suggest a scheme for the biosynthesis of stephacidin and notoamide metabolites. The work provides the first genetic and biochemical insights for understanding the structural diversity of this important family of fungal alkaloids.

  8. Cloning, expression and characterization of a nucleoside diphosphate kinase (NDPK) gene from tobacco

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nucleoside diphosphate kinase (NDPK) is a housekeeping enzyme that maintains the intracellular levels of all (d)NTPs used in biosynthesis except ATP. Here we report that a full-length cDNA encoding nucleoside diphosphate kinase (NDPK) was cloned using yeast two-hybrid approach. A tobacco NDPK gene was obtained and designated as NtNDPK1 . NtNDPK1 is 704 bp in length and encodes a putative 16.2 kD protein of 148 amino acids. Phylogenic analysis showed that NtNDPK1 is highly homologous to other plant NDPK genes and identified as type Ⅰ (NDPK1). RNA-gel blot analysis showed that there was no significant difference of NtNDPK1 expression in roots, stems, leaves and buds. And expression of NtNDPK1 was induced by ABA and PEG and repressed by NaCl, but not significantly affected by Paraquat, wounding and low temperature (4℃) treatments, indicating that NtNDPK1 may play a certain role in response to abiotic stress. In vitro phosphorylation assay demonstrated that NtNDPK1 had autophosphorylation activity.

  9. A cesium copper vanadyl-diphosphate: Synthesis, crystal structure and physical properties

    Science.gov (United States)

    Shvanskaya, Larisa; Yakubovich, Olga; Bychkov, Andrey; Shcherbakov, Vasiliy; Golovanov, Alexey; Zvereva, Elena; Volkova, Olga; Vasiliev, Alexander

    2015-02-01

    A non-centrosymmetric orthorhombic diphosphate, Cs2Cu1+x(VO)2-x(P2O7)2 (x=0.1) with a=13.7364(2) Å, b=9.2666(2) Å, c=11.5678(2) Å, Z=4, has been isolated. Its 3D framework is built from Cu atoms in square pyramidal and square planar coordination, VO5 tetragonal pyramids and P2O7 diphosphate groups, sharing vertices. Large channels are fulfilled by cesium atoms. The ESR study reveals a similarity in behaviour of two paramagnetic (Cu and V) subsystems. The temperature dependences of the ESR linewidth and static magnetic susceptibility data present evidences for a cluster type magnetic ordering in the title compound at T*=22 K. The weakness of the relevant anomalies reflects presumably obvious Cu2+ ions and (VO)2+ units disorder in the system. It is supposed that the charge and geometry of the framework are controlled by the Cu2+/(VO)2+ ratio; its variation may lead to a design of new materials.

  10. Studies on the mammary tumor-inhibiting effects of diethylstilbestrol and its mono- and diphosphate.

    Science.gov (United States)

    Schneider, M R; von Angerer, E; Prekajac, J; Brade, W P

    1986-01-01

    Diethylstilbestrol (DES), diethylstilbestrol monophosphate (DES-MP) and diethylstilbestrol diphosphate (DES-DP) were tested for their estrogen receptor affinity, estrogenic potency and mammary tumor-inhibiting activity in vitro and in vivo. DES had a much higher receptor binding affinity than its mono- or diphosphate. All three compounds inhibited the growth of the hormone-dependent MCF-7 and hormone-independent MDA-MB 231 breast cancer line only at relatively high concentrations. The estrogenic potency in the immature mouse uterine weight test decreased in the order DES greater than DES-MP much greater than DES-DP. The hormone-dependent MXT mammary tumor of the mouse was inhibited by all three compounds at a dosage of 1.0 mg/kg per week. At a dose of 0.01 mg/kg, DES, DES-MP, and DES-DP stimulated the tumor growth. Thus, for the first time, a biphasic effect on tumor growth was demonstrated in intact mature animals. As the effects of all three compounds were similar in this assay, a cleavage of the phosphate groups is likely. A decrease in estrogenic potency concomitant with a retained antitumor effect of DES-MP and DES-DP compared to DES was not demonstrable in the mature mouse using the MXT assay, only in the uterotrophic test in the immature mouse.

  11. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Jeudy, Sandra [Information Génomique et Structurale, CNRS UPR 2589, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France); Coutard, Bruno [Architecture et Fonction des Macromolecules Biologiques, CNRS UMR 6098, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France); Lebrun, Régine [IBSM, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France); Abergel, Chantal, E-mail: chantal.abergel@igs.cnrs-mrs.fr [Information Génomique et Structurale, CNRS UPR 2589, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France)

    2005-06-01

    A. polyphaga mimivirus, the largest known double-stranded DNA virus, is the first virus to exhibit a nucleoside diphosphate kinase gene. The expression and crystallization of the viral NDK are reported. The complete sequence of the largest known double-stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined [Raoult et al. (2004 ▶), Science, 306, 1344–1350] and revealed numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated [Abergel et al. (2005 ▶), Acta Cryst. F61, 212–215] both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P2{sub 1}3, with unit-cell parameter 99.425 Å. The self-rotation function confirms that there are two monomers per asymmetric unit related by a twofold non-crystallographic axis and that the unit cell thus contains four biological entities.

  12. Reversed-phase ion-pair high-performance liquid chromatography assay of polyprenyl diphosphate oligomer homologues.

    Science.gov (United States)

    Kozlov, Vyacheslav V; Danilov, Leonid L

    2016-02-01

    A reversed-phase ion-pair high-performance liquid chromatography procedure was developed for the separation of polyprenyl diphosphate oligomer homologues obtained chemically from plant polyprenols. Tetrabutylammonium phosphate was used as the ion-pair reagent, and the dependence of the separation quality on pH of ion-pair reagent was investigated for the first time. The procedure is applicable for the control of commercial available polyprenyl monophosphates (the active components of veterinary drugs Phosprenyl and Gamapren) for the possible presence of polyprenyl diphosphate byproducts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase[S

    Science.gov (United States)

    Ternes, Philipp; Brouwers, Jos F. H. M.; van den Dikkenberg, Joep; Holthuis, Joost C. M.

    2009-01-01

    Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). While sphingomyelin (SM) is the major sphingolipid in mammals, previous studies indicate that mammalian cells also produce the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or the enzyme(s) responsible for CPE biosynthesis. SM production is mediated by the SM synthases SMS1 in the Golgi and SMS2 at the PM, while a closely related enzyme, SMSr, has an unknown biochemical function. We now demonstrate that SMS family members display striking differences in substrate specificity, with SMS1 and SMSr being monofunctional enzymes with SM and CPE synthase activity, respectively, and SMS2 acting as a bifunctional enzyme with both SM and CPE synthase activity. In agreement with the PM residency of SMS2, we show that both SM and CPE synthase activities are enhanced at the surface of SMS2-overexpressing HeLa cells. Our findings reveal an unexpected diversity in substrate specificity among SMS family members that should enable the design of specific inhibitors to target the biological role of each enzyme individually. PMID:19454763

  14. Phebaclavin I, a novel 3-prenylated coumarin, and trichoclin acetate, a new natural furocoumarin, from the aerial parts of Phebalium aff. tuberculosum (Rutaceae).

    Science.gov (United States)

    Roux, Delphine; Muyard, Frédéric; Girard, Corinne; Colombain, Michel; Tillequin, François; Waterman, Peter G; Bévalot, Françoise

    2006-03-01

    Phebaclavin I, a novel 3-prenylated coumarin, was isolated from the aerial parts of Phebalium aff. tuberculosum (Rutaceae) together with five known related compounds, Phebaclavin A, B, D, G, H. The structure of phebaclavin I was established by spectroscopic methods. Several other known coumarins were obtained, including trichoclin acetate, a furocoumarin isolated for the first time from a natural source, but previously described from acetylation of trichoclin.

  15. Genome-based Characterization of Two Prenylation Steps in the Assembly of the Stephacidin and Notoamide Anticancer Agents in a Marine-derived Aspergillus sp

    OpenAIRE

    Ding, Yousong; de Wet, Jeffrey R.; Cavalcoli, James; Li, Shengying; Greshock, Thomas J.; Miller, Kenneth A.; Finefield, Jennifer M.; Sunderhaus, James D.; McAfoos, Timothy; Tsukamoto, Sachiko; Williams, Robert M.; Sherman, David H.

    2010-01-01

    Stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a core bicyclo[2.2.2]diazaoctane ring system. These bioactive fungal secondary metabolites have a range of unusual structural and stereochemical features but their biosynthesis has remained uncharacterized. Herein, we report the first biosynthetic gene cluster for this class of fungal alkaloids based on whole genome sequencing of a marine-derived Aspergillus sp. Two central pathway enzymes c...

  16. Isolation of Persicaria minor sesquiterpene synthase promoter and its deletions for transgenic Arabidopsis thaliana

    Science.gov (United States)

    Omar, Aimi Farehah; Ismail, Ismanizan

    2016-11-01

    Sesquiterpene synthase (SS) catalyzes the formation of sesquiterpenes from farnesyl diphosphate (FDP) via carbocation intermediates. In this study, the promoter region of sesquiterpene synthase was isolated from Persicaria minor to identify possible cis-acting elements in the promoter. The full-length PmSS promoter of P. minor is 1824-bp sequences. The sequence was analyzed and several putative cis-acting regulatory elements were identified. Three cis-acting regulatory elements were selected for deletion analysis which are cis-acting element involved in wound responsiveness (WUN), cis - acting element involved in defense and stress responsiveness (TC) and cis-acting element involved in ABA responsiveness (ABRE). Series of deletions were conducted to assess the promoter activity producing three truncated fragments promoter; Prom 2 1606-bp, Prom 3 1144- bp, and Prom 4 921-bp. The full-length promoter and its deletion series were cloned into the pBGWFS7 vector which contain β-glucuronidase (GUS) gene and green fluorescent protein (GFP) as the reporter gene. All constructs were successfully transformed into Arabidopsis thaliana based on PCR of positive BASTA resistance plants.

  17. Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.).

    Science.gov (United States)

    Bennett, Mark H; Mansfield, John W; Lewis, Mervyn J; Beale, Michael H

    2002-06-01

    Sesquiterpenoid lactones (SLs) from lettuce (Lactuca sativa L.) include constitutive components of latex such as lactucin and the induced phytoalexin, lettucenin A. A redundant primer strategy was used to recover two full length cDNA clones (LTC1 and LTC2) encoding sesquiterpene synthases from a cDNA library derived from seedlings with the red spot disorder, which accumulate phytoalexins. Recombinant enzymes produced from LTC1 and LTC2 in Escherichia coli catalysed the cyclisation of farnesyl diphosphate to germacrene A, potentially an early step in the biosynthesis of SLs. RT-PCR analysis showed LTC1 and LTC2 were expressed constitutively in roots, hypocotyls and true leaves but not in cotyledons. Expression in cotyledons was induced by challenge with the downy mildew pathogen Bremia lactucae in the disease resistant cultivar Diana. Southern hybridisation experiments showed that LTC1 and LTC2 were not part of a multigene family. The germacrene A synthases provide targets for modified expression to generate beneficial modifications to the SL profile in lettuce.

  18. Specificity of Ocimum basilicum geraniol synthase modified by its expression in different heterologous systems.

    Science.gov (United States)

    Fischer, Marc J C; Meyer, Sophie; Claudel, Patricia; Perrin, Mireille; Ginglinger, Jean François; Gertz, Claude; Masson, Jean E; Werck-Reinhardt, Danièle; Hugueney, Philippe; Karst, Francis

    2013-01-10

    Numerous aromatic plant species produce high levels of monoterpenols, using geranyl diphosphate (GPP) as a precursor. Sweet basil (Ocimum basilicum) geraniol synthase (GES) was used to evaluate the monoterpenol profiles arising from heterologous expressions in various plant models. Grapevine (Vitis vinifera) calli were transformed using Agrobacterium tumefasciens and the plants were regenerated. Thale cress (Arabidopsis thaliana) was transformed using the floral dip method. Tobacco (Nicotiana benthamiana) leaves were agro-infiltrated for transient expression. Although, as expected, geraniol was the main product detected in the leaves, different minor products were observed in these plants (V. vinifera: citronellol and nerol; N. benthamiana: linalool and nerol; A. thaliana: none). O. basilicum GES expression was also carried out with microbial system yeasts (Saccharomyces cerevisiae) and Escherichia coli. These results suggest that the functional properties of a monoterpenol synthase depend not only on the enzyme's amino-acidic sequence, but also on the cellular background. They also suggest that some plant species or microbial expression systems could induce the simultaneous formation of several carbocations, and could thus have a natural tendency to produce a wider spectrum of monoterpenols.

  19. Thermodynamic, dynamic and solvational properties of PDEδ binding to farnesylated cystein: a model study for uncovering the molecular mechanism of PDEδ interaction with prenylated proteins.

    Science.gov (United States)

    Suladze, S; Ismail, S; Winter, R

    2014-01-30

    The protein PDEδ is an important solubilizing factor for several prenylated proteins including the Ras subfamily members. The binding occurs mainly through the farnesyl anchor of Ras proteins, which is recognized by a hydrophobic pocket of PDEδ. In this study, we carried out a detailed study of the thermodynamic and solvational properties of PDEδ binding to farnesyl-cystein, which serves as a model for PDEδ association to prenylated proteins. Using various biophysical approaches in conjunction with theoretical considerations, we show here that binding of the largely hydrophobic ligand surprisingly has enthalpy-driven signature, and the entropy change is largely controlled by the fine balance between the hydrational and conformational terms. Moreover, binding of PDEδ to farnesyl-cystein is accompanied by an increase in thermal stability, the release of about 150 water molecules from the interacting species, a decrease in solvent accessible surface area, and a marked decrease of the volume fluctuations and hence dynamics of the protein. Altogether, our results shed more light on the molecular mechanism of PDEδ interaction with prenylated Ras proteins, which is also prerequisite for an optimization of the structure-based molecular design of drugs against Ras related diseases and for understanding the multitude of biological functions of PDEδ.

  20. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-β-farnesene

    Science.gov (United States)

    Crock, John; Wildung, Mark; Croteau, Rodney

    1997-01-01

    (E)-β-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-β-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (Km for Mg2+ ≈ 150 μM; Km for Mn2+ ≈ 7 μM). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-β-farnesene (85%), (Z)-β-farnesene (8%), and δ-cadinene (5%) with the native C15 substrate farnesyl diphosphate (Km ≈ 0.6 μM; Vrel = 100) and Mg2+ as cofactor, and (E)-β-farnesene (98%) and (Z)-β-farnesene (2%) with Mn2+ as cofactor (Vrel = 80). With the C10 analog, GDP, as substrate (Km = 1.5 μM; Vrel = 3 with Mg2+ as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced. PMID:9371761

  1. Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization.

    Science.gov (United States)

    Jackson, Alana J; Hershey, David M; Chesnut, Taylor; Xu, Meimei; Peters, Reuben J

    2014-07-01

    It has become apparent that plants have extensively diversified their arsenal of labdane-related diterpenoids (LRDs), in part via gene duplication and neo-functionalization of the ancestral ent-kaurene synthase (KS) required for gibberellin metabolism. For example, castor bean (Ricinus communis) was previously shown to produce an interesting set of biosynthetically related diterpenes, specifically ent-sandracopimaradiene, ent-beyerene, and ent-trachylobane, in addition to ent-kaurene, using four separate diterpene synthases, albeit these remain unidentified. Notably, despite mechanistic similarity of the underlying reaction to that catalyzed by KSs, ent-beyerene and ent-trachylobane synthases have not yet been identified. Given our interest in LRD biosynthesis, and the recent availability of the castor bean genome sequence, a synthetic biology approach was applied to biochemically characterize the four KS(-like) enzymes [KS(L)s] found in Ricinus communis [i.e., the RcKS(L)s]. In particular, using bacteria engineered to produce the relevant ent-copalyl diphosphate precursor and synthetic genes based on the predicted RcKS(L)s, although this ultimately required correction of a "splicing" error in one of the predicted genes, highlighting the dependence of such a synthetic biology approach on accurate gene sequences. Nevertheless, it is possible to assign each of the four RcKS(L)s to one of the previously observed diterpene synthase activities, providing access to functionally enzymes. Intriguingly, the product distribution of the RcKS(L)s seems to support the distinct diterpene synthase reaction mechanism proposed by quantum chemical calculations, rather than the classically proposed pathway.

  2. Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization

    Science.gov (United States)

    Jackson, Alana J.; Hershey, David M.; Chesnut, Taylor; Xu, Meimei; Peters, Reuben J.

    2014-01-01

    It has become apparent that plants have extensively diversified their arsenal of labdane-related diterpenoids (LRDs), in part via gene duplication and neo-functionalization of the ancestral ent-kaurene synthase (KS) required for gibberellin metabolism. For example, castor bean (Ricinus communis) was previously shown to produce an interesting set of biosynthetically related diterpenes, specifically ent-sandracopimaradiene, ent-beyerene, and ent-trachylobane, in addition to ent-kaurene, using four separate diterpene synthases, albeit these remain unidentified. Notably, despite mechanistic similarity of the underlying reaction to that catalyzed by KSs, ent-beyerene and ent-trachylobane synthases have not yet been identified. Given our interest in LRD biosynthesis, and the recent availability of the castor bean genome sequence, we applied a synthetic biology approach to biochemically characterize the four KS(-like) enzymes [KS(L)s] found in Ricinus communis [i.e., the RcKS(L)s]. In particular, using bacteria engineered to produce the relevant ent-copalyl diphosphate precursor and synthetic genes based on the predicted RcKS(L)s, although this ultimately required correction of a “splicing” error in one of the predicted genes, highlighting the dependence of such a synthetic biology approach on accurate gene sequences. Nevertheless, we can assign each of the four RcKS(L)s to one of the previously observed diterpene synthase activities, providing access to functionally novel enzymes. Intriguingly, the product distribution of the RcKS(L)s seems to support the distinct diterpene synthase reaction mechanism proposed by quantum chemical calculations, rather than the classically proposed pathway. PMID:24810014

  3. Effect of guanosine 5'-diphosphate 3'-diphosphate and related nucleoside polyphosphates on induction of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli.

    Science.gov (United States)

    Yoshimoto, A; Oki, T; Inui, T

    1978-10-04

    Exogenous addition of guanosine and adenosine 5'-(mono, di and tri) phosphate 3'-diphosphates (pppGpp, ppGpp, pGpp, pppApp, ppApp and pApp) stimulated the synthesis of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli. From the results obtained with ppGpp and pppApp, this effect appeared to be at a transcriptional level and depended greatly on the growth condition; the largest effect was observed in cells under shiftdown or grown on poor enrgy source. ppGpp and pppApp, unlike cyclic AMP, did not act to overcome the inhibition of enzyme induction by glucose, but in combination with cyclic AMP caused a synergistic stimulation effect. In the shiftdown cells, ppGpp and pppApp gave 30% or more stimulation effect on tryptophanase induction while cyclic AMP did not stimulate induction. There was therefore a pronounced difference between cyclic AMP and ppGpp or pppApp in stimulatory function.

  4. Alteration of RhoA Prenylation Ameliorates Cardiac and Vascular Remodeling in Spontaneously Hypertensive Rats

    Directory of Open Access Journals (Sweden)

    Jian Yang

    2016-06-01

    Full Text Available Background: In our previous study, farnesyl pyrophosphate synthase (FPPS was shown to be increased in spontaneously hypertensive rats (SHR and in mice with angiotensin-II induced cardiac hypertrophy. Overexpression of FPPS induced cardiac hypertrophy and fibrosis in mice, accompanied by an increase in the synthesis of farnesyl pyrophosphate (FPP and geranylgeranyl pyrophosphate (GGPP. In the present study, we investigated the mechanisms of reversing cardiovascular remodeling in SHR by inhibiting FPPS. Methods and Results: Six-week-old rats were given vehicle or an FPPS inhibitor (alendronate, 100 ug/kg/d daily for twelve weeks by osmotic mini-pump. The results demonstrated that FPPS inhibition attenuated cardiac hypertrophy and fibrosis in SHR as shown by the heart weight to body weight ratio, echocardiographic parameters, and histological examination. In addition, FPPS inhibition attenuated aortic remodeling as shown by reduced media thickness, media cross-sectional area and collagen of the aorta as well as SBP, DBP, MBP. Furthermore, 12 weeks of alendronate treatment significantly decreased FPP and GGPP levels, RhoA activation and geranylgeranylation in the heart and aorta, all of which were significantly upregulated in SHR compared with normotensive Wistar-Kyoto rats. Conclusion: Taken together, these results indicate that chronic treatment with alendronate decreases the development of cardiac and aortic remodeling, by a pathway which involves inhibition of the geranylgeranylation and activation of RhoA.

  5. Identification of a Plastid-Localized Bifunctional Nerolidol/Linalool Synthase in Relation to Linalool Biosynthesis in Young Grape Berries

    Directory of Open Access Journals (Sweden)

    Bao-Qing Zhu

    2014-12-01

    Full Text Available Monoterpenoids are a diverse class of natural products and contribute to the important varietal aroma of certain Vitis vinifera grape cultivars. Among the typical monoterpenoids, linalool exists in almost all grape varieties. A gene coding for a nerolidol/linalool (NES/LINS synthase was evaluated in the role of linalool biosynthesis in grape berries. Enzyme activity assay of this recombinant protein revealed that it could convert geranyl diphosphate and farnesyl diphosphate into linalool and nerolidol in vitro, respectively, and thus it was named VvRILinNer. However, localization experiment showed that this enzyme was only localized to chloroplasts, which indicates that VvRILinNer functions in the linalool production in vivo. The patterns of gene expression and linalool accumulation were analyzed in the berries of three grape cultivars (“Riesling”, “Cabernet Sauvignon”, “Gewurztraminer” with significantly different levels of monoterpenoids. The VvRILinNer was considered to be mainly responsible for the synthesis of linalool at the early developmental stage. This finding has provided us with new knowledge to uncover the complex monoterpene biosynthesis in grapes.

  6. Computational study on the carboligation reaction of acetohidroxyacid synthase: new approach on the role of the HEThDP- intermediate.

    Science.gov (United States)

    Jaña, Gonzalo; Jiménez, Verónica; Villà-Freixa, Jordi; Prat-Resina, Xavier; Delgado, Eduardo; Alderete, Joel

    2010-05-15

    Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate dependent enzyme that catalyses the decarboxylation of pyruvate to yield the hydroxyethyl-thiamin diphosphate (ThDP) anion/enamine intermediate (HEThDP(-)). This intermediate reacts with a second ketoacid to form acetolactate or acetohydroxybutyrate as products. Whereas the mechanism involved in the formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in controlling the carboligation reaction of HEThDP(-) has not been determined yet. In this work, molecular dynamics (MD) simulations were employed to identify the aminoacids involved in the carboligation stage. These MD studies were carried out over the catalytic subunit of yeast AHAS containing the reaction intermediate (HEThDP(-)) and a second pyruvate molecule. Our results suggest that additional acid-base ionizable groups are not required to promote the catalytic cycle, in contrast with earlier proposals. This finding leads us to postulate that the formation of acetolactate relies on the acid-base properties of the HEThDP(-) intermediate itself. PM3 semiempirical calculations were employed to obtain the energy profile of the proposed mechanism on a reduced model of the active site. These calculations confirm the role of HEThDP(-) intermediate as the ionizable group that promotes the carboligation and product formation steps of the catalytic cycle. Proteins 2010. (c) 2010 Wiley-Liss, Inc.

  7. Prenylation of a Rab1B mutant with altered GTPase activity is impaired in cell-free systems but not in intact mammalian cells.

    Science.gov (United States)

    Wilson, A L; Sheridan, K M; Erdman, R A; Maltese, W A

    1996-09-15

    Previous studies have reached differing conclusions as to whether or not guanine-nucleotide-dependent conformational changes affect the ability of Rab proteins to undergo post-translational modification by Rab:geranylgeranyltransferase (Rab-GGTase). We now show that the ability of a Rab1B mutant [Q67L (Gln-67-->Leu)] with reduced intrinsic GTPase activity to undergo geranylgeranylation in cell-free assays depends on the guanine nucleotide composition of the system. When GTP is the predominant nucleotide in the assay, Rab1BQ67L is a poor substrate. However, when GDP is present and GTP is omitted, prenylation of the Q67L mutant is comparable with that of the wild-type (WT) protein. These studies, coupled with the poor prenylation of Rab1BWT in the presence of the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate, support the notion that Rab-GGTase prefers substrates in the GDP conformation. When the abilities of Rab1BQ67L and Rab1BWT to undergo prenylation were compared by metabolic labelling of transiently expressed proteins in cultured human 293 cells, we did not observe a decline in prenylation of the mutant protein as predicted on the basis of the cell-free assays. Moreover, the Q67L mutant was comparable with the wild-type Rab1B in its ability to associate with co-expressed Rab GDP dissociation inhibitors in 293 cells. These findings raise the possibility that unidentified proteins present in intact cells may compensate for the reduced intrinsic GTPase activity of the Q67L mutant, allowing a significant proportion of the nascent Rab1BQ67L to assume a GDP conformation. The differential prenylation of Rab1BQ67L in cell-free systems versus intact cells underscores the importance of evaluating the post-translational modification of specific Rab mutants in vivo, where poorly characterized regulatory proteins may have a significant effect on GTPase activity or nucleotide exchange rates.

