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Sample records for mevalonate diphosphate decarboxylase

  1. Bacopa monniera recombinant mevalonate diphosphate decarboxylase: Biochemical characterization.

    PubMed

    Abbassi, Shakeel J; Vishwakarma, Rishi K; Patel, Parth; Kumari, Uma; Khan, Bashir M

    2015-08-01

    Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) is an important enzyme in the mevalonic acid pathway catalyzing the Mg(2+)-ATP dependant decarboxylation of mevalonate 5-diphosphate (MVAPP) to isopentenyl diphosphate (IPP). Bacopa monniera recombinant MDD (BmMDD) protein was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Km and Vmax for MVAPP were 144 μM and 52 U mg(-1) respectively. The values of turnover (kcat) and kcat/Km for mevalonate 5-diphosphate were determined to be 40s(-1) and 2.77×10(5) M(-1) s(-1) and kcat and kcat/Km values for ATP were found to be 30 s(-1) and 2.20×10(4) M(-1) s(-1), respectively. pH activity profile indicated the involvement of carboxylate ion, lysine and arginine for the activity of enzyme. The apparent activation energy for the BmMDD catalyzed reaction was 12.7 kJ mol(-1). Optimum pH and temperature for the forward reaction was found to be 8.0 and 45 °C. The enzyme was most stable at pH 7 at 20 °C with the deactivation rate constant (Kd(*)) of 1.69×10(-4) and half life (t1/2) of 68 h. The cation studies suggested that BmMDD is a cation dependant enzyme and optimum activity was achieved in the presence of Mg(2+).

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

    SciTech Connect

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

    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.

  3. Functional and conformational transitions of mevalonate diphosphate decarboxylase from Bacopa monniera.

    PubMed

    Abbassi, Shakeel; Patel, Krunal; Khan, Bashir; Bhosale, Siddharth; Gaikwad, Sushama

    2016-02-01

    Functional and conformational transitions of mevalonate diphosphate decarboxylase (MDD), a key enzyme of mevalonate pathway in isoprenoid biosynthesis, from Bacopa monniera (BmMDD), cloned and overexpressed in Escherichia coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and Circular dichroism spectroscopy. Native BmMDD is a helix dominant structure with 45% helix and 11% sheets and possesses seven tryptophan residues with two residues exposed on surface, three residues partially exposed and two situated in the interior of the protein. Thermal denaturation of BmMDD causes rapid structural transitions at and above 40°C and transient exposure of hydrophobic residues at 50°C, leading to aggregation of the protein. An acid induced molten globule like structure was observed at pH 4, exhibiting altered but compact secondary structure, distorted tertiary structure and exposed hydrophobic residues. The molten globule displayed different response at higher temperature and similar response to chemical denaturation as compared to the native protein. The surface tryptophans have predominantly positively charged amino acids around them, as indicated by higher KSV for KI as compared to that for CsCl. The native enzyme displayed two different lifetimes, τ1 (1.203±0.036 ns) and τ2 (3.473±0.12 ns) indicating two populations of tryptophan.

  4. The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene

    PubMed Central

    Hall, Stephen J.; Eastham, Graham; Licence, Peter; Stephens, Gill

    2015-01-01

    Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min−1 g cells−1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min−1 mg protein−1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway. PMID:25636853

  5. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    SciTech Connect

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V.

    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.

  6. Crystal structures of Staphylococcus epidermidis mevalonate diphosphate decarboxylase bound to inhibitory analogs reveal new insight into substrate binding and catalysis.

    PubMed

    Barta, Michael L; Skaff, D Andrew; McWhorter, William J; Herdendorf, Timothy J; Miziorko, Henry M; Geisbrecht, Brian V

    2011-07-08

    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 Å 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 Å resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 Å resolution). Comparison of these structures provides a physical basis for the significant differences in K(i) values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser(192) as making potential contributions to catalysis. Significantly, Ser → Ala substitution of this side chain decreases k(cat) by ∼10(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 Å 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.

  7. The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase is essential for viability, and a single Leu-to-Pro mutation in a conserved sequence leads to thermosensitivity.