  8. A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni).

    Science.gov (United States)

    Kumar, Hitesh; Kumar, Sanjay

    2013-09-15

    The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 araispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.

  9. Early Cessation of Adenosine Diphosphate Receptor Inhibitors Among Acute Myocardial Infarction Patients Treated With Percutaneous Coronary Intervention

    DEFF Research Database (Denmark)

    Fosbøl, Emil L; Ju, Christine; Anstrom, Kevin J

    2016-01-01

    BACKGROUND: Guidelines recommend the use of adenosine diphosphate receptor inhibitor (ADPri) therapy for 1 year postacute myocardial infarction; yet, early cessation of therapy occurs frequently in clinical practice. METHODS AND RESULTS: We examined 11 858 acute myocardial infarction patients tre...... adverse cardiovascular event risk. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01088503....

  10. Adenosine-diphosphate (ADP) receptor antagonists for the prevention of cardiovascular disease in type 2 diabetes mellitus (Review)

    NARCIS (Netherlands)

    Valentine, N.; Laar, F.A. van de; Driel, M.L. van

    2012-01-01

    BACKGROUND: Cardiovascular disease (CVD) is the most prevalent complication of type 2 diabetes with an estimated 65% of people with type 2 diabetes dying from a cause related to atherosclerosis. Adenosine-diphosphate (ADP) receptor antagonists like clopidogrel, ticlopidine, prasugrel and ticagrelor

  11. Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves.

    Science.gov (United States)

    Muñoz-Bertomeu, Jesús; Ros, Roc; Arrillaga, Isabel; Segura, Juan

    2008-01-01

    We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the limonene synthase (LS) gene from spearmint (Mentha spicata), encoding the LS enzyme that catalyzes the synthesis of limonene from geranyl diphosphate. Overexpression of the LS transgene did not consistently affect monoterpene profile in pooled leaves or flowers from transgenic T(0) plants. Analyses from cohorts of leaves sampled at different developmental stages showed that essential oil accumulation in transgenic and control plants was higher in developing than in mature leaves. Furthermore, developing leaves of transgenic plants contained increased limonene contents (more than 450% increase compared to controls) that correlated with the highest transcript accumulation of the LS gene. The levels of other monoterpene pathway components were also significantly altered. T(0) transgenic plants were grown for 2 years, self-pollinated, and the T(1) seeds obtained. The increased limonene phenotype was maintained in the progenies that inherited the LS transgene.

  12. Extending a Single Residue Switch for Abbreviating Catalysis in Plant ent-Kaurene Synthases

    Directory of Open Access Journals (Sweden)

    Meirong Jia

    2016-11-01

    Full Text Available Production of ent-kaurene as a precursor for important signaling molecules such as the gibberellins seems to have arisen early in plant evolution, with corresponding cyclase(s present in all land plants (i.e., embryophyta. The relevant enzymes seem to represent fusion of the class II diterpene cyclase that produces the intermediate ent-copalyl diphosphate (ent-CPP and the subsequently acting class I diterpene synthase that produces ent-kaurene, although the bifunctionality of the ancestral gene is only retained in certain early diverging plants, with gene duplication and sub-functionalization leading to distinct ent-CPP synthases (CPSs and ent-kaurene synthases (KSs generally observed. This evolutionary scenario implies that plant KSs should have conserved structural features uniquely required for production of ent-kaurene relative to related enzymes that have alternative function. Notably, substitution of threonine for a conserved isoleucine has been shown to short-circuit the complex bicyclization and rearrangement reaction catalyzed by KSs after initial cyclization, leading to predominant production of ent-pimaradiene, at least in KSs from angiosperms. Here this effect is shown to extend to KSs from earlier diverging plants (i.e., bryophytes, including a bifunctional CPS/KS. In addition, attribution of the dramatic effect of this single residue switch on product outcome to electrostatic stabilization of the ent-pimarenyl carbocation intermediate formed upon initial cyclization by the hydroxyl introduced by threonine substitution has been called into question by the observation of similar effects from substitution of alanine. Here further mutational analysis and detailed product analysis is reported that supports the importance of electrostatic stabilization by a hydroxyl or water.

  13. Structure of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Entamoeba histolytica.

    Science.gov (United States)

    Edwards, Thomas E; Gardberg, Anna S; Phan, Isabelle Q H; Zhang, Yang; Staker, Bart L; Myler, Peter J; Lorimer, Donald D

    2015-05-01

    Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final step in the synthesis of UDP-GlcNAc, which is involved in cell-wall biogenesis in plants and fungi and in protein glycosylation. Small-molecule inhibitors have been developed against UAP from Trypanosoma brucei that target an allosteric pocket to provide selectivity over the human enzyme. A 1.8 Å resolution crystal structure was determined of UAP from Entamoeba histolytica, an anaerobic parasitic protozoan that causes amoebic dysentery. Although E. histolytica UAP exhibits the same three-domain global architecture as other UAPs, it appears to lack three α-helices at the N-terminus and contains two amino acids in the allosteric pocket that make it appear more like the enzyme from the human host than that from the other parasite T. brucei. Thus, allosteric inhibitors of T. brucei UAP are unlikely to target Entamoeba UAPs.

  14.   Adenosine-diphosphate (ADP) reduces infarct size and improves porcine heart function after myocardial infarction

    DEFF Research Database (Denmark)

    Bune, Laurids Touborg; Larsen, Jens Kjærgaard Rolighed; Thaning, Pia;

    2013-01-01

    (UTP) are both released during myocardial ischemia, influencing hemodynamics. Both mediate the release of tissue plasminogen activator (t-PA), which can reduce infarct size (IS). The objective of this study was to investigate whether exogenous ADP and UTP administration during reperfusion could reduce......Acute myocardial infarction continues to be a major cause of morbidity and mortality. Timely reperfusion can substantially improve outcomes and the administration of cardioprotective substances during reperfusion is therefore highly attractive. Adenosine diphosphate (ADP) and uridine-5-triphoshate...... myocardial IS and whether this correlated to t-PA release or improvements in hemodynamic responses. Hemodynamic variables and t-PA were measured in 22 pigs before, during, and after 45 min of left anterior coronary artery occlusion. During reperfusion, the pigs were randomized to 240 min of intracoronary...

  15. Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast.

    Science.gov (United States)

    Dyda, F; Furey, W; Swaminathan, S; Sax, M; Farrenkopf, B; Jordan, F

    1990-10-15

    Single crystals of the thiamin diphosphate (the vitamin B1 coenzyme)-dependent enzyme pyruvate decarboxylase (EC 4.1.1.1) from brewers' yeast have been grown using polyethylene glycol as a precipitating agent. Crystals of the homotetrameric version alpha 4 of the holoenzyme are triclinic, space group P1, with cell constants a = 81.0, b = 82.4, c = 116.6 A, alpha = 69.5 beta = 72.6, gamma = 62.4 degrees. The crystals are reasonably stable in a rotating anode x-ray beam and diffract to at least 2.5 A resolution. The Vm value of 2.55 A/dalton is consistent with a unit cell containing four subunits with mass of approximately 60 kDa each. Rotation function results with native data indicate strong non-crystallographic 222 symmetry relating the four identical subunits, thus density averaging methods are likely to play a role in the structure determination.

  16. Interaction of thiamin diphosphate with phosphorylated and dephosphorylated mammalian pyruvate dehydrogenase complex.

    Science.gov (United States)

    Liu, Xiaoqing; Bisswanger, Hans

    2005-01-01

    Kinetic and binding studies were carried out on substrate and cofactor interaction with the pyruvate dehydrogenase complex from bovine heart. Fluoropyruvate and pyruvamide, previously described as irreversible and allosteric inhibitors, respectively, are strong competitive inhibitors with respect to pyruvate. Binding of thiamin diphosphate was used to study differences between the active dephosphorylated and inactive phosphorylated enzyme states by spectroscopic methods. The change in both the intrinsic tryptophan fluorescence and the fluorescence of the 6-bromoacetyl-2-dimethylaminonaphthalene-labelled enzyme complex produced on addition of the cofactor showed similar binding behaviour for both enzyme forms, with slightly higher affinity for the phosphorylated form. Changes in the CD spectrum, especially the negative Cotton effect at 330 nm as a function of cofactor concentration, both in the absence and presence of pyruvate, also revealed no drastic differences between the two enzyme forms. Thus, inactivation of the enzyme activity of the pyruvate dehydrogenase complex is not caused by impeding the binding of substrate or cofactor.

  17. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase.

    Science.gov (United States)

    Jeudy, Sandra; Coutard, Bruno; Lebrun, Régine; Abergel, Chantal

    2005-06-01

    The complete sequence of the largest known double-stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined [Raoult et al. (2004), Science, 306, 1344-1350] and revealed numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated [Abergel et al. (2005), Acta Cryst. F61, 212-215] both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P2(1)3, with unit-cell parameter 99.425 A. The self-rotation function confirms that there are two monomers per asymmetric unit related by a twofold non-crystallographic axis and that the unit cell thus contains four biological entities.

  18. Structure activity relationship of uridine 5′-diphosphate analogues at the human P2Y6 receptor

    Science.gov (United States)

    Besada, Pedro; Shin, Dae Hong; Costanzi, Stefano; Ko, Hyojin; Mathé, Christophe; Gagneron, Julien; Gosselin, Gilles; Maddileti, Savitri; Harden, T. Kendall; Jacobsona, Kenneth A.

    2012-01-01

    The structure activity relationships and molecular modeling of the uracil nucleotide-activated P2Y6 receptor have been studied. A series of UDP analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosphate group was synthesized and assayed for activity at the human P2Y6 receptor. The uracil ring was modified at the 4-position, with the synthesis of 4-substituted-thiouridine-5′-diphosphate analogues, as well as at positions 3 and 5. The effect of modifications at the level of the phosphate chain was studied by preparing a cyclic 3′,5′-diphosphate analogue, a 3′-diphosphate analogue and several dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 0.15 μM) was equipotent to UDP, while substitutions of the 2′-hydroxyl (amino, azido) greatly reduce potency. 2- and 4-Thio analogues, 20 and 21, respectively, were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency. Molecular modeling indicates that the β-phosphate of 5′-UDP and analogs is essential for the establishment of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among 4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 μM, indicative of favorable interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S) conformation (P= 126°, 1′-exo) was consistent with molecular modeling. These results provide a better understanding of molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor ligands PMID:16942026

  19. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2011-10-28

    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  20. Use of cyclodextrins in biotransformation reactions with cell cultures of Morus nigra: biosynthesis of prenylated chalcone isocordoin.

    Science.gov (United States)

    Bolasco, Adriana; Fioravanti, Rossella; Rossi, Francesca; Rossi, Paola; Vitali, Alberto

    2010-06-16

    In vivo biotransformation experiments were performed by using a cell suspension culture of Morus nigra expressing a high PT (prenyltransferase) activity, fed with the target substrate 2',4'-dihydroxychalcone. In order to improve the reaction yields by enhancing the chalcone solubility, three different cyclodextrins have been used to host the substrate. The respective complexes have been studied by means of both spectroscopic and calorimetric techniques (Fourier-transform infrared, 1H-NMR and differential scanning calorimetry) and the solution behaviours have been characterized by solubility phase studies. The hydroxypropyl-beta-cyclodextrin complex was found to be the most suitable for biotransformation, and the reaction of prenylation resulted in a 6-fold higher yield of the final product when compared with the use of the free substrate. The reaction provided as the sole product the 3'-dimethylallyl derivative isocordoin, a biologically active plant compound. The results obtained allow the development of systems based on the use of biofermentors or the use of immobilized cells in order to enhance the biotransformation yields.

  1. Enzymatic prenylation and oxime ligation for the synthesis of stable and homogeneous protein-drug conjugates for targeted therapy.

    Science.gov (United States)

    Lee, Joong-Jae; Choi, Hyo-Jung; Yun, Misun; Kang, YingJin; Jung, Ji-Eun; Ryu, Yiseul; Kim, Tae Yoon; Cha, Young-Je; Cho, Hyun-Soo; Min, Jung-Joon; Chung, Chul-Woong; Kim, Hak-Sung

    2015-10-05

    Targeted therapy based on protein-drug conjugates has attracted significant attention owing to its high efficacy and low side effects. However, efficient and stable drug conjugation to a protein binder remains a challenge. Herein, a chemoenzymatic method to generate highly stable and homogenous drug conjugates with high efficiency is presented. The approach comprises the insertion of the CaaX sequence at the C-terminal end of the protein binder, prenylation using farnesyltransferase, and drug conjugation through an oxime ligation reaction. MMAF and an EGFR-specific repebody are used as the antitumor agent and protein binder, respectively. The method enables the precisely controlled synthesis of repebody-drug conjugates with high yield and homogeneity. The utility of this approach is illustrated by the notable stability of the repebody-drug conjugates in human plasma, negligible off-target effects, and a remarkable antitumor activity in vivo. The present method can be widely used for generating highly homogeneous and stable PDCs for targeted therapy.

  2. The molecular organization of prenylated flavonoid xanthohumol in DPPC multibilayers: X-ray diffraction and FTIR spectroscopic studies.

    Science.gov (United States)

    Arczewska, Marta; Kamiński, Daniel M; Górecka, Ewa; Pociecha, Damian; Rój, Edward; Sławińska-Brych, Adrianna; Gagoś, Mariusz

    2013-02-01

    Xanthohumol (XN) is the major prenylated flavonoid found in hop resin. It has attracted considerable attention in recent years due to its wide spectrum of biological activities and the beneficial effect on human health. Since lipid membrane is first target for biologically active compounds, we decided to investigate the influence of XN on the dipalmitoylphosphatidylcholine (DPPC) multibilayers. Interactions of XN with DPPC were investigated as a function of temperature and its concentration by using X-ray diffraction and the ATR-FTIR spectroscopy techniques. The aim of understanding the mechanisms of molecular interactions between XN and DPPC was to indicate the localization of the XN with respect to the membrane and the type of interaction with phospholipids. The results revealed that XN changes the physical properties of the DPPC multibilayers in the form of dry film. A new complex formation between XN and DPPC is reported. The detailed analysis of refraction effect indicates the changes in electron density ratio between hydrophobic and hydrophilic zones of lipid at phase transition. This is in compliance with reported changes in FTIR spectra where at pretransition XN moves from interface region between polar heads to the neighborhood of phosphate groups.

  3. Xanthohumol, a Prenylated Chalcone from Hops, Inhibits the Viability and Stemness of Doxorubicin-Resistant MCF-7/ADR Cells

    Directory of Open Access Journals (Sweden)

    Ming Liu

    2016-12-01

    Full Text Available Xanthohumol is a unique prenylated flavonoid in hops (Humulus lupulus L. and beer. Xanthohumol has been shown to possess a variety of pharmacological activities. There is little research on its effect on doxorubicin-resistant breast cancer cells (MCF-7/ADR and the cancer stem-like cells exiting in this cell line. In the present study, we investigate the effect of xanthohumol on the viability and stemness of MCF-7/ADR cells. Xanthohumol inhibits viability, induces apoptosis, and arrests the cell cycle of MCF-7/ADR cells in a dose-dependent manner; in addition, xanthohumol sensitizes the inhibition effect of doxorubicin on MCF-7/ADR cells. Interestingly, we also find that xanthohumol can reduce the stemness of MCF-7/ADR cells evidenced by the xanthohumol-induced decrease in the colony formation, the migration, the percentage of side population cells, the sphere formation, and the down-regulation of stemness-related biomarkers. These results demonstrate that xanthohumol is a promising compound targeting the doxorubicin resistant breast cancer cells and regulating their stemness, which, therefore, will be applied as a potential candidate for the development of a doxorubicin-resistant breast cancer agent and combination therapy of breast cancer.

  4. Xanthohumol, a Prenylated Chalcone from Hops, Inhibits the Viability and Stemness of Doxorubicin-Resistant MCF-7/ADR Cells.

    Science.gov (United States)

    Liu, Ming; Yin, Hua; Qian, Xiaokun; Dong, Jianjun; Qian, Zhonghua; Miao, Jinlai

    2016-12-28

    Xanthohumol is a unique prenylated flavonoid in hops (Humulus lupulus L.) and beer. Xanthohumol has been shown to possess a variety of pharmacological activities. There is little research on its effect on doxorubicin-resistant breast cancer cells (MCF-7/ADR) and the cancer stem-like cells exiting in this cell line. In the present study, we investigate the effect of xanthohumol on the viability and stemness of MCF-7/ADR cells. Xanthohumol inhibits viability, induces apoptosis, and arrests the cell cycle of MCF-7/ADR cells in a dose-dependent manner; in addition, xanthohumol sensitizes the inhibition effect of doxorubicin on MCF-7/ADR cells. Interestingly, we also find that xanthohumol can reduce the stemness of MCF-7/ADR cells evidenced by the xanthohumol-induced decrease in the colony formation, the migration, the percentage of side population cells, the sphere formation, and the down-regulation of stemness-related biomarkers. These results demonstrate that xanthohumol is a promising compound targeting the doxorubicin resistant breast cancer cells and regulating their stemness, which, therefore, will be applied as a potential candidate for the development of a doxorubicin-resistant breast cancer agent and combination therapy of breast cancer.

  5. Comparison of different extraction methods and HPLC quantification of prenylated and unprenylated phenylpropanoids in raw Italian propolis.

    Science.gov (United States)

    Taddeo, Vito Alessandro; Epifano, Francesco; Fiorito, Serena; Genovese, Salvatore

    2016-09-10

    In this paper the presence of selected prenylated and unprenylated phenylpropanoids, namely ferulic acid 1, boropinic acid 2, 4'-geranyloxyferulic acid 3, umbelliferone 4, 7-isopentenyloxycoumarin 5, and auraptene 6, have been determined in Italian raw propolis after having been extracted with different methodologies. An aqueous solution of β-cyclodextrin was the best extraction method for ferulic acid 1, treatment with indifferently EtOH or aqueous β-cyclodextrin were the most effective one for umbelliferone 4, boropinic acid 2 gave the best yields either with H2O/β-cyclodextrin or olive oil treatment or in biphasic systems, maceration with biphasic mixtures of aqueous β-cyclodextrin and olive oil was seen to be the most effective procedure for 7-isopentenyloxycoumarin 5, the only method providing significant quantities of 4'-geranyloxyferulic acid 3 was the maceration of raw propolis with olive oil, and finally auraptene 4 was best extracted with absolute EtOH. "Classic" maceration in general performed better than ultrasound-assisted one.

  6. Prenyl Ammonium Salts – New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model

    Science.gov (United States)

    Grecka, Emilia; Statkiewicz, Malgorzata; Gorska, Agnieszka; Biernacka, Marzena; Grygorowicz, Monika Anna; Masnyk, Marek; Chmielewski, Marek; Gawarecka, Katarzyna; Chojnacki, Tadeusz; Swiezewska, Ewa; Malecki, Maciej

    2016-01-01

    Purpose Prenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents. Methods AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells. Results All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation—considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination. Conclusion Obtained results indicate that APs have a potential as non-viral vectors for cell transfection. PMID:27088717

  7. Prenyl Ammonium Salts--New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model.

    Science.gov (United States)

    Grecka, Emilia; Statkiewicz, Malgorzata; Gorska, Agnieszka; Biernacka, Marzena; Grygorowicz, Monika Anna; Masnyk, Marek; Chmielewski, Marek; Gawarecka, Katarzyna; Chojnacki, Tadeusz; Swiezewska, Ewa; Malecki, Maciej

    2016-01-01

    Prenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents. AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells. All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation--considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination. Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.

  8. Stathmin slows down guanosine diphosphate dissociation from tubulin in a phosphorylation-controlled fashion.

    Science.gov (United States)

    Amayed, P; Carlier, M F; Pantaloni, D

    2000-10-10

    Stathmin is an important protein that interacts with tubulin and regulates microtubule dynamics in a phosphorylation-controlled fashion. Here we show that the dissociation of guanosine 5'-diphosphate (GDP) from beta-tubulin is slowed 20-fold in the (tubulin)(2)-stathmin ternary complex (T(2)S). The kinetics of GDP or guanosine 5'-triphosphate (GTP) dissociation from tubulin have been monitored by the change in tryptophan fluorescence of tubulin upon exchanging 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-diphosphate (S6-GDP) for tubulin-bound guanine nucleotide. At molar ratios of stathmin to tubulin lower than 0.5, biphasic kinetics were observed, indicating that the dynamics of the complex is extremely slow, consistent with its high stability. The method was used to characterize the effects of phosphorylation of stathmin on its interaction with tubulin. The serine-to-glutamate substitution of all four phosphorylatable serines of stathmin (4E-stathmin) weakens the stability of the T(2)S complex by about 2 orders of magnitude. The phosphorylation of serines 16 and 63 in stathmin has a more severe effect and weakens the stability of T(2)S 10(4)-fold. The rate of GDP dissociation is lowered only 7-fold and 4-fold in the complexes of tubulin with 4E-stathmin and diphosphostathmin, respectively. Sedimentation velocity studies support the conclusions of nucleotide exchange data and show that the T(2)S complexes formed between tubulin and 4E-stathmin or diphosphostathmin are less compact than the highly stable T(2)S complex. The correlation between the effect of phosphorylation of stathmin on the stability of T(2)S complex measured in vitro and on the function of stathmin in vivo is discussed.

  9. An intersubunit disulfide bridge stabilizes the tetrameric nucleoside diphosphate kinase of Aquifex aeolicus.

    Science.gov (United States)

    Boissier, Fanny; Georgescauld, Florian; Moynié, Lucile; Dupuy, Jean-William; Sarger, Claude; Podar, Mircea; Lascu, Ioan; Giraud, Marie-France; Dautant, Alain

    2012-06-01

    The nucleoside diphosphate kinase (Ndk) catalyzes the reversible transfer of the γ-phosphate from nucleoside triphosphate to nucleoside diphosphate. Ndks form hexamers or two types of tetramers made of the same building block, namely, the common dimer. The secondary interfaces of the Type I tetramer found in Myxococcus xanthus Ndk and of the Type II found in Escherichia coli Ndk involve the opposite sides of subunits. Up to now, the few available structures of Ndk from thermophiles were hexameric. Here, we determined the X-ray structures of four crystal forms of the Ndk from the hyperthermophilic bacterium Aquifex aeolicus (Aa-Ndk). Aa-Ndk displays numerous features of thermostable proteins and is made of the common dimer but it is a tetramer of Type I. Indeed, the insertion of three residues in a surface-exposed spiral loop, named the Kpn-loop, leads to the formation of a two-turn α-helix that prevents both hexamer and Type II tetramer assembly. Moreover, the side chain of the cysteine at position 133, which is not present in other Ndk sequences, adopts two alternate conformations. Through the secondary interface, each one forms a disulfide bridge with the equivalent Cys133 from the neighboring subunit. This disulfide bridge was progressively broken during X-ray data collection by radiation damage. Such crosslinks counterbalance the weakness of the common-dimer interface. A 40% decrease of the kinase activity at 60°C after reduction and alkylation of the protein corroborates the structural relevance of the disulfide bridge on the tetramer assembly and enzymatic function.

  10. A STUDY OF THE NUCLEOSIDE TRI- AND DIPHOSPHATE ACTIVITIES OF RAT LIVER MICROSOMES

    Science.gov (United States)

    Ernster, Lars; Jones, Lois C.

    1962-01-01

    Rat liver microsomes catalyze the hydrolysis of the triphosphates of adenosine, guanosine, uridine, cytidine, and inosine into the corresponding diphosphates and inorganic orthophosphate. The activities are stimulated by Na2S2O4, and inhibited by atebrin, chlorpromazine, sodium azide, and deaminothyroxine. Sodium deoxycholate inhibits the ATPase activity in a progressive manner; the release of orthophosphate from GTP and UTP is stimulated by low, and inhibited by high, concentrations of deoxycholate, and that from CTP and ITP is unaffected by low, and inhibited by high, concentrations of deoxycholate. Subfractionation of microsomes with deoxycholate into ribosomal, membrane, and soluble fractions reveals a concentration of the triphosphatase activity in the membrane fraction. Rat liver microsomes also catalyze the hydrolysis of the diphosphates of the above nucleosides into the corresponding monophosphates and inorganic orthophosphate. Deoxycholate strongly enhances the GDPase, UDPase, and IDPase activities while causing no activation or even inhibition of the ADPase and CDPase activities. The diphosphatase is unaffected by Na2S2O4 and is inhibited by azide and deaminothyroxine but not by atebrin or chlorpromazine. Upon fractionation of the microsomes with deoxycholate, a large part of the GDPase, UDPase, and IDPase activities is recovered in the soluble fraction. Mechanical disruption of the microsomes with an Ultra Turrax Blender both activates and releases the GDPase, UDPase, and IDPase activities, and the former effect occurs more readily than the latter. The GDPase, UDPase, and IDPase activities of the rat liver cell reside almost exclusively in the microsomal fraction, as revealed by comparative assays of the mitochondrial, microsomal, and final supernatant fractions of the homogenate. The microsomes exhibit relatively low nucleoside monophosphatase and inorganic pyrophosphatase activities, and these are unaffected by deoxycholate or mechanical treatment

  11. Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects.

    Science.gov (United States)

    Chakraborty, Sumit; Nemeria, Natalia; Yep, Alejandra; McLeish, Michael J; Kenyon, George L; Jordan, Frank

    2008-03-25

    Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of ( R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at lambda max 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of ( R)-benzoin synthesis, the (1)H/ (2)H kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 +/- 0.03), indicating that ionization of the C2alphaH from C2alpha-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that while a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1',4'-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue ( E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product ( E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures.

  12. Repeated febrile convulsions impair hippocampal neurons and cause synaptic damage in immature rats:neuroprotective effect of fructose-1,6-diphosphate

    Institute of Scientific and Technical Information of China (English)

    Jianping Zhou; Fan Wang; Jun Zhang; Hui Gao; Yufeng Yang; Rongguo Fu

    2014-01-01

    Fructose-1,6-diphosphate is a metabolic intermediate that promotes cell metabolism. We hy-pothesize that fructose-1,6-diphosphate can protect against neuronal damage induced by febrile convulsions. Hot-water bathing was used to establish a repetitive febrile convulsion model in rats aged 21 days, equivalent to 3-5 years in humans. Ninety minutes before each seizure induc-tion, rats received an intraperitoneal injection of low- or high-dose fructose-1,6-diphosphate (500 or 1,000 mg/kg, respectively). Low- and high-dose fructose-1,6-diphosphate prolonged the latency and shortened the duration of seizures. Furthermore, high-dose fructose-1,6-di-phosphate effectively reduced seizure severity. Transmission electron microscopy revealed that 24 hours after the last seizure, high-dose fructose-1,6-diphosphate reduced mitochondrial swelling, rough endoplasmic reticulum degranulation, Golgi dilation and synaptic cleft size, and increased synaptic active zone length, postsynaptic density thickness, and synaptic interface cur-vature in the hippocampal CA1 area. The present findings suggest that fructose-1,6-diphosphate is a neuroprotectant against hippocampal neuron and synapse damage induced by repeated fe-brile convulsion in immature rats.

  13. Evaluation of the sorption of Eu(III) in titanium diphosphate; Evaluacion de la sorcion de Eu(III) en difosfato de titanio

    Energy Technology Data Exchange (ETDEWEB)

    Ortiz O, H.B.; Ordonez R, E.; Fernandez V, S.M. [ININ, Carretera Mexico-Toluca Km 36.5, Salazar, Estado de Mexico (Mexico)]. e-mail: hortiz@nuclear.inin.mx

    2007-07-01

    In this work its are presented: the synthesis, physicochemical characterization and the surface parameters estimation that can be related with the retention properties of the titanium diphosphate for the actinides of valence III (Pu, Am, Cm among others), using the Eu{sup 3+} like a chemical analog. The surface area, hydration time, zero charge point, density of active sites and the surface species distribution in the titanium diphosphate are reported. This information was used to explain the retention of the Eu(lll) in the surface of the titanium diphosphate. (Author)

  14. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  15. Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization-Fragmentation Reaction Sequence.

    Science.gov (United States)

    Harris, Golda G; Lombardi, Patrick M; Pemberton, Travis A; Matsui, Tsutomu; Weiss, Thomas M; Cole, Kathryn E; Köksal, Mustafa; Murphy, Frank V; Vedula, L Sangeetha; Chou, Wayne K W; Cane, David E; Christianson, David W

    2015-12-01

    Geosmin synthase from Streptomyces coelicolor (ScGS) catalyzes an unusual, metal-dependent terpenoid cyclization and fragmentation reaction sequence. Two distinct active sites are required for catalysis: the N-terminal domain catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate (PPi), and the C-terminal domain catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone through a retro-Prins reaction. A unique αα domain architecture is predicted for ScGS based on amino acid sequence: each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg(2+) for catalysis. Here, we report the X-ray crystal structure of the unliganded N-terminal domain of ScGS and the structure of its complex with three Mg(2+) ions and alendronate. These structures highlight conformational changes required for active site closure and catalysis. Although neither full-length ScGS nor constructs of the C-terminal domain could be crystallized, homology models of the C-terminal domain were constructed on the basis of ∼36% sequence identity with the N-terminal domain. Small-angle X-ray scattering experiments yield low-resolution molecular envelopes into which the N-terminal domain crystal structure and the C-terminal domain homology model were fit, suggesting possible αα domain architectures as frameworks for bifunctional catalysis.

  16. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    Science.gov (United States)

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

  17. A new dimeric dihydrochalcone and a new prenylated flavone from the bud covers of Artocarpus altilis: potent inhibitors of cathepsin K.

    Science.gov (United States)

    Patil, Ashok D; Freyer, Alan J; Killmer, Lew; Offen, Priscilla; Taylor, Paul B; Votta, Bartholomew J; Johnson, Randall K

    2002-04-01

    A MeOH/CH(2)Cl(2) extract of the bud covers of Artocarpus altilis collected in Micronesia showed activity in a cathepsin K inhibition assay. In addition to the three known flavonoids isolated from the bud covers of this species, two new compounds have been identified whose structures were determined on the basis of spectral data. These compounds include a dimeric dihydrochalcone, cycloaltilisin 6 (2), and a new prenylated flavone, cycloaltilisin 7 (3). Novel compounds 2 and 3 have IC(50) values of 98 and 840 nM, respectively, in cathepsin inhibition.

  18. A Comparative Study on the Metabolism of Epimedium koreanum Nakai-Prenylated Flavonoids in Rats by an Intestinal Enzyme (Lactase Phlorizin Hydrolase) and Intestinal Flora

    OpenAIRE

    Jing Zhou; Yan Chen; Ying Wang; Xia Gao; Ding Qu; Congyan Liu

    2013-01-01

    The aim of this study was to compare the significance of the intestinal hydrolysis of prenylated flavonoids in Herba Epimedii by an intestinal enzyme and flora. Flavonoids were incubated at 37 °C with rat intestinal enzyme and intestinal flora. HPLC-UV was used to calculate the metabolic rates of the parent drug in the incubation and LC/MS/MS was used to determine the chemical structures of metabolites generated by different flavonoid glycosides. Rates of flavonoid metabolism by rat intestin...

  19. Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

    Directory of Open Access Journals (Sweden)

    Brans Alain

    2007-08-01

    Full Text Available Abstract Background We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP, in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. Results Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried out from thiamine diphosphate (ThDP and ADP or ATP by a soluble high molecular mass nucleotidyl transferase. We partially purified this enzyme and characterized some of its functional properties. The enzyme activity had an absolute requirement for divalent metal ions, such as Mn2+ or Mg2+, as well as for a heat-stable soluble activator present in bacterial extracts. The enzyme has a pH optimum of 6.5–7.0 and a high Km for ThDP (5 mM, suggesting that, in vivo, the rate of AThTP synthesis is proportional to the free ThDP concentration. When ADP was used as the variable substrate at a fixed ThDP concentration, a sigmoid curve was obtained, with a Hill coefficient of 2.1 and an S0.5 value of 0.08 mM. The specificity of the AThTP synthesizing enzyme with respect to nucleotide substrate is restricted to ATP/ADP, and only ThDP can serve as the second substrate of the reaction. We tentatively named this enzyme ThDP adenylyl transferase (EC 2.7.7.65. Conclusion This is the first demonstration of an enzyme activity transferring a nucleotidyl group on thiamine diphosphate to produce AThTP. The existence of a mechanism for the enzymatic synthesis of this compound is in agreement with the hypothesis of a non-cofactor role for thiamine derivatives in living cells.

  20. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G

    1996-01-01

    to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic...... phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP...... of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  1. Peculiarities of the inhibition of the pyruvate dehydrogenase complex by thiamine thiazolone diphosphate in vitro and in intact mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Yakovleva, G.M.; Strumilo, S.A.; Gorenshtein, B.I.; Ostrovskii, Yu.M.

    1986-07-10

    Thiamine thiazolone diphosphate (TTPP) possesses the ability to penetrate through the mitochondrial membrane and inhibit the pyruvate dehydrogenase complex in intact mitochondria, TTPP inhibits the activity of the complex of animal origin according to a mixed type (K/sub i/ 5 x 10/sup -8/ M) and yeast pyruvate decarboxylase according to a competitive type (K/sub i/ 5 x 10/sup -6/ M) with respect to thiamine diphosphate (TPP). Decarboxylation of pyruvate in intact and lysed rat liver and brain mitochondria is inhibited in the presence of TTPP significantly more weakly than the total activity of the pyruvate dehydrogenase complex, determined according to the formation of acetyl-CoA. It is suggested that TTPP, as an analog of the transition state, acts only in dehydrogenase reactions but not at the stage of simple decarboxylation of pyruvate.

  2. Activation and modulation of cardiac poly-adenosine diphosphate ribose polymerase activity in a rat model of brain death.

    Science.gov (United States)

    Brain, John G; Rostron, Anthony J; Dark, John H; Kirby, John A

    2008-05-15

    DNA damage during transplantation can activate poly-adenosine diphosphate ribose polymerase (PARP) resulting in the generation of polymers of adenosine diphosphate-ribose (PAR). Excessive linkage of PAR to nuclear proteins can induce cell death, thereby limiting the function of transplanted organs. This study uses a rat model of brain death to determine the profile of PARP activation and whether mechanisms that lead to cell death can be ameliorated by appropriate donor resuscitation. The expression of PAR-linked nuclear proteins within cardiac myocytes was greatly increased after the induction of donor brain death. Importantly, infusion of noradrenaline or vasopressin to normalize the chronic hypotension produced by brain death reduced the expression of PAR to a level below baseline. These data suggest that chronic hypotension after donor brain death has the potential to limit cardiac function through the activation of PARP; however, this early cause of graft damage can be mitigated by appropriate donor resuscitation.

  3. Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase

    Science.gov (United States)

    Ker, De-Sheng; Pang, Sze Lei; Othman, Noor Farhan; Kumaran, Sekar; Tan, Ee Fun; Krishnan, Thiba; Chan, Kok Gan; Othman, Roohaida

    2017-01-01

    Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation

  4. Saccharomyces pastorianus as cell factory to improve production of fructose 1,6-diphosphate using novel fermentation strategies

    Directory of Open Access Journals (Sweden)

    Chiara Schiraldi

    2015-08-01

    Full Text Available Enzymatic phosphorylation of glucose with inorganic phosphate, mediated by permeabilized yeast cells, is one of the methods commonly used to manufacture fructose 1,6-diphosphate, a compound of pharmaceutical interest. This process requires high concentrations of yeast active biomass, that is the catalyst of bioconversion of glucose and inorganic phosphate into fructose 1,6-diphosphate. In this study we firstly describe the high cell density production of a brewer's Saccharomyces strain (Saccharomyces pastorianus DSM 6581, focusing on the optimization of medium composition and exploiting fed-batch strategies and novel technologies based on membrane bioreactors. In fed-batch fermentation an appropriate exponential feed profile was set up to maintain the glucose concentration in the bioreactor below 0.9 g·L-1, thus yielding reproducibly 58 g dry weight biomass per liter in 80 h fermentation, improving eight-fold batch processes output. In addition a higher final biomass density was reached when implementing a microfiltration strategy (70 g dry weight biomass, that led to a productivity of 2.1 gcdw·L-1·h-1, 2.4-fold the fed-batch one. Successively, this biomass was opportunely permeabilized and proved capable of catalyzing the bioconversion of glucose into fructose 1,6-diphosphate. Acting on critical parameters of the bioconversion (substrates molar ratio, catalyst concentration and permeabilization agent, fructose 1,6-diphosphate was produced, after 3 h of process, at 56.3 ± 1 g·L-1 with a yield of 80% of the theoretical value.

  5. Feasibility of gas/solid carboligation: conversion of benzaldehyde to benzoin using thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Mikolajek, R; Spiess, A C; Büchs, J

    2007-05-10

    A carboligation was investigated for the first time as an enzymatic gas phase reaction, where benzaldehyde was converted to benzoin using thiamine diphosphate (ThDP)-dependent enzymes, namely benzaldehyde lyase (BAL) and benzoylformate decarboxylase (BFD). The biocatalyst was immobilized per deposition on non-porous support. Some limitations of the gas/solid biocatalysis are discussed based on this carboligation and it is also demonstrated that the solid/gas system is an interesting tool for more volatile products.

  6. A tailor-made chimeric thiamine diphosphate dependent enzyme for the direct asymmetric synthesis of (S)-benzoins.

    Science.gov (United States)

    Westphal, Robert; Vogel, Constantin; Schmitz, Carlo; Pleiss, Jürgen; Müller, Michael; Pohl, Martina; Rother, Dörte

    2014-08-25

    Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to (S)-benzoins have not so far been identified. We were able to close this gap by constructing a chimeric enzyme, which catalyzes the synthesis of various (S)-benzoins with excellent enantiomeric excess (>99%) and very good conversion.

  7. Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate

    OpenAIRE

    Singleton Charles K; Song Qilin

    2002-01-01

    Abstract Background Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. Results Here we examine Th...

  8. Biosynthesis of anthecotuloide, an irregular sesquiterpene lactone from Anthemis cotula L. (Asteraceae) via a non-farnesyl diphosphate route.

    Science.gov (United States)

    van Klink, John; Becker, Hans; Andersson, Susannah; Boland, Wilhelm

    2003-05-07

    Retrobiosynthetic analysis of the allergenic sesquiterpene lactone, anthecotuloide, suggested that this natural product could be formed either by head to head condensation of geranyl diphosphate with dimethylallyl diphosphate, or from farnesyl diphosphate (FPP), the accepted regular sesquiterpene precursor via the rearrangement of a germacranolide precursor. Isotopic labelling of anthecotuloide has now been achieved by feeding [1-(13)C]-glucose, [U-13C6]-glucose and [6,6-(2)H2]-glucose to aseptically grown plantlets of Anthemis cotula(family Asteraceae). Analysis of labelling patterns and absolute 13C abundances using quantitative 13C NMR spectroscopy showed that the isoprene building blocks of this sesquiterpene are formed exclusively via the MEP terpene biosynthetic pathway. This was supported by results from an experiment using [U-13C6]-glucose. A deuterium labelling experiment using [6,6-(2)H2]-glucose supported the original proposal and showed that anthecotuloide is formed from a non FPP precursor. Isotope ratio mass spectrometry suggested that there were two pathways for sesquiterpene biosynthesis in A. cotula.

  9. Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

    Science.gov (United States)

    Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

    1993-01-01

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

  10. Evaluation of serum nucleoside diphosphate kinase A for the detection of colorectal cancer

    Science.gov (United States)

    Otero-Estévez, Olalla; De Chiara, Loretta; Barcia-Castro, Leticia; Páez de la Cadena, María; Rodríguez-Berrocal, Francisco Javier; Cubiella, Joaquín; Hernández, Vicent; Martínez-Zorzano, Vicenta Soledad

    2016-01-01

    We previously described the over-expression of nucleoside diphosphate kinase A (NDKA) in tumours and serum from colorectal cancer (CRC) patients, suggesting its use as biomarker. In this study we evaluated the diagnostic accuracy of serum NDKA to detect advanced neoplasia (CRC or advanced adenomas). Furthermore, the performance of NDKA was compared with the faecal immunochemical test (FIT). The study population included a case-control cohort and a screening cohort (511 asymptomatic first-degree relatives of CRC patients that underwent a colonoscopy and a FIT). Serum NDKA was elevated in CRC patients in the case-control cohort (p = 0.002). In the screening cohort, NDKA levels were higher for advanced adenomas (p = 0.010) and advanced neoplasia (p = 0.006) compared to no neoplasia. Moreover, elevated NDKA was associated with severe characteristics of adenomas (≥3 lesions, size ≥ 1 cm or villous component). Setting specificity to 85%, NDKA showed a sensitivity of 30.19% and 29.82% for advanced adenomas and advanced neoplasia, respectively. NDKA combined with FIT (100 ng/mL cut-off) detected advanced adenomas and advanced neoplasia with 45.28% and 49.12% sensitivity, with specificity close to 90%. The combination of serum NDKA and FIT can improve the detection of advanced neoplasia, mainly for lesions located on the proximal colon, in asymptomatic individuals with CRC family-risk. PMID:27222072

  11. Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli.

    Science.gov (United States)

    Schulz, Ryan; Zhang, Yian-Biao; Liu, Chang-Jun; Freimuth, Paul

    2007-12-01

    Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.

  12. Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca2+ signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Wenjie; Wei; Richard; Graeff; Jianbo; Yue

    2014-01-01

    Mobilization of intracellular Ca2+ stores is involved inmany diverse cell functions, including: cell proliferation;differentiation; fertilization; muscle contraction; secre-tion of neurotransmitters, hormones and enzymes;and lymphocyte activation and proliferation. Cyclic ad-enosine diphosphate ribose(cADPR) is an endogenousCa2+ mobilizing nucleotide present in many cell typesand species, from plants to animals. cADPR is formedby ADP-ribosyl cyclases from nicotinamide adenine di-nucleotide. The main ADP-ribosyl cyclase in mammalsis CD38, a multi-functional enzyme and a type Ⅱ mem-brane protein. It has been shown that many extracel-lular stimuli can induce cADPR production that leadsto calcium release or influx, establishing cADPR as asecond messenger. cADPR has been linked to a widevariety of cellular processes, but the molecular mecha-nisms regarding cADPR signaling remain elusive. Theaim of this review is to summarize the CD38/cADPR/Ca2+ signaling pathway, focusing on the recent advanc-es involving the mechanism and physiological functionsof cADPR-mediated Ca2+ mobilization.

  13. Solubility of triuranyl diphosphate tetrahydrate (TDT) and Na autunite at 23 and 50 degrees C

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, Christopher R.; Felmy, Andrew R.; Clark, Sue B.

    2010-11-01

    In this report we present experimental solubility data for well-characterized triuranyl diphosphate tetrahydrate (TDT: (UO2)(3)(PO4)(2)center dot 4H(2)O) and Na autunite (Na[UO2PO4]center dot xH(2)O) at 23 and 50 degrees C in NaClO4-HClO4 solutions at pC(H+) = 2. Duplicate samples of TDT in 0.1, 0.5, 1.0, 2.0 and 5.0 in solutions were equilibrated at 23 and 50 degrees C. TDT solid was synthesized and characterized with ICP-OES, ATR-IR and powder XRD before and after solubility experiments. The pH of the suspensions were monitored throughout the experiments. Equilibrium was achieved from undersaturation with respect to TDT and oversaturation for Na autunite. Steady-state conditions were achieved in all cases within 82 d. TDT was unstable at ionic strengths above 0.1 m, where its complete conversion to Na autunite was observed. The ion-interaction model was used to interpret the experimental solubility data. The solubility product, log K-sp, for TDT was determined to be -49.7 and -51.3 at 23 and 50 degrees C respectively. log K for Na autunite was determined to be -24.4 (23 degrees C) and -24.1 +/- 0.2 (50 degrees C).

  14. Solubility of triuranyl diphosphate tetrahydrate (TDT) and Na autunite at 23 and 50 C

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, C.R.; Clark, S.B. [Washington State Univ., Pullman, WA (United States); Felmy, A.R. [Pacific Northwest National Lab., Richland, WA (United States)

    2010-07-01

    In this report we present experimental solubility data for well-characterized triuranyl diphosphate tetrahydrate (TDT: (UO{sub 2}){sub 3}(PO{sub 4}){sub 2} . 4H{sub 2}O) and Na autunite (Na[UO{sub 2}PO{sub 4}] . xH{sub 2}O) at 23 and 50 C in NaClO{sub 4}-HClO{sub 4} solutions at pC{sub H1} = 2. Duplicate samples of TDT in 0.1, 0.5, 1.0, 2.0 and 5.0 m solutions were equilibrated at 23 and 50 C. TDT solid was synthesized and characterized with ICP-OES, ATR-IR and powder XRD before and after solubility experiments. The pH of the suspensions were monitored throughout the experiments. Equilibrium was achieved from undersaturation with respect to TDT and oversaturation for Na autunite. Steady-state conditions were achieved in all cases within 82 d. TDT was unstable at ionic strengths above 0.1 m, where its complete conversion to Na autunite was observed. The ion-interaction model was used to interpret the experimental solubility data. The solubility product, log K{sub sp}, for TDT was determined to be -49.7 and -51.3 at 23 and 50 C respectively. log K{sub sp} for Na autunite was determined to be -24.4 (23 C) and -24.1 {+-} 0.2 (50 C). (orig.)

  15. The uridine diphosphate glucuronosyltransferases: quantitative structure-activity relationships for hydroxyl polychlorinated biphenyl substrates

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Degao [Dalian University of Technology, Department of Environmental Science and Technology, Dalian (China)

    2005-10-01

    Quantitative structure-activity relationships (QSARs), which relate the glucuronidation of hydroxyl polychlorinated biphenyls (OH-PCBs) - catalyzed by the uridine diphosphate glucuronosyltransferases (UGTs) - to their physicochemical properties and molecular structural parameters, can be used to predict the rate constants and interpret the mechanism of glucuronidation. In this study, QSARs have been developed that use 23 semi-empirical calculated quantum chemical descriptors to predict the logarithms of the constants 1/K{sub m} and V{sub max}, related to enzyme kinetics. A partial least squares regression method was used to select the optimal set of descriptors to minimize the multicollinearity between the descriptors, as well as to maximize the cross-validated coefficient (Q{sup 2} {sub cum}) values. The key descriptors affecting log(1/K{sub m}) were E{sub lumo}- E{sub homo} (the energy gap between the lowest unoccupied molecular orbital and the highest occupied molecular orbital) and q{sub C}{sup -} (the largest negative net atomic charge on a carbon atom), while the key descriptors affecting log V{sub max} were the polarizability {alpha}, the Connolly solvent-excluded volume (CSEV), and logP (the logarithm of the partition coefficient for octanol/water). From the results obtained it can be concluded that hydrophobic and electronic aspects of OH-PCBs are important in the glucuronidation of OH-PCBs. (orig.)

  16. In vitro synthesis and characterization of guanosine 3',5'-bis(diphosphate).

    Science.gov (United States)

    Hardiman, Timo; Windeisen, Volker; Ewald, Jennifer C; Zibek, Susanne; Schlack, Petra; Rebell, Jochen; Reuss, Matthias; Siemann-Herzberg, Martin

    2008-12-15

    The intracellular alarmone guanosine 3',5'-bis(diphosphate) (ppGpp) has been thoroughly investigated over the past 40 years and has become one of the best-known effectors in bacterial physiology. ppGpp is also of great importance for biotechnological applications. Systems biology research, involving experimental and mathematical approaches, has contributed a great deal to uncovering the alarmone's complex regulatory effects. HPLC analysis and UV detection are used to quantify intracellular ppGpp. The samples analyzed also contain other phosphorylated guanine nucleotides and, therefore, are spiked with a standard ppGpp solution. A rapidly growing number of laboratories are turning to synthesizing the nucleotide in vitro involving time-consuming protocols and yielding only low amounts of ppGpp. The current article provides a protocol for the preparation of large quantities of a ribosome extract that contains high ppGpp synthesis activity. The demonstrated upscaling from shaking flask to bioreactor cultivation involves the continuous and refrigerated harvest of the biomass. (13)C NMR analysis enabled the structural characterization of the synthesis product and was complemented by mass spectrometry and methods that are commonly used to identify ppGpp.

  17. Antisense suppression of cucumber (Cucumis sativus L.) sucrose synthase 3 (CsSUS3) reduces hypoxic stress tolerance.

    Science.gov (United States)

    Wang, Hongyun; Sui, Xiaolei; Guo, Jinju; Wang, Zhenyu; Cheng, Jintao; Ma, Si; Li, Xiang; Zhang, Zhenxian

    2014-03-01

    Sucrose synthase (SUS; EC 2.4.1.13) plays important roles in sugar metabolism and abiotic stress response. But the genes encoding SUS in cucumber (Cucumis sativus L.) have not been well studied. Here, we isolated four cucumber sucrose synthase genes (CsSUS). Among them, CsSUS3, which highly expressed in the roots, was chosen for further study. Immunolocalization and subcellular localization analysis indicated that CsSUS3 localized in the cytosol and the plasma membrane, and mainly existed in the companion cells of phloem in the roots. When suffering hypoxia stress from flooding, CsSUS3 expression and SUS activity in roots increased, especially in the lateral roots; moreover, the soluble SUS activity increased clearly, but the membrane fraction hardly changed. Compared with the wild-type cucumbers, the transgenic lines with antisense expression of CsSUS3 were more sensitive to flooding. After 6 d of flooding, the SUS activity, soluble sugar and uridine 5'-diphosphate glucose (UDPG) content and the ratio of ATP/ADP in the roots of transgenic plants were significantly lower than that in wild-type plants. Moreover, the transgenic lines grew more slowly with more yellow necrosis in the leaves. These findings suggested CsSUS3 participated in resisting hypoxic stress. Furthermore, the mechanism of CsSUS3 in resisting hypoxic stress was also discussed.