    PubMed Central

    Bergès, T; Guyonnet, D; Karst, F

    1997-01-01

    The mevalonate diphosphate decarboxylase is an enzyme which converts mevalonate diphosphate to isopentenyl diphosphate, the building block of isoprenoids. We used the Saccharomyces cerevisiae temperature-sensitive mutant defective for mevalonate diphosphate decarboxylase previously described (C. Chambon, V. Ladeveve, M. Servouse, L. Blanchard, C. Javelot, B. Vladescu, and F. Karst, Lipids 26:633-636, 1991) to characterize the mutated allele. We showed that a single change in a conserved amino acid accounts for the temperature-sensitive phenotype of the mutant. Complementation experiments were done both in the erg19-mutated background and in a strain in which the ERG19 gene, which was shown to be an essential gene for yeast, was disrupted. Epitope tagging of the wild-type mevalonate diphosphate decarboxylase allowed us to isolate the enzyme in an active form by a versatile one-step immunoprecipitation procedure. Furthermore, during the course of this study, we observed that a high level of expression of the wild-type ERG19 gene led to a lower sterol steady-state accumulation compared to that of a wild-type strain, suggesting that this enzyme may be a key enzyme in mevalonate pathway regulation. PMID:9244250

  8. Active site binding modes of inhibitors of Staphylococcus aureus mevalonate diphosphate decarboxylase from docking and molecular dynamics simulations.

    PubMed

    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.

  9. Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production

    SciTech Connect

    Kang, Aram; George, Kevin W.; Wang, George; Baidoo, Edward; Keasling, Jay D.; Lee, Taek Soon

    2015-12-17

    Branched C 5 alcohols are promising biofuels with excellent combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C 5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C 5 alcohols and initial precursor to longer chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C 5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.

  10. Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production

    DOE PAGES

    Kang, Aram; George, Kevin W.; Wang, George; ...

    2015-12-17

    Branched C 5 alcohols are promising biofuels with excellent combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C 5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C 5 alcohols and initial precursor to longermore » chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C 5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.« less

  11. Disruption of the mevalonate pathway induces dNTP depletion and DNA damage.

    PubMed

    Martín Sánchez, Covadonga; Pérez Martín, José Manuel; Jin, Jong-Sik; Dávalos, Alberto; Zhang, Wei; de la Peña, Gema; Martínez-Botas, Javier; Rodríguez-Acebes, Sara; Suárez, Yajaira; Hazen, María José; Gómez-Coronado, Diego; Busto, Rebeca; Cheng, Yung-Chi; Lasunción, Miguel A

    2015-09-01

    The mevalonate pathway is tightly linked to cell division. Mevalonate derived non-sterol isoprenoids and cholesterol are essential for cell cycle progression and mitosis completion respectively. In the present work, we studied the effects of fluoromevalonate, a competitive inhibitor of mevalonate diphosphate decarboxylase, on cell proliferation and cell cycle progression in both HL-60 and MOLT-4 cells. This enzyme catalyzes the synthesis of isopentenyl diphosphate, the first isoprenoid in the cholesterol biosynthesis pathway, consuming ATP at the same time. Inhibition of mevalonate diphosphate decarboxylase was followed by a rapid accumulation of mevalonate diphosphate and the reduction of ATP concentrations, while the cell content of cholesterol was barely affected. Strikingly, mevalonate diphosphate decarboxylase inhibition also resulted in the depletion of dNTP pools, which has never been reported before. These effects were accompanied by inhibition of cell proliferation and cell cycle arrest at S phase, together with the appearance of γ-H2AX foci and Chk1 activation. Inhibition of Chk1 in cells treated with fluoromevalonate resulted in premature entry into mitosis and massive cell death, indicating that the inhibition of mevalonate diphosphate decarboxylase triggered a DNA damage response. Notably, the supply of exogenously deoxyribonucleosides abolished γ-H2AX formation and prevented the effects of mevalonate diphosphate decarboxylase inhibition on DNA replication and cell growth. The results indicate that dNTP pool depletion caused by mevalonate diphosphate decarboxylase inhibition hampered DNA replication with subsequent DNA damage, which may have important consequences for replication stress and genomic instability.