  18. Transgenic switchgrass (Panicum virgatum L.) biomass is increased by overexpression of switchgrass sucrose synthase (PvSUS1).

    Science.gov (United States)

    Poovaiah, Charleson R; Mazarei, Mitra; Decker, Stephen R; Turner, Geoffrey B; Sykes, Robert W; Davis, Mark F; Stewart, C Neal

    2015-04-01

    Sucrose synthase (SUS) converts sucrose and uridine di-phosphate (UDP) into UDP-glucose and fructose. UDP-glucose is used by the cellulose synthase to produce cellulose for cell wall biosynthesis. For lignocellulosic feedstocks such as switchgrass, the manipulation of cell walls to decrease lignin content is needed to reduce recalcitrance of conversion of biomass into biofuels. Of perhaps equal importance for bioenergy feedstocks is increasing biomass. Four SUS genes were identified in switchgrass. Each gene contained 14 or 15 introns. PvSUS1 was expressed ubiquitously in the tissues tested. PvSUS2 and PvSUS6 were highly expressed in internodes and roots, respectively. PvSUS4 was expressed in low levels in the tissues tested. Transgenic switchgrass plants overexpressing PvSUS1 had increases in plant height by up to 37%, biomass by up to 13.6%, and tiller number by up to 79% compared to control plants. The lignin content was increased in all lines, while the sugar release efficiency was decreased in PvSUS1-overexpressing transgenic switchgrass plants. For switchgrass and other bioenergy feedstocks, the overexpression of SUS1 genes might be a feasible strategy to increase both plant biomass and cellulose content, and to stack with other genes to increase biofuel production per land area cultivated.

  19. UPLC-PDA-QTOFMS-guided isolation of prenylated xanthones and benzoylphloroglucinols from the leaves of Garcinia oblongifolia and their migration-inhibitory activity

    Science.gov (United States)

    Zhang, Hong; Dan, Zheng; Ding, Zhi-Jie; Lao, Yuan-Zhi; Tan, Hong-Sheng; Xu, Hong-Xi

    2016-10-01

    A UPLC-PDA-QTOFMS-guided isolation strategy was employed to screen and track potentially new compounds from Garcinia oblongifolia. As a result, two new prenylated xanthones, oblongixanthones D and E (1–2), six new prenylated benzoylphloroglucinol derivatives, oblongifolins V–Z (3–7) and oblongifolin AA (8), as well as a known compound oblongifolin L (9), were isolated from the EtOAc-soluble fraction of an acetone extract of the leaves of Garcinia oblongifolia guided by UPLC-PDA-QTOFMS analysis. The structures of the new compounds were elucidated by 1D- and 2D-NMR spectroscopic analysis and mass spectrometry. Experimental and calculated ECD spectra were used to determine the absolute configurations. The results of wound healing and transwell migration assay showed that oblongixanthones D (1), E (2), and oblongifolin L (9) have the ability to inhibit cancer cell migration in lower cytotoxic concentrations. Western blotting results showed that these compounds exhibited an anti-metastasis effect mainly through downregulating RAF protein levels. In addition, 2 and 9 could inhibit phospho-MEK and phospho-ERK at downstream. Moreover, 1, 2, and 9 could inhibit snail protein level, suggesting that they could regulate the EMT pathway.

  20. Preparative Isolation of Two Prenylated Biflavonoids from the Roots and Rhizomes of Sinopodophyllum emodi by Sephadex LH-20 Column and High-Speed Counter-Current Chromatography

    Directory of Open Access Journals (Sweden)

    Yan-Jun Sun

    2015-12-01

    Full Text Available Two prenylated biflavonoids, podoverines B–C, were isolated from the dried roots and rhizomes of Sinopodophyllum emodi using a Sephadex LH-20 column (SLHC and high-speed counter-current chromatography (HSCCC. The 95% ethanol extract was partitioned with ethyl acetate in water. Target compounds from the ethyl acetate fraction were further enriched and purified by the combined application of SLHC and HSCCC. n-Hexane–ethyl acetate–methanol–water (3.5:5:3.5:5, v/v was chosen as the two phase solvent system. The flow rate of mobile phase was optimized at 2.0 mL·min−1. Finally, under optimized conditions, 13.8 mg of podoverine B and 16.2 mg of podoverine C were obtained from 200 mg of the enriched sample. The purities of podoverines B and C were 98.62% and 99.05%, respectively, as determined by HPLC. For the first time, podoverins B and C were found in the genus Sinopodophyllum. Their structures were determined by spectroscopic methods (HR-ESI-MS, 1H-NMR, 13C-NMR, HSQC, HMBC. Their absolute configurations were elucidated by comparison of their experimental and calculated ECD spectra. The cytotoxic activities were evaluated against MCF-7 and HepG2 cell lines. The separation procedures proved to be practical and economical, especially for trace prenylated biflavonoids from traditional Chinese medicine.

  1. Preparative Isolation of Two Prenylated Biflavonoids from the Roots and Rhizomes of Sinopodophyllum emodi by Sephadex LH-20 Column and High-Speed Counter-Current Chromatography.

    Science.gov (United States)

    Sun, Yan-Jun; Pei, Li-Xin; Wang, Kai-Bo; Sun, Yin-Shi; Wang, Jun-Min; Zhang, Yan-Li; Gao, Mei-Ling; Ji, Bao-Yu

    2015-12-23

    Two prenylated biflavonoids, podoverines B-C, were isolated from the dried roots and rhizomes of Sinopodophyllum emodi using a Sephadex LH-20 column (SLHC) and high-speed counter-current chromatography (HSCCC). The 95% ethanol extract was partitioned with ethyl acetate in water. Target compounds from the ethyl acetate fraction were further enriched and purified by the combined application of SLHC and HSCCC. n-Hexane-ethyl acetate-methanol-water (3.5:5:3.5:5, v/v) was chosen as the two phase solvent system. The flow rate of mobile phase was optimized at 2.0 mL·min(-1). Finally, under optimized conditions, 13.8 mg of podoverine B and 16.2 mg of podoverine C were obtained from 200 mg of the enriched sample. The purities of podoverines B and C were 98.62% and 99.05%, respectively, as determined by HPLC. For the first time, podoverins B and C were found in the genus Sinopodophyllum. Their structures were determined by spectroscopic methods (HR-ESI-MS, ¹H-NMR, (13)C-NMR, HSQC, HMBC). Their absolute configurations were elucidated by comparison of their experimental and calculated ECD spectra. The cytotoxic activities were evaluated against MCF-7 and HepG2 cell lines. The separation procedures proved to be practical and economical, especially for trace prenylated biflavonoids from traditional Chinese medicine.

  2. UPLC-PDA-QTOFMS-guided isolation of prenylated xanthones and benzoylphloroglucinols from the leaves of Garcinia oblongifolia and their migration-inhibitory activity

    Science.gov (United States)

    Zhang, Hong; Dan, Zheng; Ding, Zhi-Jie; Lao, Yuan-Zhi; Tan, Hong-Sheng; Xu, Hong-Xi

    2016-01-01

    A UPLC-PDA-QTOFMS-guided isolation strategy was employed to screen and track potentially new compounds from Garcinia oblongifolia. As a result, two new prenylated xanthones, oblongixanthones D and E (1–2), six new prenylated benzoylphloroglucinol derivatives, oblongifolins V–Z (3–7) and oblongifolin AA (8), as well as a known compound oblongifolin L (9), were isolated from the EtOAc-soluble fraction of an acetone extract of the leaves of Garcinia oblongifolia guided by UPLC-PDA-QTOFMS analysis. The structures of the new compounds were elucidated by 1D- and 2D-NMR spectroscopic analysis and mass spectrometry. Experimental and calculated ECD spectra were used to determine the absolute configurations. The results of wound healing and transwell migration assay showed that oblongixanthones D (1), E (2), and oblongifolin L (9) have the ability to inhibit cancer cell migration in lower cytotoxic concentrations. Western blotting results showed that these compounds exhibited an anti-metastasis effect mainly through downregulating RAF protein levels. In addition, 2 and 9 could inhibit phospho-MEK and phospho-ERK at downstream. Moreover, 1, 2, and 9 could inhibit snail protein level, suggesting that they could regulate the EMT pathway. PMID:27767059

  3. Induced biosynthesis of resveratrol and the prenylated stilbenoids arachidin-1 and arachidin-3 in hairy root cultures of peanut: Effects of culture medium and growth stage.

    Science.gov (United States)

    Condori, Jose; Sivakumar, Ganapathy; Hubstenberger, John; Dolan, Maureen C; Sobolev, Victor S; Medina-Bolivar, Fabricio

    2010-05-01

    Previously, we have shown that hairy root cultures of peanut provide a controlled, sustainable and scalable production system that can be induced to produce stilbenoids. However to leverage peanut hairy roots to study the biosynthesis of this polyphenolic biosynthetic pathway, growing conditions and elicitation kinetics of these tissue cultures must be defined and understood. To this end, a new peanut cv. Hull hairy root (line 3) that produces resveratrol and its prenylated analogues arachidin-1 and arachidin-3 upon sodium acetate-mediated elicitation was established. Two culture media were compared for impact on root growth and stilbenoid biosynthesis/secretion. The levels of ammonium, nitrate, phosphate and residual sugars were monitored along growth and elicitation period. A modified MS (MSV) medium resulted in higher root biomass when compared to B5 medium. The stilbenoid profile after elicitation varied depending on the age of the culture (6, 9, 12, and 15-day old). After elicitation at day 9 (exponential growth in MSV medium), over 90% of the total resveratrol, arachidin-1 and arachidin-3 accumulated in the medium. Our studies demonstrate the benefits of the hairy root culture system to study the biosynthesis of stilbenoids including valuable prenylated polyphenolic compounds.

  4. Cyclohexane-1,2-dione hydrolase from denitrifying Azoarcus sp. strain 22Lin, a novel member of the thiamine diphosphate enzyme family.

    Science.gov (United States)

    Steinbach, Alma K; Fraas, Sonja; Harder, Jens; Tabbert, Anja; Brinkmann, Henner; Meyer, Axel; Ermler, Ulrich; Kroneck, Peter M H

    2011-12-01

    Alicyclic compounds with hydroxyl groups represent common structures in numerous natural compounds, such as terpenes and steroids. Their degradation by microorganisms in the absence of dioxygen may involve a C-C bond ring cleavage to form an aliphatic intermediate that can be further oxidized. The cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) from denitrifying Azoarcus sp. strain 22Lin, grown on cyclohexane-1,2-diol as a sole electron donor and carbon source, is the first thiamine diphosphate (ThDP)-dependent enzyme characterized to date that cleaves a cyclic aliphatic compound. The degradation of cyclohexane-1,2-dione (CDO) to 6-oxohexanoate comprises the cleavage of a C-C bond adjacent to a carbonyl group, a typical feature of reactions catalyzed by ThDP-dependent enzymes. In the subsequent NAD(+)-dependent reaction, 6-oxohexanoate is oxidized to adipate. CDH has been purified to homogeneity by the criteria of gel electrophoresis (a single band at ∼59 kDa; calculated molecular mass, 64.5 kDa); in solution, the enzyme is a homodimer (∼105 kDa; gel filtration). As isolated, CDH contains 0.8 ± 0.05 ThDP, 1.0 ± 0.02 Mg(2+), and 1.0 ± 0.015 flavin adenine dinucleotide (FAD) per monomer as a second organic cofactor, the role of which remains unclear. Strong reductants, Ti(III)-citrate, Na(+)-dithionite, and the photochemical 5-deazaflavin/oxalate system, led to a partial reduction of the FAD chromophore. The cleavage product of CDO, 6-oxohexanoate, was also a substrate; the corresponding cyclic 1,3- and 1,4-diones did not react with CDH, nor did the cis- and trans-cyclohexane diols. The enzymes acetohydroxyacid synthase (AHAS) from Saccharomyces cerevisiae, pyruvate oxidase (POX) from Lactobacillus plantarum, benzoylformate decarboxylase from Pseudomonas putida, and pyruvate decarboxylase from Zymomonas mobilis were identified as the closest relatives of CDH by comparative amino acid sequence analysis, and a ThDP binding motif and a 2-fold Rossmann fold

  5. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity

    Science.gov (United States)

    Banerjee, Aparajita; Preiser, Alyssa L.

    2016-01-01

    Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity. PMID:27548482

  6. Molecular Cloning and Functional Analysis of Squalene Synthase 2(SQS2) in Salvia miltiorrhiza Bunge.

    Science.gov (United States)

    Rong, Qixian; Jiang, Dan; Chen, Yijun; Shen, Ye; Yuan, Qingjun; Lin, Huixin; Zha, Liangping; Zhang, Yan; Huang, Luqi

    2016-01-01

    Salvia miltiorrhiza Bunge, which is also known as a traditional Chinese herbal medicine, is widely studied for its ability to accumulate the diterpene quinone Tanshinones. In addition to producing a variety of diterpene quinone, S. miltiorrhiza Bunge also accumulates sterol, brassinosteroid and triterpenoids. During their biosynthesis, squalene synthase (SQS, EC 2.5.1.21) converts two molecules of the hydrophilic substrate farnesyl diphosphate (FPP) into a hydrophobic product, squalene. In the present study, cloning and characterization of S. miltiorrhiza Bunge squalene synthase 2 (SmSQS2, Genbank Accession Number: KM408605) cDNA was investigated subsequently followed by its recombinant expression and preliminary enzyme activity. The full-length cDNA of SmSQS2 was 1 597 bp in length, with an open reading frame of 1 245 bp encoding 414 amino acids. The deduced amino acid sequence of SmSQS2 shared high similarity with those of SQSs from other plants. To obtain soluble recombinant enzymes, the truncated SmSQS2 in which 28 amino acids were deleted from the carboxy terminus was expressed as GST-Tag fusion protein in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western Blot analysis, and the resultant bacterial crude extract was incubated with FPP and NADPH. Gas chromatograph-mass spectrometer analysis showed that squalene was detected in the in vitro reaction mixture. The gene expression level was analyzed through Quantitative real-time PCR, and was found to be higher in roots as compared to the leaves, and was up-regulated upon YE+ Ag(+) treatment. These results could serve as an important to understand the function of the SQS family. In addition, the identification of SmSQS2 is important for further studies of terpenoid and sterol biosynthesis in S. miltiorrhiza Bunge.

  7. Structural Basis for Iron-Mediated Sulfur Transfer in Archael and Yeast Thiazole Synthases.

    Science.gov (United States)

    Zhang, Xuan; Eser, Bekir E; Chanani, Prem K; Begley, Tadhg P; Ealick, Steven E

    2016-03-29

    Thiamin diphosphate is an essential cofactor in all forms of life and plays a key role in amino acid and carbohydrate metabolism. Its biosynthesis involves separate syntheses of the pyrimidine and thiazole moieties, which are then coupled to form thiamin monophosphate. A final phosphorylation produces the active form of the cofactor. In most bacteria, six gene products are required for biosynthesis of the thiamin thiazole. In yeast and fungi only one gene product, Thi4, is required for thiazole biosynthesis. Methanococcus jannaschii expresses a putative Thi4 ortholog that was previously reported to be a ribulose 1,5-bisphosphate synthase [Finn, M. W. and Tabita, F. R. (2004) J. Bacteriol., 186, 6360-6366]. Our structural studies show that the Thi4 orthologs from M. jannaschii and Methanococcus igneus are structurally similar to Thi4 from Saccharomyces cerevisiae. In addition, all active site residues are conserved except for a key cysteine residue, which in S. cerevisiae is the source of the thiazole sulfur atom. Our recent biochemical studies showed that the archael Thi4 orthologs use nicotinamide adenine dinucleotide, glycine, and free sulfide to form the thiamin thiazole in an iron-dependent reaction [Eser, B., Zhang, X., Chanani, P. K., Begley, T. P., and Ealick, S. E. (2016) J. Am. Chem. Soc. , DOI: 10.1021/jacs.6b00445]. Here we report X-ray crystal structures of Thi4 from M. jannaschii complexed with ADP-ribulose, the C205S variant of Thi4 from S. cerevisiae with a bound glycine imine intermediate, and Thi4 from M. igneus with bound glycine imine intermediate and iron. These studies reveal the structural basis for the iron-dependent mechanism of sulfur transfer in archael and yeast thiazole synthases.

  8. Role of a Highly Conserved and Catalytically Important Glutamate-49 in the Enterococcus faecalis Acetolactate Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Miyoung; Lee, Sangchoon; Cho, Junehaeng; Ryu, Seong Eon; Yoon, Moonyoung [Hanyang Univ., Seoul (Korea, Republic of); Koo, Bonsung [Rural Development Administration, Suwon (Korea, Republic of)

    2013-02-15

    Acetolactate synthase (ALS) is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylation of pyruvate and then condenses the hydroxyethyl moiety with another molecule of pyruvate to give 2-acetolactate (AL). AL is a key metabolic intermediate in various metabolic pathways of microorganisms. In addition, AL can be converted to acetoin, an important physiological metabolite that is excreted by many microorganisms. There are two types of ALSs reported in the literature, anabolic aceto-hydroxyacid synthase (AHAS) and catabolic ALSs (cALS). The anabolic AHAS is primarily found in plants, fungi, and bacteria, is involved in the biosynthesis of branched-chain amino acids (BCAAs), and contains flavin adenine dinucleotide (FAD), whereas the cALS is found only in some bacteria and is involved in the butanediol fermentation pathway. Both of the enzymes are ThDP-dependent and require a divalent metal ion for catalytic activity. Despite the similarities of the reactions catalyzed, the cALS can be distinguished from anabolic AHAS by a low optimal pH of about 6.0, FAD-independent functionality, a genetic location within the butanediol operon, and lack of a regulatory subunit. It is noteworthy that the structural and functional features of AHAS have been extensively studied, in contrast to those of cALS, for which only limited information is available. To date, the only crystal structure of cALS reported is from Klebsiella pneumonia, which revealed that the overall structure of K. pneumonia ALS is similar to that of AHAS except for the FAD binding region found in AHAS.

  9. Molecular Cloning and Functional Analysis of Squalene Synthase 2(SQS2) in Salvia miltiorrhiza Bunge

    Science.gov (United States)

    Rong, Qixian; Jiang, Dan; Chen, Yijun; Shen, Ye; Yuan, Qingjun; Lin, Huixin; Zha, Liangping; Zhang, Yan; Huang, Luqi

    2016-01-01

    Salvia miltiorrhiza Bunge, which is also known as a traditional Chinese herbal medicine, is widely studied for its ability to accumulate the diterpene quinone Tanshinones. In addition to producing a variety of diterpene quinone, S. miltiorrhiza Bunge also accumulates sterol, brassinosteroid and triterpenoids. During their biosynthesis, squalene synthase (SQS, EC 2.5.1.21) converts two molecules of the hydrophilic substrate farnesyl diphosphate (FPP) into a hydrophobic product, squalene. In the present study, cloning and characterization of S. miltiorrhiza Bunge squalene synthase 2 (SmSQS2, Genbank Accession Number: KM408605) cDNA was investigated subsequently followed by its recombinant expression and preliminary enzyme activity. The full-length cDNA of SmSQS2 was 1 597 bp in length, with an open reading frame of 1 245 bp encoding 414 amino acids. The deduced amino acid sequence of SmSQS2 shared high similarity with those of SQSs from other plants. To obtain soluble recombinant enzymes, the truncated SmSQS2 in which 28 amino acids were deleted from the carboxy terminus was expressed as GST-Tag fusion protein in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western Blot analysis, and the resultant bacterial crude extract was incubated with FPP and NADPH. Gas chromatograph-mass spectrometer analysis showed that squalene was detected in the in vitro reaction mixture. The gene expression level was analyzed through Quantitative real-time PCR, and was found to be higher in roots as compared to the leaves, and was up-regulated upon YE+ Ag+ treatment. These results could serve as an important to understand the function of the SQS family. In addition, the identification of SmSQS2 is important for further studies of terpenoid and sterol biosynthesis in S. miltiorrhiza Bunge. PMID:27605932

  10. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    OpenAIRE

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic ch...

  11. Functional characterization of wheat ent-kaurene(-like) synthases indicates continuing evolution of labdane-related diterpenoid metabolism in the cereals.

    Science.gov (United States)

    Zhou, Ke; Xu, Meimei; Tiernan, Mollie; Xie, Qian; Toyomasu, Tomonobu; Sugawara, Chizu; Oku, Madoka; Usui, Masami; Mitsuhashi, Wataru; Chono, Makiko; Chandler, Peter M; Peters, Reuben J

    2012-12-01

    Wheat (Triticum aestivum) and rice (Oryza sativa) are two of the most agriculturally important cereal crop plants. Rice is known to produce numerous diterpenoid natural products that serve as phytoalexins and/or allelochemicals. Specifically, these are labdane-related diterpenoids, derived from a characteristic labdadienyl/copalyl diphosphate (CPP), whose biosynthetic relationship to gibberellin biosynthesis is evident from the relevant expanded and functionally diverse family of ent-kaurene synthase-like (KSL) genes found in rice the (OsKSLs). Herein reported is the biochemical characterization of a similarly expansive family of KSL from wheat (the TaKSLs). In particular, beyond ent-kaurene synthases (KS), wheat also contains several biochemically diversified KSLs. These react either with the ent-CPP intermediate common to gibberellin biosynthesis or with the normal stereoisomer of CPP that also is found in wheat (as demonstrated by the accompanying paper describing the wheat CPP synthases). Comparison with a barley (Hordeum vulgare) KS indicates conservation of monocot KS, with early and continued expansion and functional diversification of KSLs in at least the small grain cereals. In addition, some of the TaKSLs that utilize normal CPP also will react with syn-CPP, echoing previous findings with the OsKSL family, with such enzymatic promiscuity/elasticity providing insight into the continuing evolution of diterpenoid metabolism in the cereal crop plant family, as well as more generally, which is discussed here.

  12. Evident and latent plasticity across the rice diterpene synthase family with potential implications for the evolution of diterpenoid metabolism in the cereals

    Science.gov (United States)

    Morrone, Dana; Hillwig, Matthew L.; Mead, Matthew E.; Lowry, Luke; Fulton, D. Bruce; Peters, Reuben J.

    2013-01-01

    SYNOPSIS The evolution of natural products biosynthetic pathways can be envisioned to occur via a number of mechanisms. Here we provide evidence that latent plasticity plays a role in such metabolic evolution. In particular, rice (Oryza sativa) produces both ent- and syn-copalyl diphosphate (CPP), which are substrates for downstream diterpene synthases. Here we report that several members of this enzymatic family exhibit dual reactivity with some pairing of ent-, syn-, or normal CPP stereochemistry. Evident plasticity was observed, as a previously reported ent-sandaracopimaradiene synthase also converts syn-CPP to syn-labda-8(17),12E,14-triene, which can be found in planta. Notably, normal CPP is not naturally found in rice. Thus, the presence of diterpene synthases that react with this non-native metabolite reveals latent enzymatic/metabolic plasticity, providing biochemical capacity for utilization of such a novel substrate (i.e., normal CPP) that may arise during evolution, the implications of which are discussed. PMID:21323642

  13. Crystal structures of two novel sulfonylurea herbicides in complex with Arabidopsis thaliana acetohydroxyacid synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jian-Guo; Lee, Patrick K.-M.; Dong, Yu-Hui; Pang, Siew Siew; Duggleby, Ronald G.; Li, Zheng-Ming; Guddat, Luke W.; (Queensland); (Nankai); (IHEP-Beijing)

    2009-08-17

    Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids. It catalyzes the conversion of two molecules of pyruvate into 2-acetolactate or one molecule of pyruvate and one molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. AHAS requires the cofactors thiamine diphosphate (ThDP), Mg{sup 2+} and FAD for activity. The herbicides that target this enzyme are effective in protecting a broad range of crops from weed species. However, resistance in the field is now a serious problem worldwide. To address this, two new sulfonylureas, monosulfuron and monosulfuron ester, have been developed as commercial herbicides in China. These molecules differ from the traditional sulfonylureas in that the heterocyclic ring attached to the nitrogen atom of the sulfonylurea bridge is monosubstituted rather than disubstituted. The structures of these compounds in complex with the catalytic subunit of Arabidopsis thaliana AHAS have been determined to 3.0 and 2.8 {angstrom}, respectively. In both complexes, these molecules are bound in the tunnel leading to the active site, such that the sole substituent of the heterocyclic ring is buried deepest and oriented towards the ThDP. Unlike the structures of Arabidopsis thaliana AHAS in complex with the classic disubstituted sulfonylureas, where ThDP is broken, this cofactor is intact and present most likely as the hydroxylethyl intermediate.

  14. Crystal structures of two novel sulfonylurea herbicides in complex with Arabidopsis thaliana acetohydroxyacid synthase.

    Science.gov (United States)

    Wang, Jian-Guo; Lee, Patrick K-M; Dong, Yu-Hui; Pang, Siew Siew; Duggleby, Ronald G; Li, Zheng-Ming; Guddat, Luke W

    2009-03-01

    Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids. It catalyzes the conversion of two molecules of pyruvate into 2-acetolactate or one molecule of pyruvate and one molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. AHAS requires the cofactors thiamine diphosphate (ThDP), Mg(2+) and FAD for activity. The herbicides that target this enzyme are effective in protecting a broad range of crops from weed species. However, resistance in the field is now a serious problem worldwide. To address this, two new sulfonylureas, monosulfuron and monosulfuron ester, have been developed as commercial herbicides in China. These molecules differ from the traditional sulfonylureas in that the heterocyclic ring attached to the nitrogen atom of the sulfonylurea bridge is monosubstituted rather than disubstituted. The structures of these compounds in complex with the catalytic subunit of Arabidopsis thaliana AHAS have been determined to 3.0 and 2.8 A, respectively. In both complexes, these molecules are bound in the tunnel leading to the active site, such that the sole substituent of the heterocyclic ring is buried deepest and oriented towards the ThDP. Unlike the structures of Arabidopsis thaliana AHAS in complex with the classic disubstituted sulfonylureas, where ThDP is broken, this cofactor is intact and present most likely as the hydroxylethyl intermediate.

  15. The lumazine synthase/riboflavin synthase complex: shapes and functions of a highly variable enzyme system.

    Science.gov (United States)

    Ladenstein, Rudolf; Fischer, Markus; Bacher, Adelbert

    2013-06-01

    The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

  16. Mammalian N-acetylglutamate synthase.

    Science.gov (United States)

    Morizono, Hiroki; Caldovic, Ljubica; Shi, Dashuang; Tuchman, Mendel

    2004-04-01

    N-Acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI). The mouse and human NAGS genes have been identified based on similarity to regions of NAGS from Neurospora crassa and cloned from liver cDNA libraries. These genes were shown to complement an argA- (NAGS) deficient Escherichia coli strain, and enzymatic activity of the proteins was confirmed by a new stable isotope dilution assay. The deduced amino acid sequence of mammalian NAGS contains a putative mitochondrial-targeting signal at the N-terminus. The mouse NAGS preprotein was overexpressed in insect cells to determine post-translational modifications and two processed proteins with different N-terminal truncations have been identified. Sequence analysis using a hidden Markov model suggests that the vertebrate NAGS protein contains domains with a carbamate kinase fold and an acyl-CoA N-acyltransferase fold, and protein crystallization experiments are currently underway. Inherited NAGS deficiency results in hyperammonemia, presumably due to the loss of CPSI activity. We, and others, have recently identified mutations in families with neonatal and late-onset NAGS deficiency and the identification of the gene has now made carrier testing and prenatal diagnosis feasible. A structural analog of NAG, carbamylglutamate, has been shown to bind and activate CPSI, and several patients have been reported to respond favorably to this drug (Carbaglu).

  17. Terpene synthases from Cannabis sativa.

    Science.gov (United States)

    Booth, Judith K; Page, Jonathan E; Bohlmann, Jörg

    2017-01-01

    Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  18. Mycobacterium tuberculosis nucleoside diphosphate kinase inactivates small GTPases leading to evasion of innate immunity.

    Directory of Open Access Journals (Sweden)

    Jim Sun

    Full Text Available Defining the mechanisms of Mycobacterium tuberculosis (Mtb persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2 dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk and obtained a stable mutant (Mtb Ndk-AS that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk

  19. Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

    Science.gov (United States)

    Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

    2013-12-01

    Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed.

  20. Flagellar Radial Spokes Contain a Ca2+-stimulated Nucleoside Diphosphate Kinase

    Science.gov (United States)

    Patel-King, Ramila S.; Gorbatyuk, Oksana; Takebe, Sachiko; King, Stephen M.

    2004-01-01

    The radial spokes are required for Ca2+-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca2+-independent manner, whereas IQ2 and IQ3 show Ca2+-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca2+. This Ca2+-responsive enzyme, which accounts for ∼45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca2+-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes. PMID:15194815

  1. Bacterial and plant HAD enzymes catalyse a missing phosphatase step in thiamin diphosphate biosynthesis.

    Science.gov (United States)

    Hasnain, Ghulam; Roje, Sanja; Sa, Na; Zallot, Rémi; Ziemak, Michael J; de Crécy-Lagard, Valérie; Gregory, Jesse F; Hanson, Andrew D

    2016-01-15

    The penultimate step of thiamin diphosphate (ThDP) synthesis in plants and many bacteria is dephosphorylation of thiamin monophosphate (ThMP). Non-specific phosphatases have been thought to mediate this step and no genes encoding specific ThMP phosphatases (ThMPases) are known. Comparative genomic analysis uncovered bacterial haloacid dehalogenase (HAD) phosphatase family genes (from subfamilies IA and IB) that cluster on the chromosome with, or are fused to, thiamin synthesis genes and are thus candidates for the missing phosphatase (ThMPase). Three typical candidates (from Anaerotruncus colihominis, Dorea longicatena and Syntrophomonas wolfei) were shown to have efficient in vivo ThMPase activity by expressing them in an Escherichia coli strain engineered to require an active ThMPase for growth. In vitro assays confirmed that these candidates all preferred ThMP to any of 45 other phosphate ester substrates tested. An Arabidopsis thaliana ThMPase homologue (At4g29530) of unknown function whose expression pattern and compartmentation fit with a role in ThDP synthesis was shown to have in vivo ThMPase activity in E. coli and to prefer ThMP to any other substrate tested. However, insertional inactivation of the At4g29530 gene did not affect growth or the levels of thiamin or its phosphates, indicating that Arabidopsis has at least one other ThMPase gene. The Zea mays orthologue of At4g29530 (GRMZM2G035134) was also shown to have ThMPase activity. These data identify HAD genes specifying the elusive ThMPase activity, indicate that ThMPases are substrate-specific rather than general phosphatases and suggest that different evolutionary lineages have recruited ThMPases independently from different branches of the HAD family.

  2. Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium.

    Science.gov (United States)

    Vaccaro, J A; Crane, E J; Harris, T K; Washabaugh, M W

    1995-10-01

    Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) exhibits biphasic kinetics with 52 +/- 11% of the PDC reacting with k1 = (1.0 +/- 0.3) x 10(-2) s-1 and 48 +/- 12% of the PDC reacting with k2 = (1.1 +/- 0.6) x 10(-1) s-1 when TDP (KTDP = 0.5 +/- 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 +/- 0.5) x 10(-2) s-1 upon addition of Mg2+ (KMg2+ = 0.2 +/- 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 < or = 3-fold and has no effect on the value of k1.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Heterologous expression of active human uridine diphosphate glucuronosyltransferase 1A3 in Chinese hamster lung cells

    Institute of Scientific and Technical Information of China (English)

    Ya-Kun Chen; Xin Li; Shu-Qing Chen; Su Zeng

    2005-01-01

    AIM: To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.METHODS: The full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR)using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with β-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR.RESULTS: The gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418.The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected.CONCLUSION: Correct sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells.

  4. Mycobacterium tuberculosis nucleoside diphosphate kinase inactivates small GTPases leading to evasion of innate immunity.

    Directory of Open Access Journals (Sweden)

    Jim Sun

    Full Text Available Defining the mechanisms of Mycobacterium tuberculosis (Mtb persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2 dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk and obtained a stable mutant (Mtb Ndk-AS that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk

  5. P2X receptors regulate adenosine diphosphate release from hepatic cells.

    Science.gov (United States)

    Chatterjee, Cynthia; Sparks, Daniel L

    2014-12-01

    Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2-4 h. Nucleotide release from hepatic cells is stimulated by the Ca(2+) ionophore, ionomycin, and by the P2 receptor agonist, 2'3'-O-(4-benzoyl-benzoyl)-adenosine 5'-triphosphate (BzATP). Ionomycin (10 μM) has a minimal effect on ATP release, but doubles media ADP levels at 5 min. In contrast, BzATP (10-100 μM) increases both ATP and ADP levels by over 100-fold at 5 min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100 μM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca(2+) levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.

  6. The antihyperalgesic effect of cytidine-5'-diphosphate-choline in neuropathic and inflammatory pain models.

    Science.gov (United States)

    Bagdas, Deniz; Sonat, Fusun Ak; Hamurtekin, Emre; Sonal, Songul; Gurun, Mine Sibel

    2011-09-01

    This study was designed to test the effects of intracerebroventricularly (i.c.v.) administered CDP-choline (cytidine-5'-diphosphate-choline; citicoline) and its metabolites in rat models of inflammatory and neuropathic pain. The i.c.v. administration of CDP-choline (0.5, 1.0 and 2.0 µmol) produced a dose and time-dependent reversal of mechanical hyperalgesia in both carrageenan-induced inflammatory and chronic constriction injury-induced neuropathic pain models in rats. The antihyperalgesic effect of CDP-choline was similar to that observed with an equimolar dose of choline (1 µmol). The CDP-choline-induced antihyperalgesic effect was prevented by central administration of the neuronal high-affinity choline uptake inhibitor hemicholinium-3 (1 µg), the nonselective nicotinic receptor antagonist mecamylamine (50 µg), the α7-selective nicotinic ACh receptor antagonist, α-bungarotoxin (2 µg) and the γ-aminobutyric acid B receptor antagonist CGP-35348 (20 µg). In contrast, i.c.v. pretreatment with the nonselective opioid receptor antagonist naloxone (10 µg) only prevented the CDP-choline-induced antihyperalgesic effect in the neuropathic pain model while the nonselective muscarinic receptor antagonist atropine (10 µg) did not alter the antihyperalgesic effect in the two models. These results indicate that CDP-choline-elicited antihyperalgesic effect in different models of pain occurs through mechanisms that seem to involve an interaction with supraspinal α7-selective nicotinic ACh receptors, and γ-aminobutyric acid B receptors, whereas central opioid receptors have a role only in the neuropathic pain model.

  7. The 2.0 Å X-ray structure for yeast acetohydroxyacid synthase provides new insights into its cofactor and quaternary structure requirements

    Science.gov (United States)

    Lonhienne, Thierry; Garcia, Mario D.; Fraser, James A.; Williams, Craig M.; Guddat, Luke W.

    2017-01-01

    Acetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid biosynthesis, a pathway essential to the life-cycle of plants and micro-organisms. The catalytic subunit has thiamin diphosphate (ThDP) and flavin adenine dinucleotide (FAD) as indispensable co-factors. A new, high resolution, 2.0 Å crystal structure of Saccharomyces cerevisiae AHAS reveals that the dimer is asymmetric, with the catalytic centres having distinct structures where FAD is trapped in two different conformations indicative of different redox states. Two molecules of oxygen (O2) are bound on the surface of each active site and a tunnel in the polypeptide appears to passage O2 to the active site independently of the substrate. Thus, O2 appears to play a novel “co-factor” role in this enzyme. We discuss the functional implications of these features of the enzyme that have not previously been described. PMID:28178302

  8. Genome mining in Streptomyces avermitilis: cloning and characterization of SAV_76, the synthase for a new sesquiterpene, avermitilol.

    Science.gov (United States)

    Chou, Wayne K W; Fanizza, Immacolata; Uchiyama, Takuma; Komatsu, Mamoru; Ikeda, Haruo; Cane, David E

    2010-07-07

    The terpene synthase encoded by the sav76 gene of Streptomyces avermtilis was expressed in Escherichia coli as an N-terminal-His(6)-tag protein, using a codon-optimized synthetic gene. Incubation of the recombinant protein, SAV_76, with farnesyl diphosphate (1, FPP) in the presence of Mg(2+) gave a new sesquiterpene alcohol avermitilol (2), whose structure and stereochemistry were determined by a combination of (1)H, (13)C, COSY, HMQC, HMBC, and NOESY NMR, along with minor amounts of germacrene A (3), germacrene B (4), and viridiflorol (5). The absolute configuration of 2 was assigned by (1)H NMR analysis of the corresponding (R)- and (S)-Mosher esters. The steady state kinetic parameters were k(cat) 0.040 +/- 0.001 s(-1) and K(m) 1.06 +/- 0.11 microM. Individual incubations of recombinant avermitilol synthase with [1,1-(2)H(2)]FPP (1a), (1S)-[1-(2)H]-FPP (1b), and (1R)-[1-(2)H]-FPP (1c) and NMR analysis of the resulting avermitilols supported a cyclization mechanism involving the loss of H-1(re) to generate the intermediate bicyclogermacrene (7), which then undergoes proton-initiated anti-Markovnikov cyclization and capture of water to generate 2. A copy of the sav76 gene was reintroduced into S. avermitilis SUKA17, a large deletion mutant from which the genes for the major endogenous secondary metabolites had been removed, and expressed under control of the native S. avermitilis promoter rpsJp (sav4925). The resultant transformants generated avermitilol (2) as well as the derived ketone, avermitilone (8), along with small amounts of 3, 4, and 5. The biochemical function of all four terpene synthases found in the S. avermtilis genome have now been determined.

  9. Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex.

    Science.gov (United States)

    Sümegi, B; Alkonyi, I

    1983-11-02

    In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.

  10. Characterization of the plastidial geraniol synthase from Madagascar periwinkle which initiates the monoterpenoid branch of the alkaloid pathway in internal phloem associated parenchyma.

    Science.gov (United States)

    Simkin, Andrew J; Miettinen, Karel; Claudel, Patricia; Burlat, Vincent; Guirimand, Grégory; Courdavault, Vincent; Papon, Nicolas; Meyer, Sophie; Godet, Stéphanie; St-Pierre, Benoit; Giglioli-Guivarc'h, Nathalie; Fischer, Marc J C; Memelink, Johan; Clastre, Marc

    2013-01-01

    Madagascar periwinkle (Catharanthus roseus [L.] G. Don, Apocynaceae) produces monoterpene indole alkaloids (MIAs), secondary metabolites of high interest due to their therapeutic value. A key step in the biosynthesis is the generation of geraniol from geranyl diphosphate (GPP) in the monoterpenoid branch of the MIA pathway. Here we report on the cloning and functional characterization of C. roseus geraniol synthase (CrGES). The full-length CrGES was over-expressed in Escherichia coli and the purified recombinant protein catalyzed the conversion of GPP into geraniol with a K(m) value of 58.5 μM for GPP. In vivo CrGES activity was evaluated by heterologous expression in a Saccharomyces cerevisiae strain mutated in the farnesyl diphosphate synthase gene. Analysis of culture extracts by gas chromatography-mass spectrometry confirmed the excretion of geraniol into the growth medium. Transient transformation of C. roseus cells with a Yellow Fluorescent Protein-fusion construct revealed that CrGES is localized in plastid stroma and stromules. In aerial plant organs, RNA in situ hybridization showed specific labeling of CrGES transcripts in the internal phloem associated parenchyma as observed for other characterized genes involved in the early steps of MIA biosynthesis. Finally, when cultures of Catharanthus cells were treated with the alkaloid-inducing hormone methyl jasmonate, an increase in CrGES transcript levels was observed. This observation coupled with the tissue-specific expression and the subcellular compartmentalization support the idea that CrGES initiates the monoterpenoid branch of the MIA biosynthetic pathway.

  11. Critical aspartic acid residues in pseudouridine synthases.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  12. Regeneration of hemopoietic and lymphoid tissues following total-body irradiation and therapeutic administration of thiamin diphosphate. [Mice, gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Vavrova, J.; Nouza, K.; Petjrek, P.

    1977-01-01

    Analysis was made of the mechanism of therapeutic application of thiamine diphosphate (TDP) in radiation sickness in mice. This agent increased the number of endogenous colonies in the spleen and incorporation of /sup 59/Fe in spleen and bone marrow with sublethal doses of radiation (500 and 600 R) and has no effect with a lethal dose (750 R). After administration of TDP to mice exposed to 500 R radiation, there is faster DNA synthesis in the spleen, thymus and bone marrow, as well as reliable increase in number of nuclear cells in femoral marrow.

  13. Four gene products are required for the fungal synthesis of the indole-diterpene, paspaline.

    Science.gov (United States)

    Saikia, Sanjay; Parker, Emily J; Koulman, Albert; Scott, Barry

    2006-03-06

    Paspaline belongs to a large, structurally and functionally diverse group of indole-diterpenes synthesized by filamentous fungi. However, the identity of the gene products required for the biosynthesis of paspaline, a key intermediate for the synthesis of paxilline and other indole-diterpenes, is not known. Transfer of constructs containing different pax gene combinations into a paxilline negative deletion derivative of Penicillium paxilli demonstrated that just four proteins, PaxG, a geranylgeranyl diphosphate synthase, PaxM, a FAD-dependent monooxygenase, PaxB, a putative membrane protein, and PaxC, a prenyl transferase, are required for the biosynthesis of paspaline.

  14. The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily

    DEFF Research Database (Denmark)

    Hansen, Nikolaj L; Heskes, Allison M; Hamberger, Britta;

    2017-01-01

    in the formation of four C-20 diphosphate intermediates, precursors of both generalized and specialized metabolism and a novel scaffold for Celastraceae. Functional pairs of the class I and II enzymes resulted in formation of three scaffolds, accounting for some of the terpenoid diversity found in T. wilfordii...

  15. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...

  16. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose 5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-D-ribosyl α-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...

  17. Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions.

    Science.gov (United States)

    Gillette, William K; Esposito, Dominic; Abreu Blanco, Maria; Alexander, Patrick; Bindu, Lakshman; Bittner, Cammi; Chertov, Oleg; Frank, Peter H; Grose, Carissa; Jones, Jane E; Meng, Zhaojing; Perkins, Shelley; Van, Que; Ghirlando, Rodolfo; Fivash, Matthew; Nissley, Dwight V; McCormick, Frank; Holderfield, Matthew; Stephen, Andrew G

    2015-11-02

    Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

  18. An investigation into eukaryotic pseudouridine synthases.

    Science.gov (United States)

    King, Ross D; Lu, Chuan

    2014-08-01

    A common post-transcriptional modification of RNA is the conversion of uridine to its isomer pseudouridine. We investigated the biological significance of eukaryotic pseudouridine synthases using the yeast Saccharomyces cerevisiae. We conducted a comprehensive statistical analysis on growth data from automated perturbation (gene deletion) experiments, and used bi-logistic curve analysis to characterise the yeast phenotypes. The deletant strains displayed different alteration in growth properties, including in some cases enhanced growth and/or biphasic growth curves not seen in wild-type strains under matched conditions. These results demonstrate that disrupting pseudouridine synthases can have a significant qualitative effect on growth. We further investigated the significance of post-transcriptional pseudouridine modification through investigation of the scientific literature. We found that (1) In Toxoplasma gondii, a pseudouridine synthase gene is critical in cellular differentiation between the two asexual forms: Tachyzoites and bradyzoites; (2) Mutation of pseudouridine synthase genes has also been implicated in human diseases (mitochondrial myopathy and sideroblastic anemia (MLASA); dyskeratosis congenita). Taken together, these results are consistent with pseudouridine synthases having a Gene Ontology function of "biological regulation".

  19. A comparative study on the metabolism of Epimedium koreanum Nakai-prenylated flavonoids in rats by an intestinal enzyme (lactase phlorizin hydrolase) and intestinal flora.

    Science.gov (United States)

    Zhou, Jing; Chen, Yan; Wang, Ying; Gao, Xia; Qu, Ding; Liu, Congyan

    2013-12-24

    The aim of this study was to compare the significance of the intestinal hydrolysis of prenylated flavonoids in Herba Epimedii by an intestinal enzyme and flora. Flavonoids were incubated at 37 °C with rat intestinal enzyme and intestinal flora. HPLC-UV was used to calculate the metabolic rates of the parent drug in the incubation and LC/MS/MS was used to determine the chemical structures of metabolites generated by different flavonoid glycosides. Rates of flavonoid metabolism by rat intestinal enzyme were quicker than those of intestinal flora. The sequence of intestinal flora metabolic rates was icariin>epimedin B>epimedin A>epimedin C>baohuoside I, whereas the order of intestinal enzyme metabolic rates was icariin>epimedin A>epimedin C>epimedin B>baohuoside I. Meanwhile, the LC/MS/MS graphs showed that icariin produced three products, epimedin A/B/C had four and baohuoside I yielded one product in incubations of both intestinal enzyme and flora, which were more than the results of HPLC-UV due to the fact LC/MS/MS has lower detectability and higher sensitivity. Moreover, the outcomes indicated that the rate of metabolization of flavonoids by intestinal enzyme were faster than those of intestinal flora, which was consistent with the HPLC-UV results. In conclusion, the metabolic pathways of the same components by intestinal flora and enzyme were the same. What's more, an intestinal enzyme such as lactase phlorizin hydrolase exhibited a more significant metabolic role in prenylated flavonoids of Herba Epimedi compared with intestinal flora.

  20. Quantitative and qualitative characteristics of cell wall components and prenyl lipids in the leaves of Tilia x euchlora trees growing under salt stress.

    Science.gov (United States)

    Milewska-Hendel, Anna; Baczewska, Aneta H; Sala, Katarzyna; Dmuchowski, Wojciech; Brągoszewska, Paulina; Gozdowski, Dariusz; Jozwiak, Adam; Chojnacki, Tadeusz; Swiezewska, Ewa; Kurczynska, Ewa

    2017-01-01

    The study was focused on assessing the presence of arabinogalactan proteins (AGPs) and pectins within the cell walls as well as prenyl lipids, sodium and chlorine content in leaves of Tilia x euchlora trees. The leaves that were analyzed were collected from trees with and without signs of damage that were all growing in the same salt stress conditions. The reason for undertaking these investigations was the observations over many years that indicated that there are trees that present a healthy appearance and trees that have visible symptoms of decay in the same habitat. Leaf samples were collected from trees growing in the median strip between roadways that have been intensively salted during the winter season for many years. The sodium content was determined using atomic spectrophotometry, chloride using potentiometric titration and poly-isoprenoids using HPLC/UV. AGPs and pectins were determined using immunohistochemistry methods. The immunohistochemical analysis showed that rhamnogalacturonans I (RG-I) and homogalacturonans were differentially distributed in leaves from healthy trees in contrast to leaves from injured trees. In the case of AGPs, the most visible difference was the presence of the JIM16 epitope. Chemical analyses of sodium and chloride showed that in the leaves from injured trees, the level of these ions was higher than in the leaves from healthy trees. Based on chromatographic analysis, four poly-isoprenoid alcohols were identified in the leaves of T. x euchlora. The levels of these lipids were higher in the leaves from healthy trees. The results suggest that the differences that were detected in the apoplast and symplasm may be part of the defensive strategy of T. x euchlora trees to salt stress, which rely on changes in the chemical composition of the cell wall with respect to the pectic and AGP epitopes and an increased synthesis of prenyl lipids.

  1. Quantitative and qualitative characteristics of cell wall components and prenyl lipids in the leaves of Tilia x euchlora trees growing under salt stress

    Science.gov (United States)

    Milewska-Hendel, Anna; Baczewska, Aneta H.; Sala, Katarzyna; Dmuchowski, Wojciech; Brągoszewska, Paulina; Gozdowski, Dariusz; Jozwiak, Adam; Chojnacki, Tadeusz; Swiezewska, Ewa; Kurczynska, Ewa

    2017-01-01

    The study was focused on assessing the presence of arabinogalactan proteins (AGPs) and pectins within the cell walls as well as prenyl lipids, sodium and chlorine content in leaves of Tilia x euchlora trees. The leaves that were analyzed were collected from trees with and without signs of damage that were all growing in the same salt stress conditions. The reason for undertaking these investigations was the observations over many years that indicated that there are trees that present a healthy appearance and trees that have visible symptoms of decay in the same habitat. Leaf samples were collected from trees growing in the median strip between roadways that have been intensively salted during the winter season for many years. The sodium content was determined using atomic spectrophotometry, chloride using potentiometric titration and poly-isoprenoids using HPLC/UV. AGPs and pectins were determined using immunohistochemistry methods. The immunohistochemical analysis showed that rhamnogalacturonans I (RG-I) and homogalacturonans were differentially distributed in leaves from healthy trees in contrast to leaves from injured trees. In the case of AGPs, the most visible difference was the presence of the JIM16 epitope. Chemical analyses of sodium and chloride showed that in the leaves from injured trees, the level of these ions was higher than in the leaves from healthy trees. Based on chromatographic analysis, four poly-isoprenoid alcohols were identified in the leaves of T. x euchlora. The levels of these lipids were higher in the leaves from healthy trees. The results suggest that the differences that were detected in the apoplast and symplasm may be part of the defensive strategy of T. x euchlora trees to salt stress, which rely on changes in the chemical composition of the cell wall with respect to the pectic and AGP epitopes and an increased synthesis of prenyl lipids. PMID:28234963

  2. Active site binding modes of inhibitors of Staphylococcus aureus mevalonate diphosphate decarboxylase from docking and molecular dynamics simulations.