  12. Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis.

    PubMed Central

    Campos, N; Rodríguez-Concepción, M; Sauret-Güeto, S; Gallego, F; Lois, L M; Boronat, A

    2001-01-01

    Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway. PMID:11115399

  13. Molecular cloning, characterization, and function analysis of a mevalonate pyrophosphate decarboxylase gene from Ganoderma lucidum.

    PubMed

    Shi, Liang; Qin, Lei; Xu, Yingjie; Ren, Ang; Fang, Xing; Mu, Dashuai; Tan, Qi; Zhao, Mingwen

    2012-05-01

    This study investigated the role of the mevalonate pyrophosphate decarboxylase gene in the triterpene biosynthetic pathway of Ganoderma lucidum. The mevalonate pyrophosphate decarboxylase gene (mvd) was isolated using a degenerate primer-PCR technique. An analysis of the Gl-mvd transcription profile revealed a positive correlation between the expression of the Gl-mvd gene and triterpene content changes in G. lucidum during development. Furthermore, a promoter deletion analysis was conducted in G. lucidum to investigate the promoter activity and the role of methyl jasmonate (MeJA) responsive elements in the mvd promoter under the MeJA elicitor. The overexpression of Gl-mvd increased triterpene accumulation compared with the wild-type strain and increased the expression of several genes involved in the triterpene biosynthetic pathway. The findings of this study suggest that mvd may play an important role in triterpene biosynthesis regulation. Moreover, there may be the interactions among the genes involved in the triterpene biosynthetic pathway in the G. lucidum. Additionally, this study provides an approach for improving triterpene content through the overexpression of a key gene.

  14. Mevalonate-derived isopentenyl diphosphate is the biosynthetic precursor of ubiquinone prenyl side chain in tobacco BY-2 cells.

    PubMed Central

    Disch, A; Hemmerlin, A; Bach, T J; Rohmer, M

    1998-01-01

    Study of the incorporation of 13C-labelled glucose or pyruvate into the isoprenoids of tobacco BY-2 cells allowed the biosynthetic origin of isopentenyl diphosphate to be determined. Sterols synthesized in the cytoplasm and the prenyl chain of ubiquinone Q10 located in mitochondria were derived from the same isopentenyl diphosphate pool, synthesized from acetyl-CoA through mevalonate, whereas the prenyl chain of plastoquinone was obtained from the mevalonate-independent glyceraldehyde 3-phosphate/pyruvate route, like all chloroplast isoprenoids from higher plants. These results are in accord with the compartmentation and complete enzymic independence of the biosynthesis of long-chain all-trans polyprenols in mitochondria and chloroplasts. PMID:9531505

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

    PubMed

    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.

  16. An unusual isopentenyl diphosphate isomerase found in the mevalonate pathway gene cluster from Streptomyces sp. strain CL190

    PubMed Central

    Kaneda, Kazuhide; Kuzuyama, Tomohisa; Takagi, Motoki; Hayakawa, Yoichi; Seto, Haruo

    2001-01-01

    A gene cluster encoding five enzymes of the mevalonate pathway had been cloned from Streptomyces sp. strain CL190. This gene cluster contained an additional ORF, orfD, encoding an unknown protein that was detected in some archaebacteria and some Gram-positive bacteria including Staphylococcus aureus. The recombinant product of orfD was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 37 kDa by SDS-polyacrylamide gel electrophoresis and 155 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a tetramer. The purified enzyme contained flavin mononucleotide (FMN) with the amount per tetramer being 1.4 to 1.6 mol/mol. The enzyme catalyzed the isomerization of isopentenyl diphosphate (IPP) to produce dimethylallyl diphosphate (DMAPP) in the presence of both FMN and NADPH. The Escherichia coli plasmid expressing orfD could complement the disrupted IPP isomerase gene in E. coli. These results indicate that orfD encodes an unusual IPP isomerase showing no sequence similarity to those of IPP isomerases identified to date. Based on the difference in enzymatic properties, we classify the IPP isomerases into two types: Type 2 for FMN- and NAD(P)H-dependent enzymes, and type 1 for the others. In view of the critical role of this isomerase in S. aureus and of the different enzymatic properties of mammalian (type 1) and S. aureus (type 2) isomerases, this unusual enzyme is considered to be a suitable molecular target for the screening of antibacterial drugs specific to S. aureus. PMID:11158573

  17. Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase.