    Science.gov (United States)

    Addo, James K; Skaff, D Andrew; Miziorko, Henry M

    2016-01-01

    Bacterial mevalonate diphosphate decarboxylase (MDD) is an attractive therapeutic target for antibacterial drug development. In this work, we discuss a combined docking and molecular dynamics strategy toward inhibitor binding to bacterial MDD. The docking parameters utilized in this study were first validated with observations for the inhibitors 6-fluoromevalonate diphosphate (FMVAPP) and diphosphoglycolylproline (DPGP) using existing structures for the Staphylococcus epidermidis enzyme. The validated docking protocol was then used to predict structures of the inhibitors bound to Staphylococcus aureus MDD using the unliganded crystal structure of Staphylococcus aureus MDD. We also investigated a possible interactions improvement by combining this docking method with molecular dynamics simulations. Thus, the predicted docking structures were analyzed in a molecular dynamics trajectory to generate dynamic models and reinforce the predicted binding modes. FMVAPP is predicted to make more extensive contacts with S. aureus MDD, forming stable hydrogen bonds with Arg144, Arg193, Lys21, Ser107, and Tyr18, as well as making stable hydrophobic interactions with Tyr18, Trp19, and Met196. The differences in predicted binding are supported by experimentally determined Ki values of 0.23 ± 0.02 and 34 ± 8 μM, for FMVAPP and DPGP, respectively. The structural information coupled with the kinetic characterization obtained from this study should be useful in defining the requirements for inhibition as well as in guiding the selection of active compounds for inhibitor optimization.

  3. The Thiamine diphosphate dependent Enzyme Engineering Database: A tool for the systematic analysis of sequence and structure relations

    Directory of Open Access Journals (Sweden)

    Radloff Robert

    2010-02-01

    Full Text Available Abstract Background Thiamine diphosphate (ThDP-dependent enzymes form a vast and diverse class of proteins, catalyzing a wide variety of enzymatic reactions including the formation or cleavage of carbon-sulfur, carbon-oxygen, carbon-nitrogen, and especially carbon-carbon bonds. Although very diverse in sequence and domain organisation, they share two common protein domains, the pyrophosphate (PP and the pyrimidine (PYR domain. For the comprehensive and systematic comparison of protein sequences and structures the Thiamine diphosphate (ThDP-dependent Enzyme Engineering Database (TEED was established. Description The TEED http://www.teed.uni-stuttgart.de contains 12048 sequence entries which were assigned to 9443 different proteins and 379 structure entries. Proteins were assigned to 8 different superfamilies and 63 homologous protein families. For each family, the TEED offers multisequence alignments, phylogenetic trees, and family-specific HMM profiles. The conserved pyrophosphate (PP and pyrimidine (PYR domains have been annotated, which allows the analysis of sequence similarities for a broad variety of proteins. Human ThDP-dependent enzymes are known to be involved in many diseases. 20 different proteins and over 40 single nucleotide polymorphisms (SNPs of human ThDP-dependent enzymes were identified in the TEED. Conclusions The online accessible version of the TEED has been designed to serve as a navigation and analysis tool for the large and diverse family of ThDP-dependent enzymes.

  4. Protein (Cyanobacteria): 495455270 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available farnesyl-diphosphate synthase Moorea producens MVATEGRPTPKRESPSLDLLTYLKERKALIESALDQALPRMEPDKIYEAMRYSLLAGGKR...LRPILCLATCELMGGTVEMAMPTSCALEMIHTMSLIHDDLPAMDNDDYRRGKLTNHKVFGEDIAILAGDGLLAYAFEYAAAQTKNVPAPQVLQVVAWLAKAVGASGLV

  5. Molecular characterization of the thi3 gene involved in thiamine biosynthesis in Zea mays: cDNA sequence and enzymatic and structural properties of the recombinant bifunctional protein with 4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate) kinase and thiamine monophosphate synthase activities.

    Science.gov (United States)

    Rapala-Kozik, Maria; Olczak, Mariusz; Ostrowska, Katarzyna; Starosta, Agata; Kozik, Andrzej

    2007-12-01

    A thiamine biosynthesis gene, thi3, from maize Zea mays has been identified through cloning and sequencing of cDNA and heterologous overexpression of the encoded protein, THI3, in Escherichia coli. The recombinant THI3 protein was purified to homogeneity and shown to possess two essentially different enzymatic activities of HMP(-P) [4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate)] kinase and TMP (thiamine monophosphate) synthase. Both activities were characterized in terms of basic kinetic constants, with interesting findings that TMP synthase is uncompetitively inhibited by excess of one of the substrates [HMP-PP (HMP diphosphate)] and ATP. A bioinformatic analysis of the THI3 sequence suggested that these activities were located in two distinct, N-terminal kinase and C-terminal synthase, domains. Models of the overall folds of THI3 domains and the arrangements of active centre residues were obtained with the SWISS-MODEL protein modelling server, on the basis of the known three-dimensional structures of Salmonella enterica serotype Typhimurium HMP(-P) kinase and Bacillus subtilis TMP synthase. The essential roles of Gln98 and Met134 residues for HMP kinase activity and of Ser444 for TMP synthase activity were experimentally confirmed by site-directed mutagenesis.

  6. Study of the thorium phosphate-diphosphate (TPD) dissolution: kinetic aspect - thermodynamic aspect: analysis of the neo-formed phases; Etude de la dissolution du phosphate diphosphate de thorium: - aspect cinetique - aspect thermodynamique: analyse des phases neoformees

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, A.Ch

    2000-10-06

    The aim of this work is to study the aqueous corrosion of the thorium phosphate-diphosphate (TPD), of the formula Th{sub 4}(PO{sub 4}){sub 4}P{sub 2}O{sub 7}, in the framework of the actinides immobilization. In order to complete the anterior studies concerning solid solutions where thorium is substituted by a tetravalent ion (uranium (IV) or plutonium (IV)) in the TPD structure, compounds of thorium and neptunium phosphate-diphosphate, of formula Th{sub 4-x}Np{sub x}(PO{sub 4}){sub 4}P{sub 2}O{sub 7}, have been prepared. Furthermore, a new chemical way of synthesis has been investigated in order to sinter solids solution of thorium and uranium phosphate-diphosphate (TUPD) in good conditions. The TPD dissolution study showed two principals steps. The first one corresponds to the control of element concentration by the material dissolution whereas the second corresponds to the formation of secondary precipitates for which thermodynamic equilibrium controls the concentration of the species in solution. Leaching tests have been performed varying several independent parameters in order to determine the TPD dissolution rate. The partial orders related to the protons or to the hydroxide ions have been found between 0.35 and 0.45 whereas the apparent dissolution rate constants are in the range 1.10{sup -5} for 9.10{sup -5} g.m{sup -2}.j{sup -1} for acidic and basic media. The neo-formed phases have been characterized after the dissolution of TPD and TUPD. We found that the TPD leaching in acidic medium leads to the formation of the crystallized thorium phosphate-hydrogen-phosphate (TPHP), of formula Th{sub 2}(PO{sub 4}){sub 2}(HPO{sub 4}), x H{sub 2}O, whereas the TUPD dissolution leads to the TPHP and an other compound, of formula (UO{sub 2}){sub 3}(PO{sub 4}){sub 2}, 5 H{sub 2}O. We calculated its solubility product which is in good agreement with those found in the literature. The phases formed during the leaching of solids containing plutonium; americium or curium (Th

  7. The tomato terpene synthase gene family

    NARCIS (Netherlands)

    Falara, V.; Akhtar, T.A.; Nguyen, T.T.H.; Spyropoulou, E.A.; Bleeker, P.M.; Schauvinhold, I.; Matsuba, Y.; Bonini, M.E.; Schilmiller, A.L.; Last, R.L.; Schuurink, R.C.; Pichersky, E.

    2011-01-01

    Compounds of the terpenoid class play many roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of Solanum lycopersicum (cultivated tomato) contains 40 terpene synthase (TPS) genes, including 28

  8. Cloning of parsley flavone synthase I.

    Science.gov (United States)

    Martens, S; Forkmann, G; Matern, U; Lukacin, R

    2001-09-01

    A cDNA encoding flavone synthase I was amplified by RT-PCR from leaflets of Petroselinum crispum cv. Italian Giant seedlings and functionally expressed in yeast cells. The identity of the recombinant, 2-oxoglutarate-dependent enzyme was verified in assays converting (2S)-naringenin to apigenin.

  9. Inducible nitric oxide synthase in renal transplantation

    NARCIS (Netherlands)

    Joles, JA; Vos, IH; Grone, HJ; Rabelink, TJ

    2002-01-01

    The importance of the endothelial isoform of nitric oxide synthase (eNOS) has been well established. Endothelium-derived nitric oxide has been shown to be essential for vascular homeostasis and modulation of eNOS has thus become a target in prevention of cardiovascular disease. The role of the induc

  10. Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver

    Directory of Open Access Journals (Sweden)

    Chen Ren

    2012-10-01

    Full Text Available Abstract Background Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production. Results To develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629 encoding isopentenyl diphosphate isomerase (IPI was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line control. Conclusions Increasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our

  11. Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

    Science.gov (United States)

    Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

    2011-06-01

    Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.

  12. New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

    Science.gov (United States)

    Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

    2009-04-03

    Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

  13. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence.

    Science.gov (United States)

    Jagielska, Elżbieta; Płucienniczak, Andrzej; Dąbrowska, Magdalena; Dowierciał, Anna; Rode, Wojciech

    2014-04-09

    Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.

  14. Facilitated geranylgeranylation of shrimp ras-encoded p25 fusion protein by the binding with guanosine diphosphate.

    Science.gov (United States)

    Huang, C F; Chuang, N N

    1999-05-01

    A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. Similar to the mammalian Ras proteins, this shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian KB-Ras proteins and demonstrates identity in the guanine nucleotide binding domains. Expression of the cDNA of shrimp in Escherichia coli yielded a 25-kDa polypeptide with positive reactivity toward the monoclonal antibodies against Ras of mammals. As judged by nitrocellulose filtration assay, the specific GTP binding activity of ras-encoded p25 fusion protein was approximately 30,000 units/mg of protein, whereas that of GDP was 5,000 units/mg of protein. In other words, the GTP bound form of ras-encoded p25 fusion protein prevails. Fluorography analysis demonstrated that the prenylation of both shrimp Ras-GDP and shrimp Ras-GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded that of nucleotide-free form of Ras by 10-fold and four-fold, respectively. That is, the protein geranylgeranyl transferase I prefers to react with ras-encoded p25 fusion protein in the GDP bound form.

  15. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...... endothelium is consistent with a role for NO in the control of blood flow in human skeletal muscle....

  16. HbIDI, SlIDI and EcIDI: A comparative study of isopentenyl diphosphate isomerase activity and structure.

    Science.gov (United States)

    Berthelot, Karine; Estevez, Yannick; Quiliano, Miguel; Baldera-Aguayo, Pedro A; Zimic, Mirko; Pribat, Anne; Bakleh, Marc-Elias; Teyssier, Emeline; Gallusci, Philippe; Gardrat, Christian; Lecomte, Sophie; Peruch, Frédéric

    2016-08-01

    In this study, we cloned, expressed and purified the isopentenyl diphosphate isomerases (IDIs) from two plants, Hevea brasiliensis and Solanum lycopersicum, and compared them to the already well characterized Escherichia coli IDI. Phylogenetic analysis showed high homology between the three enzymes. Their catalytic activity was investigated in vitro with recombinant purified enzymes and in vivo by complementation colorimetric tests. The three enzymes displayed consistent activities both in vitro and in vivo. In term of structure, studied by ATR-FTIR and molecular modeling, it is clear that both plant enzymes are more related to their human homologue than to E. coli IDI. But it is assumed that EcIDI represent the minimalistic part of the catalytic core, as both plant enzymes present a supplementary sequence forming an extra α-helice surrounding the catalytic site that could facilitate the biocatalysis. New potential biotechnological applications may be envisaged.

  17. Interaction of hexa-His tag with acidic amino acids results in facilitated refolding of halophilic nucleoside diphosphate kinase.

    Science.gov (United States)

    Ishibashi, Matsujiro; Ida, Keiko; Tatsuda, Shuhei; Arakawa, Tsutomu; Tokunaga, Masao

    2011-11-01

    We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.

  18. THE REGULATORY EFFECT OF NUCLEOSIDE DIPHOSPHATE KINASE ON G-PROTEIN AND G-PROTEIN MEDIATED PHOSPHOLIPASE C

    Institute of Scientific and Technical Information of China (English)

    张德昌; 张宽仁

    1995-01-01

    The effect of nueleoside diphosphate kinase (NDPK) on the activity of guanine nueleotide regulatory protein (G-protein) mediated phospholipase C (PLC) and on the [35S ] GTPTτS binding of G-protein was investigated in this work in order to demonstrate the mechanism behind the regulation of G-protein and its effector PLC by NDPK. The stimulation of PLC in turkey erythrocyte membrane by both GTP and GTPτS indicated that the PLC stimulation was msdiated by G-protein, NDPK alone stimulated PLC activity, as well as the stimulation in the presence of GTP and GDP, in a dose-dependent manner. However, NDPK inhibited GTPτS-stimulated PLC, Furthermore, NDPK inhibited [35S] GTPτS binding of purified Gi-protein in a non-competitive manner. A hypothesis implying an important role of direct interaction of G-protein and NDPK in the regulation of their functions is suggested and discussed.

  19. Diethylstilbestrol-diphosphate induces chromosomal aberrations but not sister chromatid exchanges in murine bone marrow cells in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Ivett, J.L. (North Carolina State Univ., Raleigh); Tice, R.R.

    1981-01-01

    Diethylstilbestrol diphosphate (DES-dp) clastogenesis was examined in the bone marrow of C57B1/6 male and female mice. Significant and sex-related dose effects were observed for the induction of chromatid-type chromosomal aberrations and for the inhibition of cellular proliferation. Females were more sensitive to the effects of DES-dp than males when assessed for either induced chromosomal aberrations or proliferative inhibition. Contrary to other published results, we did not observe either an increase in sister chromatid exchanges or an increased incidence of aneuploidy. Ovariectomy reduced the ability of DES-dp to inhibit cellular proliferation and decreased the high degree of variability between animals at high doses of DES-dp. The results of our studies show that DES is a clastogenic agent in vivo which may relate to its carcinogenicity.

  20. α-Hydroxy-β-keto acid rearrangement-decarboxylation: impact on thiamine diphosphate-dependent enzymatic transformations.

    Science.gov (United States)

    Beigi, Maryam; Loschonsky, Sabrina; Lehwald, Patrizia; Brecht, Volker; Andrade, Susana L A; Leeper, Finian J; Hummel, Werner; Müller, Michael

    2013-01-14

    The thiamine diphosphate (ThDP) dependent MenD catalyzes the reaction of α-ketoglutarate with pyruvate to selectively form 4-hydroxy-5-oxohexanoic acid 2, which seems to be inconsistent with the assumed acyl donor role of the physiological substrate α-KG. In contrast the reaction of α-ketoglutarate with acetaldehyde gives exclusively the expected 5-hydroxy-4-oxo regioisomer 1. These reactions were studied by NMR and CD spectroscopy, which revealed that with pyruvate the observed regioselectivity is due to the rearrangement-decarboxylation of the initially formed α-hydroxy-β-keto acid rather than a donor-acceptor substrate role variation. Further experiments with other ThDP-dependent enzymes, YerE, SucA, and CDH, verified that this degenerate decarboxylation can be linked to the reduced enantioselectivity of acyloins often observed in ThDP-dependent enzymatic transformations.

  1. Theoretical pKa prediction of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc

    Science.gov (United States)

    Vipperla, Bhavaniprasad; Griffiths, Thomas M.; Wang, Xingyong; Yu, Haibo

    2017-01-01

    The pKa value of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc (UDP-GlcNAc) has been successfully calculated using density functional theory methods in conjunction with the Polarizable Continuum Models. Theoretical methods were benchmarked over a dataset comprising of alkyl phosphates. B3LYP/6-31+G(d,p) calculations using SMD solvation model provide excellent agreement with the experimental data. The predicted pKa for UDP-GlcNAc is consistent with most recent NMR studies but much higher than what it has long been thought to be. The importance of this study is evident that the predicted pKa for UDP-GlcNAc supports its potential role as a catalytic base in the substrate-assisted biocatalysis.

  2. Distribution of the thiamin diphosphate C(2)-proton during catalysis of acetaldehyde formation by brewers' yeast pyruvate decarboxylase.

    Science.gov (United States)

    Harris, T K; Washabaugh, M W

    1995-10-31

    The distribution of tritium derived from enzyme-bound [thiazole-2-T]thiamin diphosphate (TDP) during the reaction of pyruvate to form acetaldehyde catalyzed by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was determined under single-turnover conditions ([E] > [S]) in the presence of the nonsubstrate allosteric effector pyruvamide. The specific radioactivity of the [1-L]acetaldehyde product and solvent ([L]H2O) was 43 +/- 4% and 54 +/- 2%, respectively, of the initial specific radioactivity of PDC-bound [thiazole-2-T]TDP and was independent of the extent of the single-turnover reaction. There is little (< or = 3%) or no return of the abstracted C(2)-hydron to the C(2) position of PDC-bound TDP. This provides evidence that the abstracted C(2)-hydron is involved in the specific protonation of the C(alpha) position of the PDC-bound intermediate 2-(1-hydroxyethyl)thiamin diphosphate (HETDP), which is cleaved to form [1-L]acetaldehyde and PDC-bound [thiazole-2-H]TDP. The partial exchange of C(2)-derived tritium into solvent requires that (1) hydron transfer from C(2) occurs to a catalytic-base in which the conjugate catalytic acid is partially shielded from hydron exchange with the solvent, (2) the conjugate catalytic acid transfers the C(2)-derived hydron to the C(alpha) position of HETDP, and (3) hydron transfer to C(2) to regenerate the coenzyme occurs either from solvent directly or from a second catalytic acid of the enzyme that undergoes rapid hydron exchange with the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Nature of the main contaminant in the drug primaquine diphosphate: SFC and SFC-MS methods of analysis.

    Science.gov (United States)

    Brondz, Ilia; Ekeberg, Dag; Bell, David S; Annino, Amy R; Hustad, Jan Arild; Svendsen, Robert; Vlachos, Vaso; Oakley, Paul; Langley, G John; Mohini, Thite; Amaury, Cazenave-Gassiot; Mikhalitsyn, Felix

    2007-02-19

    The drug primaquine diphosphate is used for causative treatment of malaria. Using HPLC-MS and GC-MS, this research group was previously able to show that the main contaminant of primaquine is the positional isomer quinocide [I. Brondz, D. Mantzilas, U. Klein, D. Ekeberg, E. Hvattum, M.N. Lebedeva, F.S. Mikhailitsyn, G.D. Soulimanov, J. Roe, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 800 (2004) 211-223; I. Brondz, U. Klein, D. Ekeberg, D. Mantzilas, E. Hvattum, H. Schultz, F. S. Mikhailitsyn, Asian J. Chem. 17 (2005) 1678-1688]. Primaquine and quinocide are highly toxic substances which can have a number of side effects upon use in medical treatment. A standard for quinocide is not typically commercially available. In the present work, supercritical fluid chromatography-mass spectrometry (SFC-MS) with two different columns was used to achieve a shorter analysis time for the separation between the positional isomers quinocide and primaquine in primaquine diphosphate and to elucidate additional information about differences in their MS fragmentation. Unlike using HPLC-MS, it was possible to achieve the differential fragmentation of positional isomers at branching points using the SFC-MS technique. The desired short analysis time was achieved using SFC equipped with a Discovery HS F5 column and the differential fragmentation of positional isomers during SFC-MS provides information on the differences in the structure of these substances. Using a Chiralpak AD-H chiral column, it was possible to resolve the enantiomers in primaquine and separate quinocide from those enantiomers.

  4. Zoledronate derivatives as potential inhibitors of uridine diphosphate-galactose ceramide galactosyltransferase 8: A combined molecular docking and dynamic study.

    Science.gov (United States)

    Pannuzzo, Giovanna; Graziano, Adriana Carol Eleonora; Pannuzzo, Martina; Masman, Marcelo Fabricio; Avola, Rosanna; Cardile, Venera

    2016-11-01

    Krabbe's disease is a neurodegenerative disorder caused by deficiency of galactocerebrosidase activity that affects the myelin sheath of the nervous system, involving dysfunctional metabolism of sphingolipids. It has no cure. Because substrate inhibition therapy has been shown to be effective in some human lysosomal storage diseases, we hypothesize that a substrate inhibition therapeutic approach might be appropriate to allow correction of the imbalance between formation and breakdown of glycosphingolipids and to prevent pathological storage of psychosine. The enzyme responsible for the biosynthesis of galactosylceramide and psychosine is uridine diphosphate-galactose ceramide galactosyltransferase (2-hydroxyacylsphingosine 1-β-galactosyltransferase; UGT8; EC 2.4.1.45), which catalyzes the transferring of galactose from uridine diphosphate-galactose to ceramide or sphingosine, an important step of the biosynthesis of galactosphingolipids. Because some bisphosphonates have been identified as selective galactosyltransferase inhibitors, we verify the binding affinity to a generated model of the enzyme UGT8 and investigate the molecular mechanisms of UGT8-ligand interactions of the bisphosphonate zoledronate by a multistep framework combining homology modeling, molecular docking, and molecular dynamics simulations. From structural information on UGTs' active site stereochemistry, charge density, and access through the hydrophobic environment, the molecular docking procedure allowed us to identify zoledronate as a potential inhibitor of human ceramide galactosyltransferase. More importantly, zoledronate derivates were designed through computational modeling as putative new inhibitors. Experiments in vivo and in vitro have been planned to verify the possibility of using zoledronate and/or the newly identified inhibitors of UGT8 for a substrate inhibition therapy useful for treatment of Krabbe's disease and/or other lysosomal disorders. © 2016 Wiley Periodicals, Inc.

  5. Deciphering the genomic structure, function and evolution of carotenogenesis related phytoene synthases in grasses

    Directory of Open Access Journals (Sweden)

    Dibari Bianca

    2012-06-01

    Full Text Available Abstract Background Carotenoids are isoprenoid pigments, essential for photosynthesis and photoprotection in plants. The enzyme phytoene synthase (PSY plays an essential role in mediating condensation of two geranylgeranyl diphosphate molecules, the first committed step in carotenogenesis. PSY are nuclear enzymes encoded by a small gene family consisting of three paralogous genes (PSY1-3 that have been widely characterized in rice, maize and sorghum. Results In wheat, for which yellow pigment content is extremely important for flour colour, only PSY1 has been extensively studied because of its association with QTLs reported for yellow pigment whereas PSY2 has been partially characterized. Here, we report the isolation of bread wheat PSY3 genes from a Renan BAC library using Brachypodium as a model genome for the Triticeae to develop Conserved Orthologous Set markers prior to gene cloning and sequencing. Wheat PSY3 homoeologous genes were sequenced and annotated, unravelling their novel structure associated with intron-loss events and consequent exonic fusions. A wheat PSY3 promoter region was also investigated for the presence of cis-acting elements involved in the response to abscisic acid (ABA, since carotenoids also play an important role as precursors of signalling molecules devoted to plant development and biotic/abiotic stress responses. Expression of wheat PSYs in leaves and roots was investigated during ABA treatment to confirm the up-regulation of PSY3 during abiotic stress. Conclusions We investigated the structural and functional determinisms of PSY genes in wheat. More generally, among eudicots and monocots, the PSY gene family was found to be associated with differences in gene copy numbers, allowing us to propose an evolutionary model for the entire PSY gene family in Grasses.

  6. Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, Anopheles gambiae

    Institute of Scientific and Technical Information of China (English)

    Xin Zhang; Kun Yan Zhu

    2013-01-01

    Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control.However,our understanding of biochemical properties of insect CHSs has been very limited.We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito,Anopheles gambiae.Our study,which represents the first time to use a nonradioactive method to assay CHS activity in an insect species,determined the optimal conditions for measuring the enzyme activity,including pH,temperature,and concentrations of the substrate uridine diphosphate N-acetyl-D-glucosamine (UDPGlcNAc) and Mg++.The optimal pH was about 6.5-7.0,and the highest activity was detected at temperatures between 37℃ and 44℃.Dithithreitol is required to prevent melanization of the enzyme extract.CHS activity was enhanced at low concentration of GlcNAc,but inhibited at high concentrations.Proteolytic activation of the activity is significant both in the 500×g supernatant and the 40 000×g pellet.Our study revealed only slight in vitro inhibition ofA.gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5μmol/L) examined.There was no in vitro inhibition by polyoxin D at any concentration examined.Furthermore,we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined.Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A.gambiae.

  7. Transcriptome profiling, and cloning and characterization of the main monoterpene synthases of Coriandrum sativum L.

    Science.gov (United States)

    Galata, Mariana; Sarker, Lukman S; Mahmoud, Soheil S

    2014-06-01

    Terpenoids are a large and diverse class of specialized metabolites that are essential for the growth and development of plants, and have tremendous industrial applications. The mericarps of Coriandrum sativum L. (coriander) produce an essential oil (EO) rich in monoterpenes, volatile C10 terpenoids. To investigate EO metabolism, the transcriptome of coriander mericarps, at three developmental stages (early, mid, late) was sequenced via Illumina technology and a transcript library was produced. To validate the usability of the transcriptome sequences, two terpene synthase candidate genes, CsγTRPS and CsLINS, encoding 558 and 562 amino acid proteins were expressed in bacteria, and the recombinant proteins purified by Ni-NTA affinity chromatography. The 65.16 (CsγTRPS) and 65.91 (CsLINS)kDa recombinant proteins catalyzed the conversion of geranyl diphosphate, the precursor to monoterpenes, to γ-terpinene and (S)-linalool, respectively, with apparent Vmax and Km values of 2.24±0.16 (CsγTRPS); 19.63±1.05 (CsLINS)pkat/mg and 66.25±13 (CsγTRPS); 2.5±0.6 (CsLINS)μM, respectively. Together, CsγTRPS and CsLINS account for the majority of EO constituents in coriander mericarps. Investigation of the coriander transcriptome, and knowledge gained from these experiments will facilitate future studies concerning essential and fatty acid oil production in coriander. They also enable efforts to improve the coriander oils through metabolic engineering or plant breeding.

  8. Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis: probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233-Mg2+B motif.

    Science.gov (United States)

    Vedula, L Sangeetha; Jiang, Jiaoyang; Zakharian, Tatiana; Cane, David E; Christianson, David W

    2008-01-15

    Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the "aspartate-rich" motif D(100)DXX(D/E) that coordinates to Mg2+A and Mg2+C, and the "NSE/DTE" motif N(225)DXXSXXXE that chelates Mg2+B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.

  9. Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP-glucose production with sucrose synthase.

    Science.gov (United States)

    Schmölzer, Katharina; Lemmerer, Martin; Gutmann, Alexander; Nidetzky, Bernd

    2017-04-01

    Nucleotide sugar-dependent ("Leloir") glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine-5'-diphosphate glucose (UDP-glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (∼480 U/gcell dry weight ) suitable for whole-cell biotransformation. The UDP-glc production had excellent performance metrics of ∼100 gproduct /L, 86% yield (based on UDP), and a total turnover number of 103 gUDP-glc /gcell dry weight at a space-time yield of 10 g/L/h. Using efficient chromatography-free DSP, the UDP-glc was isolated in a single batch with ≥90% purity and in 73% isolated yield. Overall, the process would allow production of ∼0.7 kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924-928. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Halavaty, Andrei S. [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States); Kim, Youngchang [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Minasov, George; Shuvalova, Ludmilla; Dubrovska, Ievgeniia; Winsor, James [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States); Zhou, Min [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Onopriyenko, Olena; Skarina, Tatiana [Center for Structural Genomics of Infectious Diseases, (United States); University of Toronto, Toronto, Ontario M5G 1L6 (Canada); Papazisi, Leka; Kwon, Keehwan; Peterson, Scott N. [Center for Structural Genomics of Infectious Diseases, (United States); J. Craig Venter Institute, Rockville, MD 20850 (United States); Joachimiak, Andrzej [Center for Structural Genomics of Infectious Diseases, (United States); Argonne National Laboratory, Argonne, IL 60439 (United States); University of Chicago, Chicago, IL 60637 (United States); Savchenko, Alexei [Center for Structural Genomics of Infectious Diseases, (United States); University of Toronto, Toronto, Ontario M5G 1L6 (Canada); Anderson, Wayne F., E-mail: wf-anderson@northwestern.edu [Center for Structural Genomics of Infectious Diseases, (United States); Northwestern University, Chicago, IL 60611 (United States)

    2012-10-01

    The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS{sub SA}), Vibrio cholerae (AcpS{sub VC}) and Bacillus anthracis (AcpS{sub BA}) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS{sub BA} is emphasized because of the two 3′, 5′-adenosine diphosphate (3′, 5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′, 5′-ADP is bound as the 3′, 5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′, 5′-ADP–AcpS binary complexes. The position of the second 3′, 5′-ADP has never been described before. It is in close proximity to the first 3′, 5′-ADP and the ACP-binding site. The coordination of two ADPs in AcpS{sub BA} may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.

  11. Cellulose Synthases and Synthesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Anne Endler; Staffan Persson

    2011-01-01

    Plant cell walls are complex structures composed of high-molecular-weight polysaccharides,proteins,and lignins. Among the wall polysaccharides,cellulose,a hydrogen-bonded β-1,4-linked glucan microfibril,is the main load-bearing wall component and a key precursor for industrial applications. Cellulose is synthesized by large multi-meric cellulose synthase (CesA) complexes,tracking along cortical microtubules at the plasma membrane. The only known components of these complexes are the cellulose synthase proteins. Recent studies have identified tentative interaction partners for the CesAs and shown that the migratory patterns of the CesA complexes depend on phosphorylation status. These advances may become good platforms for expanding our knowledge about cellulose synthesis in the near future. In addition,our current understanding of cellulose chain polymerization in the context of the CesA complex is discussed.

  12. Caffeine synthase and related methyltransferases in plants.

    Science.gov (United States)

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region.

  13. Building-block selectivity of polyketide synthases.

    Science.gov (United States)

    Liou, Grace F; Khosla, Chaitan

    2003-04-01

    For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.

  14. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  15. Toxic and antifeedant activities of prenylated flavonoids isolated from Tephrosia apollinea L. against three major coleopteran pests of stored grains with reference to their structure-activity relationship.

    Science.gov (United States)

    Nenaah, Gomah E

    2014-01-01

    Four prenylated flavonoids, isoglabratephrin, (+)-glabratephrin, tephroapollin-F and lanceolatin-A, were isolated from Tephrosia apollinea L. and tested against three stored grain insects. Using the filter paper bioassay, compounds showed adulticidal activity against Sitophilus oryzae (L), Rhyzopertha dominica (F) and Tribolium castaneum (Herbst) at concentrations of 0.875, 1.75 and 3.5 mg mL(- 1). At 3.5 mg mL(- 1), tephroapollin-F was the most toxic (78.6%, 64.6% and 60.7% mortality was recorded after 10 days exposure of S. oryzae, R. dominica and T. castaneum, respectively). The F1 progeny production of insects was affected after parental exposure to flavonoids, where S. oryzae was the most susceptible. A nutritional bioassay, employing a flour disc and test concentrations of 0.65, 1.3 and 2.6 mg g(- 1), revealed a significant reduction in the relative growth rate, relative consumption rate and efficiency of conversion of ingested food by all insects. The structure-activity relationship among the tested flavonoids was discussed.

  16. Xanthohumol, a main prenylated chalcone from hops, reduces liver damage and modulates oxidative reaction and apoptosis in hepatitis C virus infected Tupaia belangeri.

    Science.gov (United States)

    Yang, Mingbo; Li, Na; Li, Fang; Zhu, Qianqian; Liu, Xi; Han, Qunying; Wang, Yawen; Chen, Yanping; Zeng, Xiaoyan; Lv, Yi; Zhang, Pingping; Yang, Cuiling; Liu, Zhengwen

    2013-08-01

    Hepatitis C virus (HCV) infection in Tupaia belangeri (Tupaia) represents an important model of HCV infection. Xanthohumol (XN), a major prenylated chalcone from hops, has various biological activities including hepatopreventive and anti-viral activities. In this study, Tupaias infected with HCV RNA positive serum were used to evaluate the effects of XN on liver damage, oxidative reaction, apoptosis and viral protein expression in liver tissues. The Tupaias inoculated with HCV positive serum had elevated serum aminotransferase levels and inflammation, especially hepatic steatosis, and HCV core protein expression in liver tissue. In the animals inoculated with HCV positive serum, XN significantly decreased aminotransferase levels, histological activity index, hepatic steatosis score and transforming growth factor β1 expression in liver tissue compared with the animals without XN intervention. XN reduced HCV core protein expression in liver tissue compared with those without XN intervention but the difference was not significant. XN significantly decreased malondialdehyde, potentiated superoxide dismutase and glutathione peroxidase, reduced Bax expression, promoted Bcl-xL and inhibited caspase 3 activity in liver tissues compared with the animals without XN intervention. These results indicate that XN may effectively improve hepatic inflammation, steatosis and fibrosis induced by HCV in Tupaias primarily through inhibition of oxidative reaction and regulation of apoptosis and possible suppression of hepatic stellate cell activation. The anti-HCV potential of XN needs further investigation.

  17. Prenylated flavonoids from the stems and leaves of Desmodium caudatum and evaluation of their inhibitory activity against the film-forming growth of Zygosaccharomyces rouxii F51.

    Science.gov (United States)

    Sasaki, Hisako; Shibata, Hirofumi; Imabayashi, Kiyoshi; Takaishi, Yoshihisa; Kashiwada, Yoshiki

    2014-07-09

    In order to provide scientific evidence for the relationship between the traditional usage, stems and leaves of Desmodium caudatum being used for protecting miso from spoilage, and its Japanese name (miso-naoshi), phytochemical study on the stems and leaves of this plant was carried out. Seven new prenylated flavonoids (1-3, 15-18), together with 19 known compounds (4-14, 19-26), were isolated, and the structures of new compounds were elucidated by extensive spectroscopic analyses. The minimum inhibitory concentrations (MICs) of 28 flavonoids, including 17 compounds (1, 2, 4, 5, 7-14, 20-22, 24, 25) isolated in this study and 11 flavonoids (27-37) previously isolated from the roots of this plant, against the film-forming yeast of Zygosaccharomyces rouxii F51 were determined. Fifteen compounds (2, 4, 5, 11, 12, 14, 21, 22, 25, 27, 28, 32-35) inhibited the film-forming growth of Z. rouxii F51 (MIC values, 7.8-62.5 μg/mL), among which 2",2"-dimethylpyran-(5",6":7,8)-5,2'-dihydroxy-4'-methoxy-(2R,3R)-dihydroflavonol (11) demonstrated potent inhibitory activity with an MIC value of 7.8 μg/mL.

  18. Comparative inhibitory effect of prenylated coumarins, ferulenol and ferprenin, contained in the 'poisonous chemotype' of Ferula communis on mammal liver microsomal VKORC1 activity.

    Science.gov (United States)

    Louvet, Marie-Sophie; Gault, Gilbert; Lefebvre, Sébastien; Popowycz, Florence; Boulven, Manon; Besse, Stéphane; Benoit, Etienne; Lattard, Virginie; Grancher, Denis

    2015-10-01

    Two distinguishable chemotypes of Ferula communis have been described: the 'nonpoisonous' chemotype, containing as main constituents the daucane esters; and the 'poisonous' chemotype containing prenylated coumarins, such as ferulenol and ferprenin. Ferulenol and ferprenin are 4-oxygenated molecules such as dicoumarol and warfarin, the first developed antivitamin K molecules. Antivitamin K molecules specifically inhibit VKORC1, an enzyme essential for recycling vitamin K. This latest is involved in the activation of clotting factors II, VII, IX, X. The inhibiting effect of ferulenol on VKORC1 was shown in rat, but not for species exposed to F. communis while in vivo studies suggest differences between animal susceptibility to ferulenol. The inhibiting effect of ferprenin on VKORC1 was never demonstrated. The aim of this study was to compare the inhibiting effect of both compounds on VKORC1 of different species exposed to F. communis. Vitamin K epoxide activity was evaluated for each species from liver microsomes and inhibiting effect of ferulenol and ferprenin was characterized. Ferulenol and ferprenin were shown to be able to inhibit VKORC1 from all analyzed species. Nevertheless, susceptibility to ferulenol and ferprenin presented differences between species, suggesting a different susceptibility to 'poisonous' chemotypes of F. communis.

  19. Neuroprotective effect of prenylated arylbenzofuran and flavonoids from morus alba fruits on glutamate-induced oxidative injury in HT22 hippocampal cells.

    Science.gov (United States)

    Seo, Kyeong-Hwa; Lee, Dae-Young; Jeong, Rak-Hun; Lee, Dong-Sung; Kim, Young-Eon; Hong, Eock-Kee; Kim, Youn-Chul; Baek, Nam-In

    2015-04-01

    A prenylated arylbenzofuran and six flavonoids were isolated from the fruits of Morus alba L. through silica gel, octadecyl silica gel, and Diaion HP-20 column chromatography. Based on the nuclear magnetic resonance, mass spectrometry, and infrared spectroscopic data, the chemical structures of the compounds were determined to be artoindonesianin O (1), isobavachalcone (2), morachalcone A (3), quercetin (4), astragalin (5), isoquercetin (6), and rutin (7). The isolated compounds were evaluated for protection of HT22-immortalized hippocampal cells against glutamate-induced oxidative stress. Compounds 1 and 3 exhibited protective effects with EC(50) values of 19.7±1.2 and 35.5±2.1 μM, respectively. The major compounds 1-3 and 7 were quantified using liquid chromatography/mass spectrometry analysis and were determined to be 1.88±2.1, 1.90±1.8, 0.78±1.5, and 37.29±2.2 mg/kg, respectively, in the ethanol extract of M. alba L. fruits.

  20. RNA interference-mediated repression of SmCPS (copalyldiphosphate synthase) expression in hairy roots of Salvia miltiorrhiza causes a decrease of tanshinones and sheds light on the functional role of SmCPS.

    Science.gov (United States)

    Cheng, Qiqing; Su, Ping; Hu, Yating; He, Yunfei; Gao, Wei; Huang, Luqi

    2014-02-01

    Tanshinones are a group of bioactive abietane-type norditerpenoid quinone compounds in Salvia miltiorrhiza. Copalyldiphosphate synthase of S. miltiorrhiza (SmCPS) is the first key enzyme in tanshinone biosynthesis from the universal diterpene precursor geranylgeranyl diphosphate. Hairy roots of S. miltiorrhiza were transformed with Agrobacterium rhizogenes carrying an RNA interference (RNAi) construct designed to silence SmCPS, and we examined the resulting SmCPS expression and tanshinone accumulation. In SmCPS–RNAi hairy roots, the transcript level of SmCPS was reduced to 26 % while the dihydrotanshinone I and cryptotanshinone levels were decreased by 53 and 38 % compared to those of the vector control hairy roots; tanshinone IIA was not detected. Therefore, the decreased expression of SmCPS caused a decrease in tanshinone levels which verifies that SmCPS is a key enzyme for tanshinone biosynthesis in S. miltiorrhiza.

  1. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.

    Science.gov (United States)

    Hyatt, David C; Croteau, Rodney

    2005-07-15

    Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.

  2. Enhancing sucrose synthase activity in transgenic potato (Solanum tuberosum L.) tubers results in increased levels of starch, ADPglucose and UDPglucose and total yield.

    Science.gov (United States)

    Baroja-Fernández, Edurne; Muñoz, Francisco José; Montero, Manuel; Etxeberria, Ed; Sesma, María Teresa; Ovecka, Miroslav; Bahaji, Abdellatif; Ezquer, Ignacio; Li, Jun; Prat, Salomé; Pozueta-Romero, Javier

    2009-09-01

    Sucrose synthase (SuSy) is a highly regulated cytosolic enzyme that catalyzes the conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. To determine the impact of SuSy activity in starch metabolism and yield in potato (Solanum tuberosum L.) tubers we measured sugar levels and enzyme activities in tubers of SuSy-overexpressing potato plants grown in greenhouse and open field conditions. We also transcriptionally characterized tubers of SuSy-overexpressing and -antisensed potato plants. SuSy-overexpressing tubers exhibited a substantial increase in starch, UDPglucose and ADPglucose content when compared with controls. Tuber dry weight, starch content per plant and total yield of SuSy-overexpressing tubers increased significantly over those of control plants. In contrast, activities of enzymes directly involved in starch metabolism in SuSy-overexpressing tubers were normal when compared with controls. Transcriptomic analyses using POCI arrays and the MapMan software revealed that changes in SuSy activity affect the expression of genes involved in multiple biological processes, but not that of genes directly involved in starch metabolism. These analyses also revealed a reverse correlation between the expressions of acid invertase and SuSy-encoding genes, indicating that the balance between SuSy- and acid invertase-mediated sucrolytic pathways is a major determinant of starch accumulation in potato tubers. Results presented in this work show that SuSy strongly determines the intracellular levels of UDPglucose, ADPglucose and starch, and total yield in potato tubers. We also show that enhancement of SuSy activity represents a useful strategy for increasing starch accumulation and yield in potato tubers.

  3. Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture.

    Science.gov (United States)

    Caniard, Anne; Zerbe, Philipp; Legrand, Sylvain; Cohade, Allison; Valot, Nadine; Magnard, Jean-Louis; Bohlmann, Jörg; Legendre, Laurent

    2012-07-26

    Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They

  4. Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L. and their relevance for perfume manufacture

    Directory of Open Access Journals (Sweden)

    Caniard Anne

    2012-07-01

    Full Text Available Abstract Background Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Results Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S-copalyl diphosphate. A class I diTPS (SsSS then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae. Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. Conclusions SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid

  5. Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture

    Science.gov (United States)

    2012-01-01

    Background Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Results Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. Conclusions SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol

  6. Blocking Cyclic Adenosine Diphosphate Ribose-mediated Calcium Overload Attenuates Sepsis-induced Acute Lung Injury in Rats

    Institute of Scientific and Technical Information of China (English)

    Qian-yi Peng; Yu Zou; Li-Na Zhang; Mei-Lin Ai; Wei Liu; Yu-Hang Ai

    2016-01-01

    Background:Acute lung injury (ALI) is a common complication of sepsis that is associated with high mortality.Intracellular Ca2+ overload plays an important role in the pathophysiology of sepsis-induced ALI,and cyclic adenosine diphosphate ribose (cADPR) is an important regulator of intracellular Ca2+ mobilization.The cluster of differentiation 38 (CD38)/cADPR pathway has been found to play roles in multiple inflammatory processes but its role in sepsis-induced ALI is still unknown.This study aimed to investigate whether the CD38/cADPR signaling pathway is activated in sepsis-induced ALI and whether blocking cADPR-mediated calcium overload attenuates ALI.Methods:Septic rat models were established by cecal ligation and puncture (CLP).Rats were divided into the sham group,the CLP group,and the CLP+ 8-bromo-cyclic adenosine diphosphate ribose (8-Br-cADPR) group.Nicotinamide adenine dinucleotide (NAD+),cADPR,CD38,and intracellular Ca2+ levels in the lung tissues were measured at 6,12,24,and 48 h after CLP surgery.Lung histologic injury,tumor necrosis factor (TNF)-α,malondialdehyde (MDA) levels,and superoxide dismutase (SOD) activities were measured.Results:NAD+,cADPR,CD38,and intracellular Ca2+ levels in the lungs of septic rats increased significantly at 24 h after CLP surgery.Treatment with 8-Br-cADPR,a specific inhibitor of cADPR,significantly reduced intracellular Ca2+ levels (P =0.007),attenuated lung histological injury (P =0.023),reduced TNF-α and MDA levels (P < 0.001 and P =0.002,respectively) and recovered SOD activity (P =0.031) in the lungs of septic rats.Conclusions:The CD38/cADPR pathway is activated in the lungs of septic rats,and blocking cADPR-mediated calcium overload with 8-Br-cADPR protects against sepsis-induced ALI.

  7. Functional and evolutionary relationships between terpene synthases from Australian Myrtaceae.

    Science.gov (United States)

    Keszei, Andras; Brubaker, Curt L; Carter, Richard; Köllner, Tobias; Degenhardt, Jörg; Foley, William J

    2010-06-01

    Myrtaceae is one of the chemically most variable and most significant essential oil yielding plant families. Despite an abundance of chemical information, very little work has focussed on the biochemistry of terpene production in these plants. We describe 70 unique partial terpene synthase transcripts and eight full-length cDNA clones from 21 myrtaceous species, and compare phylogenetic relationships and leaf oil composition to reveal clades defined by common function. We provide further support for the correlation between function and phylogenetic relationships by the first functional characterisation of terpene synthases from Myrtaceae: a 1,8-cineole synthase from Eucalyptus sideroxylon and a caryophyllene synthase from Eucalyptusdives.

  8. Temperature effects on the interaction mechanisms between the europium (III) and uranyl ions and zirconium diphosphate; Effets de la temperature sur les mecanismes d'interaction entre les ions europium (3) et uranyle et le diphosphate de zirconium

    Energy Technology Data Exchange (ETDEWEB)

    Finck, N

    2006-10-15

    Temperature should remain higher than 25 C in the near field environment of a nuclear waste repository for thousands years. In this context, the aim of this work is to study the temperature influence on the interaction mechanisms between europium (III) and uranyl ions and zirconium diphosphate, as well as the influence of a complexing medium (nitrate) on the sorption of the lanthanide. The experimental definition of the equilibria was achieved by combining a structural investigation with the macroscopic sorption data. Surface complexes were characterized at all temperatures (25 C to 90 C) by TRLFS experiments carried out on dry and in situ samples using an oven. This characterization was completed by XPS experiments carried out at 25 C on samples prepared at 25 C and 90 C. The reaction constants (surface hydration and cations sorption) were obtained by simulating the experimental data with the constant capacitance surface complexation model. The reaction constants temperature dependency allowed one to characterize thermodynamically the different reactions by application of the van't Hoff relation. The validity of this law was tested by performing microcalorimetric measurements of the sorption heat for both cations. (author)

  9. Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

    Science.gov (United States)

    Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

    2015-11-15

    The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes.

  10. 7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

    Science.gov (United States)

    Wypijewska, Anna; Bojarska, Elzbieta; Lukaszewicz, Maciej; Stepinski, Janusz; Jemielity, Jacek; Davis, Richard E; Darzynkiewicz, Edward

    2012-10-09

    Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

  11. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    OpenAIRE

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity ...

  12. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Science.gov (United States)

    Alquézar, Berta; Rodríguez, Ana; de la Peña, Marcos; Peña, Leandro

    2017-01-01

    Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z)-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays. PMID:28883829

  13. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Berta Alquézar

    2017-08-01

    Full Text Available Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck genome sequence allowed us to characterize for the first time the terpene synthase (TPS family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays.

  14. Structures of Bacteroides fragilis uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (BfLpxA).

    Science.gov (United States)

    Ngo, Alice; Fong, Kai T; Cox, Daniel L; Chen, Xi; Fisher, Andrew J

    2015-05-01

    Uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes a reversible reaction for adding an O-acyl group to the GlcNAc in UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A constitutes a major component of lipopolysaccharides, also referred to as endotoxins, which form the outer monolayer of the outer membrane of Gram-negative bacteria. Ligand-free and UDP-GlcNAc-bound crystal structures of LpxA from Bacteroides fragilis NCTC 9343, the most common pathogenic bacteria found in abdominal abscesses, have been determined and are presented here. The enzyme crystallizes in a cubic space group, with the crystallographic threefold axis generating the biological functional homotrimer and with each monomer forming a nine-rung left-handed β-helical (LβH) fold in the N-terminus followed by an α-helical motif in the C-terminus. The structure is highly similar to LpxA from other organisms. Yet, despite sharing a similar LβH structure with LpxAs from Escherichia coli and others, previously unseen calcium ions are observed on the threefold axis in B. fragilis LpxA to help stabilize the trimeric assembly.

  15. Synthesis and Crystal Structure of a New Potassium Yttrium Diphosphate Dihydrate KYP2O7·2H2O

    Institute of Scientific and Technical Information of China (English)

    Férid M. Horchani-Naifer K

    2008-01-01

    The dihydrated potassium yttrium diphosphate, KYP2O7·2H2O, has been synthe-sized for the first time. Single-crystal XRD study has allowed the determination of its structure and correct formula. It crystallizes in the monoclinic system, space group P21/c, with the cell parameters a = 7.7069(3), b = 10.5801(4), c = 10.0204(5) ?, β = 93.24(3)°, V = 815.76(6) ?3, Z = 4, Mr = 337.98, F(000) = 656, Dc = 2.752 g/cm3 and μ = 8.073 mm-1. The single-crystal structure was solved from 1856 unique reflections with the final R = 0.0365 and wR = 0.0772, refined with 119 parameters. The atomic arrangement of KYP2O7·2H2O is built by the P2O7 groups, the YO7 and the KO10 polyhedrons which are connected by corner and edge-sharing to form a three-dimensional framework. Water molecules are directly bonded to the Y and K atoms, and they are located in channels running along the a direction.

  16. Vanadium-Binding Ability of Nucleoside Diphosphate Kinase from the Vanadium-Rich Fan Worm, Pseudopotamilla occelata.

    Science.gov (United States)

    Yamaguchi, Nobuo; Yoshinaga, Masafumi; Kamino, Kei; Ueki, Tatsuya

    2016-06-01

    Polychaete fan worms and ascidians accumulate high levels of vanadium ions. Several vanadiumbinding proteins, known as vanabins, have been found in ascidians. However, no vanadium-binding factors have been isolated from the fan worm. In the present study, we sought to identify vanadiumbinding proteins in the branchial crown of the fan worm using immobilized metal ion affinity chromatography. A nucleoside diphosphate kinase (NDK) homolog was isolated and determined to be a vanadium-binding protein. Kinase activity of the NDK homologue, PoNDK, was suppressed by the addition of V(IV), but was unaffected by V(V). The effect of V(IV) on PoNDK precedes its activation by Mg(II). This is the first report to describe the relationship between NDK and V(IV). PoNDK is located in the epidermis of the branchial crown, and its distribution is very similar to that of vanadium. These results suggest that PoNDK is associated with vanadium accumulation and metabolism in P. occelata.