    PubMed

    Kim, Yong-Kyoung; Kim, Yeon Bok; Uddin, Md Romij; Lee, Sanghyun; Kim, Soo-Un; Park, Sang Un

    2014-10-17

    To elucidate the function of mevalonate-5-pyrophosphate decarboxylase (MVD) and farnesyl pyrophosphate synthase (FPS) in triterpene biosynthesis, the genes governing the expression of these enzymes were transformed into Panax ginseng hairy roots. All the transgenic lines showed higher expression levels of PgMVD and PgFPS than that by the wild-type control. Among the hairy root lines transformed with PgMVD, M18 showed the highest level of transcription compared to the control (14.5-fold higher). Transcriptions of F11 and F20 transformed with PgFPS showed 11.1-fold higher level compared with control. In triterpene analysis, M25 of PgMVD produced 4.4-fold higher stigmasterol content (138.95 μg/100 mg, dry weight [DW]) than that by the control; F17 of PgFPS showed the highest total ginsenoside (36.42 mg/g DW) content, which was 2.4-fold higher compared with control. Our results indicate that metabolic engineering in P. ginseng was successfully achieved through Agrobacterium rhizogenes-mediated transformation and that the accumulation of phytosterols and ginsenosides was enhanced by introducing the PgMVD and PgFPS genes into the hairy roots of the plant. Our results suggest that PgMVD and PgFPS play an important role in the triterpene biosynthesis of P. ginseng.

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

    PubMed

    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.

  19. Characterisation of a thiamine diphosphate-dependent alpha-keto acid decarboxylase from Proteus mirabilis JN458.

    PubMed

    Wang, Biying; Bai, Yajun; Fan, Taiping; Zheng, Xiaohui; Cai, Yujie

    2017-10-01

    Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40-50°C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg(2+) as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. Km and kcat values for phenyl pyruvate were 0.62mM and 77.38s(-1), respectively, and the kcat/Km value was 124.81mM(-1)s(-1). The enzyme properties suggest it may act effectively under cheese ripening conditions. Copyright © 2017. Published by Elsevier Ltd.

  20. Peroxisomal localisation of the final steps of the mevalonic acid pathway in planta.

    PubMed

    Simkin, Andrew J; Guirimand, Grégory; Papon, Nicolas; Courdavault, Vincent; Thabet, Insaf; Ginis, Olivia; Bouzid, Sadok; Giglioli-Guivarc'h, Nathalie; Clastre, Marc

    2011-11-01

    In plants, the mevalonic acid (MVA) pathway provides precursors for the formation of triterpenes, sesquiterpenes, phytosterols and primary metabolites important for cell integrity. Here, we have cloned the cDNA encoding enzymes catalysing the final three steps of the MVA pathway from Madagascar periwinkle (Catharanthus roseus), mevalonate kinase (MVK), 5-phosphomevalonate kinase (PMK) and mevalonate 5-diphosphate decarboxylase (MVD). These cDNA were shown to functionally complement MVA pathway deletion mutants in the yeast Saccharomyces cerevisiae. Transient transformations of C. roseus cells with yellow fluorescent protein (YFP)-fused constructs reveal that PMK and MVD are localised to the peroxisomes, while MVK was cytosolic. These compartmentalisation results were confirmed using the Arabidopsis thaliana MVK, PMK and MVD sequences fused to YFP. Based on these observations and the arguments raised here we conclude that the final steps of the plant MVA pathway are localised to the peroxisome.

  1. Discovery of a metabolic alternative to the classical mevalonate pathway

    PubMed Central

    Dellas, Nikki; Thomas, Suzanne T; Manning, Gerard; Noel, Joseph P

    2013-01-01

    Eukarya, Archaea, and some Bacteria encode all or part of the essential mevalonate (MVA) metabolic pathway clinically modulated using statins. Curiously, two components of the MVA pathway are often absent from archaeal genomes. The search for these missing elements led to the discovery of isopentenyl phosphate kinase (IPK), one of two activities necessary to furnish the universal five-carbon isoprenoid building block, isopentenyl diphosphate (IPP). Unexpectedly, we now report functional IPKs also exist in Bacteria and Eukarya. Furthermore, amongst a subset of species within the bacterial phylum Chloroflexi, we identified a new enzyme catalyzing the missing decarboxylative step of the putative alternative MVA pathway. These results demonstrate, for the first time, a functioning alternative MVA pathway. Key to this pathway is the catalytic actions of a newly uncovered enzyme, mevalonate phosphate decarboxylase (MPD) and IPK. Together, these two discoveries suggest that unforeseen variation in isoprenoid metabolism may be widespread in nature. DOI: http://dx.doi.org/10.7554/eLife.00672.001 PMID:24327557

  2. High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

    DOE PAGES

    Kang, Aram; Meadows, Corey W.; Canu, Nicolas; ...

    2017-04-05

    Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass” isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonatemore » monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either kcat or Ki of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.« less

  3. Cloning and Characterization of 1-Deoxy-d-Xylulose 5-Phosphate Synthase from Streptomyces sp. Strain CL190, Which Uses both the Mevalonate and Nonmevalonate Pathways for Isopentenyl Diphosphate Biosynthesis

    PubMed Central

    Kuzuyama, Tomohisa; Takagi, Motoki; Takahashi, Shunji; Seto, Haruo

    2000-01-01

    In addition to the ubiquitous mevalonate pathway, Streptomyces sp. strain CL190 utilizes the nonmevalonate pathway for isopentenyl diphosphate biosynthesis. The initial step of this nonmevalonate pathway is the formation of 1-deoxy-d-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase. The corresponding gene, dxs, was cloned from CL190 by using PCR with two oligonucleotide primers synthesized on the basis of two highly conserved regions among dxs homologs from six genera. The dxs gene of CL190 encodes 631 amino acid residues with a predicted molecular mass of 68 kDa. The recombinant enzyme overexpressed in Escherichia coli was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 130 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of 9.0, with a Vmax of 370 U per mg of protein and Kms of 65 μM for pyruvate and 120 μM for d-glyceraldehyde 3-phosphate. The purified enzyme catalyzed the formation of 1-deoxyxylulose by condensation of pyruvate and glyceraldehyde as well, with a Km value of 35 mM for d-glyceraldehyde. To compare the enzymatic properties of CL190 and E. coli DXP synthases, the latter enzyme was also overexpressed and purified. Although these two enzymes had different origins, they showed the same enzymatic properties. PMID:10648511

  4. Detection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase

    SciTech Connect

    Chakraborty, Sumit; Nemeria, Natalia S.; Balakrishnan, Anand; Brandt, Gabriel S.; Kneen, Malea M.; Yep, Alejandra; McLeish, Michael J.; Kenyon, George L.; Petsko, Gregory A.; Ringe, Dagmar; Jordan, Frank

    2009-04-02

    The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with {lambda}{sub max} at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with {lambda}{sub max} at 437 nm. The time course of formation/depletion of the PKB-ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB-ThDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with {lambda}{sub max} at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 {angstrom}) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2{alpha} atom, rather than of the enamine, and supporting the assignment of the {lambda}{sub max} at 473 nm to the PAA-ThDP adduct. The structure of the PAA-ThDP covalent complex is the first example of a product-ThDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.

  5. Mevalonate-Farnesal Biosynthesis in Ticks: Comparative Synganglion Transcriptomics and a New Perspective

    PubMed Central

    Zhu, Jiwei; Khalil, Sayed M.; Mitchell, Robert D.; Bissinger, Brooke W.; Egekwu, Noble; Sonenshine, Daniel E.; Roe, R. Michael