  17. Consensus and future directions on the definition of high on-treatment platelet reactivity to adenosine diphosphate.

    Science.gov (United States)

    Bonello, Laurent; Tantry, Udaya S; Marcucci, Rossella; Blindt, Ruediger; Angiolillo, Dominick J; Becker, Richard; Bhatt, Deepak L; Cattaneo, Marco; Collet, Jean Philippe; Cuisset, Thomas; Gachet, Christian; Montalescot, Gilles; Jennings, Lisa K; Kereiakes, Dean; Sibbing, Dirk; Trenk, Dietmar; Van Werkum, Jochem W; Paganelli, Franck; Price, Matthew J; Waksman, Ron; Gurbel, Paul A

    2010-09-14

    The addition of clopidogrel to aspirin treatment reduces ischemic events in a wide range of patients with cardiovascular disease. However, recurrent ischemic event occurrence during dual antiplatelet therapy, including stent thrombosis, remains a major concern. Platelet function measurements during clopidogrel treatment demonstrated a variable and overall modest level of P2Y(12) inhibition. High on-treatment platelet reactivity to adenosine diphosphate (ADP) was observed in selected patients. Multiple studies have now demonstrated a clear association between high on-treatment platelet reactivity to ADP measured by multiple methods and adverse clinical event occurrence. However, the routine measurement of platelet reactivity has not been widely implemented and recommended in the guidelines. Reasons for the latter include: 1) a lack of consensus on the optimal method to quantify high on-treatment platelet reactivity and the cutoff value associated with clinical risk; and 2) limited data to support that alteration of therapy based on platelet function measurements actually improves outcomes. This review provides a consensus opinion on the definition of high on-treatment platelet reactivity to ADP based on various methods reported in the literature and proposes how this measurement may be used in the future care of patients. Copyright © 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  18. Separation and purification of uridine diphosphate-glucuronosyltransferases by chromatofocusing on a high-performance liquid chromatograph.

    Science.gov (United States)

    Takanashi, H; Homma, H; Matsui, M

    1989-06-01

    A rapid method for the separation and purification of uridine diphosphate-glucuronosyltransferases (GT) was developed with the use of chromatofocusing on a high-performance liquid chromatograph. GT isoenzymes solubilized from hepatic microsomes of Wistar rats were separated on a Mono P column, a pre-packed column for chromatofocusing. Using 4-nitrophenol, testosterone and androsterone as substrates, four fractions with different GT activities were separated in a pH gradient from 9.5 to 7.0. Two isoenzymes, testosterone GT and androsterone GT were purified to apparent homogeneity. They were eluted at pH 8.9 and 8.0 and had subunit molecular weight values of 50000 and 52000, respectively. Approximately 10 mg of solubilized microsomal proteins was applied and the elution was completed within 2 h. Addition of N-nitrodiethylamine, an in vitro activator of GT activity, enhanced the GT activity toward 4-nitrophenol in the three fractions. This chromatographic analysis confirmed the absence of androsterone GT isoenzyme in LA Wistar rats, a mutant strain in terms of androsterone glucuronidation.

  19. Altered expression and activities of enzymes involved in thiamine diphosphate biosynthesis in Saccharomyces cerevisiae under oxidative and osmotic stress.

    Science.gov (United States)

    Kowalska, Ewa; Kujda, Marta; Wolak, Natalia; Kozik, Andrzej

    2012-08-01

    Thiamine diphosphate (TDP) serves as a cofactor for enzymes engaged in pivotal carbohydrate metabolic pathways, which are known to be modulated under stress conditions to ensure the cell survival. Recent reports have proven a protective role of thiamine (vitamin B(1)) in the response of plants to abiotic stress. This work aimed at verifying a hypothesis that also baker's yeast, which can synthesize thiamine de novo similarly to plants and bacteria, adjust thiamine metabolism to adverse environmental conditions. Our analyses on the gene expression and enzymatic activity levels generally showed an increased production of thiamine biosynthesis enzymes (THI4 and THI6/THI6), a TDP synthesizing enzyme (THI80/THI80) and a TDP-requiring enzyme, transketolase (TKL1/TKL) by yeast subjected to oxidative (1 mM hydrogen peroxide) and osmotic (1 M sorbitol) stress. However, these effects differed in magnitude, depending on yeast growth phase and presence of thiamine in growth medium. A mutant thi4Δ with increased sensitivity to oxidative stress exhibited enhanced TDP biosynthesis as compared with the wild-type strain. Similar tendencies were observed in mutants yap1Δ and hog1Δ defective in the signaling pathways of the defense against oxidative and osmotic stress, respectively, suggesting that thiamine metabolism can partly compensate damages of yeast general defense systems.

  20. Insights into the Thiamine Diphosphate Enzyme Activation Mechanism: Computational Model for Transketolase Using a Quantum Mechanical/Molecular Mechanical Method.

    Science.gov (United States)

    Nauton, Lionel; Hélaine, Virgil; Théry, Vincent; Hecquet, Laurence

    2016-04-12

    We propose the first computational model for transketolase (TK), a thiamine diphosphate (ThDP)-dependent enzyme, using a quantum mechanical/molecular mechanical method on the basis of crystallographic TK structures from yeast and Escherichia coli, together with experimental kinetic data reported in the literature with wild-type and mutant TK. This model allowed us to define a new route for ThDP activation in the enzyme environment. We evidenced a strong interaction between ThDP and Glu418B of the TK active site, itself stabilized by Glu162A. The crucial point highlighted here is that deprotonation of ThDP C2 is not performed by ThDP N4' as reported in the literature, but by His481B, involving a HOH688A molecule bridge. Thus, ThDP N4' is converted from an amino form to an iminium form, ensuring the stabilization of the C2 carbanion or carbene. Finally, ThDP activation proceeds via an intermolecular process and not by an intramolecular one as reported in the literature. More generally, this proposed ThDP activation mechanism can be applied to some other ThDP-dependent enzymes and used to define the entire TK mechanism with donor and acceptor substrates more accurately.

  1. Progress in the experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations.

    Science.gov (United States)

    Jordan, Frank; Nemeria, Natalia S

    2014-12-01

    Thiamin diphosphate (ThDP), the vitamin B1 coenzyme is an excellent representative of coenzymes, which carry out electrophilic catalysis by forming a covalent complex with their substrates. The function of ThDP is to greatly increase the acidity of two carbon acids by stabilizing their conjugate bases, the ylide/carbene/C2-carbanion of the thiazolium ring and the C2α-carbanion/enamine, once the substrate binds to ThDP. In recent years, several ThDP-bound intermediates on such pathways have been characterized by both solution and solid-state methods. Prominent among these advances are X-ray crystallographic results identifying both oxidative and non-oxidative intermediates, rapid chemical quench followed by NMR detection of several intermediates which are stable under acidic conditions, solid-state NMR and circular dichroism detection of the states of ionization and tautomerization of the 4'-aminopyrimidine moiety of ThDP in some of the intermediates. These methods also enabled in some cases determination of the rate-limiting step in the complex series of steps. This review is an update of a review with the same title published by the authors in 2005 in this Journal. Much progress has been made in the intervening decade in the identification of the intermediates and their application to gain additional mechanistic insight.

  2. Simultaneous liquid chromatographic assessment of thiamine, thiamine monophosphate and thiamine diphosphate in human erythrocytes: a study on alcoholics.

    Science.gov (United States)

    Mancinelli, Rosanna; Ceccanti, Mauro; Guiducci, Maria Soccorsa; Sasso, Guido Francesco; Sebastiani, Gemma; Attilia, Maria Luisa; Allen, John Paul

    2003-06-15

    An isocratic HPLC procedure for the assessment of thiamine (T), thiamine monophosphate (TMP) and thiamine diphosphate (TDP) in human erythrocytes is described. Several aspects of the procedure make it suitable for both clinical and research purposes: limits of detection and quantification of 1 and 2.5 nmol/l, respectively, recovery of 102% on average (range 93-112%), intra- and inter-day precisions within 5 and 9%, respectively, total elution time 15 min. This analytical methodology was applied to a case-control study on erythrocyte samples from 103 healthy subjects and 36 alcohol-dependent patients at risk of thiamine deficiency. Mean control values obtained were: T=89.6+/-22.7 nmol/l, TMP=4.4+/-6.6 nmol/l and TDP=222.23+/-56.3 nmol/l. T and TDP mean values of alcoholics were significantly lower than those of control cases: T=69.4+/-35.9 nmol/l (Pthiamine was established in the study of alcohol related problems.

  3. Cooperation of divalent ions and thiamin diphosphate in regulation of the function of pig heart pyruvate dehydrogenase complex.

    Science.gov (United States)

    Czerniecki, J; Czygier, M

    2001-12-01

    The role of Mg2+, Ca2+, and Mn2+ in regulation of purified pig heart pyruvate dehydrogenase complex (PDC) containing endogenous thiamin diphosphate (TDP) was studied. It was found that the effects of the cations depended on the presence of exogenous TDP. In the absence of added TDP, the divalent cations led to a shortening of a lag phase of the PDC reaction and a strong reduction of the Km value for pyruvate. The relative efficiency of the three types of ions are presented as follows: Mn2+>Ca2+>Mg2+. The other sources claim that in the presence of exogenous TDP, which alone strongly increased the affinity of PDC for pyruvate, any significant additional effects of the cations were not observed. However, Mg2+, Ca2+, and Mn2+ decreased the Km value for CoA in both cases, the absence and presence of exogenous TDP, in approximately a similar extent (about twofold). The affinity of PDC for NAD+ seems to be not sensitive to the presence of the divalent cations. The data obtained suggest that Mg2+, Ca2+, and Mn2+ can cooperate with TDP as positive regulatory effectors of pig heart PDC on the level of pyruvate dehydrogenase and lipoamide acetyltransferase components of the complex.

  4. Preparation and in vitro evaluation of liposomal chloroquine diphosphate loaded by a transmembrane pH-gradient method.

    Science.gov (United States)

    Qiu, Liyan; Jing, Na; Jin, Yi

    2008-09-01

    This study developed an active loading method for encapsulating chloroquine diphosphate (CQ) into liposomes. The effects of different formulation factors on the encapsulation efficiency (EE) and the size of CQ liposomes were investigated. These factors included the internal phase of liposomes, the external phase of liposomes, the ratio of drug to soybean phosphatidylcholine (drug/SPC), the ratio of cholesterol to soybean phosphatidylcholine (Chol/SPC), and the incubation temperature and time. The EE (93%) was obtained when using drug/SPC (1:50 mass ratio), SPC/Chol (1:5 mass ratio) at 0.10 M citrate-sodium citrate buffer (pH 3.6). As 5 mol% methoxypoly(ethylene glycol)(2,000) cholesteryl succinate (CHS-PEG(2000)) or distearoyl phosphatidylethanolamine-poly (ethylene glycol)(2,000) (DSPE-PEG(2000)) was added, the size of particle was reduced and the EE was improved. Freeze-drying with 5% trehalose as a cryoprotectant was carried out to achieve long-term stability. The drug release studies were performed in vitro simulating the desired application conditions, such as physiological fluids (pH 7.4), tumor tissues (pH 6.5) and endosomal compartments (pH 5.5). The release of CQ from the liposomes prepared via remote loading showed the significant pH-sensitivity and retention properties, which favored the application of liposomal CQ at tumor tissues and endosomal compartments.

  5. Effects of cumene hydroperoxide on adenosine diphosphate ribosyl transferase in mononuclear leukocytes of patients with adenomatous polyps in the colon.

    Science.gov (United States)

    Markowitz, M M; Johnson, D B; Pero, R W; Winawer, S J; Miller, D G

    1988-03-01

    We have studied the effects of plasma and of cumene hydroperoxide (CUM) on adenosine diphosphate ribosyl transferase (ADPRT) from mononuclear leukocytes (HML) of patients with colonic adenomatous polyps (n = 22), with colonic hyperplastic polyps (n = 5) and with neither type of polyp (controls) (n = 6). ADPRT was measured after incubation of HML with plasma alone (termed the plasma value), and with plasma plus CUM (50 microM) (the activated value); the difference elicited by CUM was termed the induced value. There was no significant difference in values between the control and hyperplastic polyp groups: these were combined for further analysis. The plasma (P = 0.038), activated (P = 0.009) and induced (P = 0.0024) values of the combined group all differed significantly from those of the adenoma group. At low exposures, CUM stimulated both ADPRT and unscheduled DNA synthesis and, at higher exposures, inactivated both. Pretreatment of HML with vitamin E protected against these effects of CUM, while pretreatment with diamide (which depletes GSH) accentuated the effects. This study demonstrates a differential reaction of ADPRT in patients harboring colonic adenomas and suggests that the origin of this difference may lie in cellular responses to oxidative stress.

  6. Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in sycamore and poplar.

    Science.gov (United States)

    Dalessandro, G; Northcote, D H

    1977-02-15

    During the transition from primary wall formation to secondary thickening there is a marked shift in the synthesis of pectin, hemicellulose and cellulose. The activities of the enzymes [UDP-D-galactose 4-epimerase (EC 5.1.3.2)8 UDP-l-arabinose 4-epimerase (EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D--glucuronate decarboxylase (EC 4.1.1.35)] were measured in cambial cells, differentiating xylem cells and differentiated xylem cells isolated from sycamore and poplar trees, and phloem cells from poplar. At the final stage of the differentiation of cambium to xylem there was a decrease in activity of the enzymes directly involved in producing the soluble precursors of pectin (DUP-D-galactose 4-epimerase and UDP-L-arabinose 4-epimerase and an increase in those producing the precursors of hemicellulose (UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase). These results strongly suggest ahat the changes were correlated with the differences observed in the chemical composition of the wall during development. The changes found in the catalytic activity of the enzymes of nucleoside diphosphate sugar interconversion exert a coarse control over the synthesis of pectin and hemicelluloses. The tissues at all stages of development contained the necessary enzyme activities to produce all the precursors of pectin and hemicellulose, even at the final stage of differentiation when no pectin was formed.

  7. Global warming, plant paraquat resistance, and light signal transduction through nucleoside diphosphate kinase as a paradigm for increasing food supply.

    Science.gov (United States)

    Hasunuma, Kohji; Yoshida, Yusuke; Haque, Mohamed Emdadul; Wang, Ni-yan; Fukamatsu, Yosuke; Miyoshi, Osamu; Lee, Bumkyu

    2011-10-01

    Light signal transduction was studied in extracts of mycelia of the fungus Neurospora crassa, and the third internodes of dark-grown Pisum sativum cv Alaska. Both processes increased the phosphorylation of nucleoside diphosphate kinase (NDPK). NDPK may function as a carrier of reduction equivalents, as it binds NADH, thereby providing electrons to transform singlet oxygen to superoxide by catalases (CAT). As the C-termini of NDPK interact with CAT which receive singlet oxygen, emitted from photoreceptors post light perception (which is transmitted to ambient triplet oxygen), we hypothesize that this may increase phospho-NDPK. Singlet oxygen, emitted from the photoreceptor, also reacts with unsaturated fatty acids in membranes thereby forming malonedialdehyde, which in turn could release ions from, e.g., the thylacoid membrane thereby reducing the rate of photosynthesis. A mutant of Alaska pea, which exhibited two mutations in chloroplast NDPK-2 and one mutation in mitochondrial localized NDPK-3, was resistant to reactive oxygen species including singlet oxygen and showed an increase in the production of carotenoids, anthocyanine, and thereby could reduce the concentration of singlet oxygen. The reduction of the concentration of singlet oxygen is predicted to increase the yield of crop plants, such as Alaska pea, soybean, rice, wheat, barley, and sugarcane. This approach to increase the yield of crop plants may contribute not only to enhance food supply, but also to reduce the concentration of CO(2) in the atmosphere.

  8. Metabolic engineering of Escherichia coli to produce 2'-fucosyllactose via salvage pathway of guanosine 5'-diphosphate (GDP)-l-fucose.

    Science.gov (United States)

    Chin, Young-Wook; Seo, Nari; Kim, Jae-Han; Seo, Jin-Ho

    2016-11-01

    2'-Fucosyllactose (2-FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2-FL. Introduction of the fkp gene coding for fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α-1,2-fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2-FL from fucose, lactose and glycerol. To enhance the lactose flux to 2-FL production, the attenuated, and deleted mutants of β-galactosidase were employed. Moreover, the 2-FL yield and productivity were further improved by deletion of the fucI-fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed-batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI-fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2-FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443-2452. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Growth inhibitory effect of paratocarpin E, a prenylated chalcone isolated from Euphorbia humifusa Wild., by induction of autophagy and apoptosis in human breast cancer cells.

    Science.gov (United States)

    Gao, Suyu; Sun, Dejuan; Wang, Guan; Zhang, Jin; Jiang, Yingnan; Li, Guoyu; Zhang, Ke; Wang, Lei; Huang, Jian; Chen, Lixia

    2016-12-01

    Five flavones, including four flavonoids and one prenylated chalcone (paratocarpin E), were isolated from E. humifusa. and their chemical structures were established by spectroscopic analyses. We assessed the efficacy of these compounds against the growth of human breast cancer, leukemic, kidney cancer cell lines. Among them, paratocarpin E showed significant cytotoxicity against these cancer cell lines with an IC50 of 19.6μM on the growth of MCF-7 cells. Paratocarpin E treatment of MCF-7 cells resulted in typical apoptotic features via increasing expression of activated caspase-8 and -9 and PARP cleavage. Moreover, paratocarpin E altered the expression of Bax and Bcl-2, leading to the release of cytochrome c from the mitochondria into the cytosol, suggesting that the mitochondria-mediated apoptosis was initiated. In addition, paratocarpin E increased the MDC-positive autophagic vacuoles, the ratio of LC3-II/LC3-I protein levels of Beclin-1, but decreased p62 expression, indicating the potent pro-autophagic effects of paratocarpin E in MCF-7 cells. Mechanistically, cell death induced by paratocarpin E is able to induce apoptosis of MCF-7 cells by activating p38 and JNK signaling pathway while inhibiting Erk pathway. Furthermore, paratocarpin E promotes the activation and nuclear translocation of NF-κB, which plays an important role in balancing paratocarpin E-mediated apoptosis and autophagy. The molecular docking study also revealed that paratocarpin E bound to Fas and NF-κB complex. These findings provide initial evidences that paratocarpin E can be used as a potential anti-cancer drug in future for breast cancer therapy.

  10. Evolution and function of phytochelatin synthases.

    Science.gov (United States)

    Clemens, Stephan

    2006-02-01

    Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed.

  11. Torque generation mechanism of ATP synthase

    Science.gov (United States)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  12. [Four cases of aldosterone synthase deficiency in childhood].

    Science.gov (United States)

    Collinet, E; Pelissier, P; Richard, O; Gay, C; Pugeat, M; Morel, Y; Stephan, J-L

    2012-11-01

    Neonatal salt-wasting syndromes are rare but potentially serious conditions. Isolated hypoaldosteronism is an autosomal recessive inherited disorder of terminal aldosterone synthesis, leading to selective aldosterone deficiency. Two different biochemical forms of this disease have been described, called aldosterone synthase deficiency or corticosterone methyl oxydase, types I and II. In type I, there is no aldosterone synthase activity and the 18 hydroxycorticosterone (18 OHB) level is low, whereas in type II, a residual activity of aldosterone synthase persists and 18 OHB is overproduced. We report on four patients with isolated hypoaldosteronism. In 2 of them, who were recently diagnosed with aldosterone synthase deficit, we discuss the symptoms and treatment. The 2 other patients are now adults. We discuss the long-term outcome, the quality of adult life, aldosterone synthase deficits, as well as the pathophysiology and molecular analysis.

  13. The thorium phosphate diphosphate as matrix for radioactive waste conditioning: radionuclide immobilization and behavior under irradiation; Le phosphate diphosphate de thorium, matrice pour le conditionnement des dechets radioactifs: immobilisation de radionucleides, comportement sous irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Pichot, Erwan [Inst. de Physique Nucleaire, Paris-11 Univ., 91 - Orsay (France)

    1999-04-13

    The aim of this work was to perform successively the decontamination of liquid solutions and the final immobilization of radionuclide storage using the same matrix. For this, thorium phosphate-diphosphate (TPD) of the formula Th{sub 4}P{sub 6}O{sub 23}, is proposed as a very resistant to water corrosion matrix. A new compound, thorium phosphate hydrogeno-phosphate (TPHP) of the formula Th{sub 2}(PO{sub 4}){sub 2}(HPO{sub 4}), nH{sub 2}O with n=3-7 was synthesized and characterized. Heated at 1100 deg.C it is transformed into the TDP. Ion exchange properties of TPHP were investigated. The exchange yields of imponderable caesium, strontium and americium ion onto TPHP (NaNO{sub 3} 0.1 M media at pH=6) are equal to 60% for the first one and 100% for the two others. The results interpreted in terms of ion-exchange led to determine selectivity coefficient values for each cation and suggested that only hydrated ions are exchanged. While the TPD is proposed for the high level nuclear waste storage, the irradiation effects, particularly structural modifications were studied using both {gamma} irradiation and charged particle irradiation. ESR and TL methods were carried out in order to identify radicals created during gamma radiation exposure. Correlation between ESR and TL experiments performed at room temperature clearly show three of PO{sub 3}{sup 2-} species and one POO{center_dot} species of free radicals. We have shown that Au-ion irradiation in the range of MeV energy involved TPD structure and chemical modifications. Important sputtering was interpreted in terms of local thermal chemical decomposition. We have shown, at room temperature, that the amorphization dose for heavy ion irradiation is between 0.1 to 0.4 dpa. (author) 146 refs., 46 figs., 21 tabs.

  14. Study of the irradiation effects on thorium phosphate diphosphate ({beta}-TPD): consequences on its chemical durability; Etude des effets d'irradiation sur le phosphate diphosphate de thorium ({beta}-PDT): consequences sur la durabilite chimique

    Energy Technology Data Exchange (ETDEWEB)

    Tamain, C

    2005-12-15

    Since Thorium Phosphate Diphosphate (beta-TPD) can be considered as a potential host matrix for long-term storage in underground repository, it is necessary to study the irradiation effects on the structure of this ceramics and the consequences on its chemical durability. Sintered samples of beta-TPD and of associated solid solutions of beta-TUPD were irradiated under ion beams and then altered in aqueous solutions. Depending on the electronic LET value, beta-TPD can be completely or partly amorphized. Furthermore, the ability of recrystallization of the amorphous material by thermal annealing was also demonstrated. Some leaching tests, realized on these irradiated samples, have shown a significant effect of the amorphous fraction on the normalized dissolution rate which was increased by a factor of 10 from the crystallized to the fully amorphized material. Correlatively, the amorphous fraction also modified the delay to reach the saturation conditions associated to the thermodynamic equilibria involved. On the other hand, it exhibited no influence neither on other kinetic parameters, such as activation energy of the dissolution process or partial order related to the proton concentration, nor on the nature of the neo-formed phase formed at the saturation of the leachate and identified as Thorium Phosphate Hydrogeno-Phosphate Hydrate (TPHPH). Beta-TUPD samples were also irradiated by gamma and alpha rays during leaching tests to study the effects of radiolysis in the leaching medium on the normalized leaching rate. It appeared that the radiolytic species occurring in the dissolution mechanism were unstable, disappearing quickly when stopping the irradiation. (author)

  15. Pseudouridines and pseudouridine synthases of the ribosome.

    Science.gov (United States)

    Ofengand, J; Malhotra, A; Remme, J; Gutgsell, N S; Del Campo, M; Jean-Charles, S; Peil, L; Kaya, Y

    2001-01-01

    psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes

  16. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    Directory of Open Access Journals (Sweden)

    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  17. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway.

    Science.gov (United States)

    Taura, Futoshi; Tanaka, Shinji; Taguchi, Chiho; Fukamizu, Tomohide; Tanaka, Hiroyuki; Shoyama, Yukihiro; Morimoto, Satoshi

    2009-06-18

    Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile.

  18. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  19. The nitric oxide synthase of mouse spermatozoa.

    Science.gov (United States)

    Herrero, M B; Goin, J C; Boquet, M; Canteros, M G; Franchi, A M; Perez Martinez, S; Polak, J M; Viggiano, J M; Gimeno, M A

    1997-07-01

    Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 10(7) spermatozoa synthesized 7 +/- 2 pmol of L-[14C]citrulline. Besides, L-citrulline formation depended on the incubation time and on the concentration of L-arginine present in the incubation medium. Different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) but not aminoguanidine, inhibited L-[14C]citrulline formation. Western-blot analysis of solubilized sperm proteins revealed a unique band of M(r)=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.

  20. Endothelial nitric oxide synthase in the microcirculation.

    Science.gov (United States)

    Shu, Xiaohong; Keller, T C Stevenson; Begandt, Daniela; Butcher, Joshua T; Biwer, Lauren; Keller, Alexander S; Columbus, Linda; Isakson, Brant E

    2015-12-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.