    2016-01-01

    Juvenile hormone (JH) controls the growth, development, metamorphosis, and reproduction of insects. For many years, the general assumption has been that JH regulates tick and other acarine development and reproduction the same as in insects. Although researchers have not been able to find the common insect JHs in hard and soft tick species and JH applications appear to have no effect on tick development, it is difficult to prove the negative or to determine whether precursors to JH are made in ticks. The tick synganglion contains regions which are homologous to the corpora allata, the biosynthetic source for JH in insects. Next-gen sequencing of the tick synganglion transcriptome was conducted separately in adults of the American dog tick, Dermacentor variabilis, the deer tick, Ixodes scapularis, and the relapsing fever tick, Ornithodoros turicata as a new approach to determine whether ticks can make JH or a JH precursor. All of the enzymes that make up the mevalonate pathway from acetyl-CoA to farnesyl diphosphate (acetoacetyl-CoA thiolase, HMG-S, HMG-R, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, and farnesyl diphosphate synthase) were found in at least one of the ticks studied but most were found in all three species. Sequence analysis of the last enzyme in the mevalonate pathway, farnesyl diphosphate synthase, demonstrated conservation of the seven prenyltransferase regions and the aspartate rich motifs within those regions typical of this enzyme. In the JH branch from farnesyl diphosphate to JH III, we found a putative farnesol oxidase used for the conversion of farnesol to farnesal in the synganglion transcriptome of I. scapularis and D. variabilis. Methyltransferases (MTs) that add a methyl group to farnesoic acid to make methyl farnesoate were present in all of the ticks studied with similarities as high as 36% at the amino acid level to insect JH acid methyltransferase (JHAMT). However, when the tick MTs were compared to

  6. Mevalonate-Farnesal Biosynthesis in Ticks: Comparative Synganglion Transcriptomics and a New Perspective.

    PubMed

    Zhu, Jiwei; Khalil, Sayed M; Mitchell, Robert D; Bissinger, Brooke W; Egekwu, Noble; Sonenshine, Daniel E; Roe, R Michael

    2016-01-01

    Juvenile hormone (JH) controls the growth, development, metamorphosis, and reproduction of insects. For many years, the general assumption has been that JH regulates tick and other acarine development and reproduction the same as in insects. Although researchers have not been able to find the common insect JHs in hard and soft tick species and JH applications appear to have no effect on tick development, it is difficult to prove the negative or to determine whether precursors to JH are made in ticks. The tick synganglion contains regions which are homologous to the corpora allata, the biosynthetic source for JH in insects. Next-gen sequencing of the tick synganglion transcriptome was conducted separately in adults of the American dog tick, Dermacentor variabilis, the deer tick, Ixodes scapularis, and the relapsing fever tick, Ornithodoros turicata as a new approach to determine whether ticks can make JH or a JH precursor. All of the enzymes that make up the mevalonate pathway from acetyl-CoA to farnesyl diphosphate (acetoacetyl-CoA thiolase, HMG-S, HMG-R, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, and farnesyl diphosphate synthase) were found in at least one of the ticks studied but most were found in all three species. Sequence analysis of the last enzyme in the mevalonate pathway, farnesyl diphosphate synthase, demonstrated conservation of the seven prenyltransferase regions and the aspartate rich motifs within those regions typical of this enzyme. In the JH branch from farnesyl diphosphate to JH III, we found a putative farnesol oxidase used for the conversion of farnesol to farnesal in the synganglion transcriptome of I. scapularis and D. variabilis. Methyltransferases (MTs) that add a methyl group to farnesoic acid to make methyl farnesoate were present in all of the ticks studied with similarities as high as 36% at the amino acid level to insect JH acid methyltransferase (JHAMT). However, when the tick MTs were compared to

  7. Synthesis of ATP derivatives of compounds of the mevalonate pathway (isopentenyl di- and triphosphate; geranyl di- and triphosphate, farnesyl di- and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases Potential relationship with the effect of bisphosphonates on osteoclasts.

    PubMed

    Sillero, Maria A Günther; de Diego, Anabel; Tavares, Janeth E F; Silva, Joana A D Catanho da; Pérez-Zúñiga, Francisco J; Sillero, Antonio

    2009-08-15

    Compounds of the mevalonate pathway containing a terminal di- or triphosphate (mev-PP or mev-PPP) were tested as substrates of several enzyme ligases (T4 RNA ligase, T4 DNA ligase, firefly luciferase and other ligases) for the synthesis of ATP derivatives of the mev-pppA or mev-ppppA type. T4 RNA ligase, in the presence of ATP and the substrates: geranyl, farnesyl or isopentenyl triphosphates, and geranyl, farnesyl, dimethylallyl or isopentenyl diphosphates, all at 0.3 mM concentration, catalyzed the synthesis of the corresponding ATP derivatives at a relative rate of activity of: 7.6+/-1.4 mU/mg or 100%; 39%; 42%; 24%; 18%; 12% and 6%, respectively. Inhibition (%) of the synthesis by excess of substrate (0.8 mM vs. 0.3 mM) was observed with farnesyl diphosphate (99%); farnesyl triphosphate (96%) and geranyl triphosphate (32%). V(max), K(m), K(cat) and K(cat)/K(m) values were also determined. The K(cat)/K(m) values calculated were for: farnesyl triphosphate, 166; geranyl triphosphate, 52.2; farnesyl diphosphate, 12.1; geranyl diphosphate, 8.6; isopentenyl triphosphate, 6.7; dimethylallyl diphosphate, 3.1 and isopentenyl diphosphate, 0.9. Similar results were obtained with T4 DNA ligase. The above-mentioned compounds were also substrates of firefly luciferase synthesizing the mev-pppA or mev-ppppA derivatives. In our hands, neither the acyl- or acetyl-CoA synthetases nor the ubiquiting activating enzyme (E1) catalyzed the synthesis of ATP derivatives of these compounds. The results here presented could be related with the mechanism of action of bisphosphonates on osteoclasts or tumor cells.

  8. An adaptation to life in acid through a novel mevalonate pathway

    DOE PAGES

    Vinokur, Jeffrey M.; Cummins, Matthew C.; Korman, Tyler P.; ...

    2016-12-22

    Here, extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previouslymore » identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.« less

  9. An adaptation to life in acid through a novel mevalonate pathway

    SciTech Connect

    Vinokur, Jeffrey M.; Cummins, Matthew C.; Korman, Tyler P.; Bowie, James U.

    2016-12-22

    Here, extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previously identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.

  10. An Adaptation To Life In Acid Through A Novel Mevalonate Pathway

    PubMed Central

    Vinokur, Jeffrey M.; Cummins, Matthew C.; Korman, Tyler P.; Bowie, James U.

    2016-01-01

    Extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previously identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments. PMID:28004831

  11. Bifunctionality of the thiamin diphosphate cofactor: assignment of tautomeric/ionization states of the 4'-aminopyrimidine ring when various intermediates occupy the active sites during the catalysis of yeast pyruvate decarboxylase.

    PubMed

    Balakrishnan, Anand; Gao, Yuhong; Moorjani, Prerna; Nemeria, Natalia S; Tittmann, Kai; Jordan, Frank

    2012-02-29

    Thiamin diphosphate (ThDP) dependent enzymes perform crucial C-C bond forming and breaking reactions in sugar and amino acid metabolism and in biosynthetic pathways via a sequence of ThDP-bound covalent intermediates. A member of this superfamily, yeast pyruvate decarboxylase (YPDC) carries out the nonoxidative decarboxylation of pyruvate and is mechanistically a simpler ThDP enzyme. YPDC variants created by substitution at the active center (D28A, E51X, and E477Q) and on the substrate activation pathway (E91D and C221E) display varying activity, suggesting that they stabilize different covalent intermediates. To test the role of both rings of ThDP in YPDC catalysis (the 4'-aminopyrimidine as acid-base, and thiazolium as electrophilic covalent catalyst), we applied a combination of steady state and time-resolved circular dichroism experiments (assessing the state of ionization and tautomerization of enzyme-bound ThDP-related intermediates), and chemical quench of enzymatic reaction mixtures followed by NMR characterization of the ThDP-bound intermediates released from YPDC (assessing occupancy of active centers by these intermediates and rate-limiting steps). Results suggest the following: (1) Pyruvate and analogs induce active site asymmetry in YPDC and variants. (2) The rare 1',4'-iminopyrimidine ThDP tautomer participates in formation of ThDP-bound intermediates. (3) Propionylphosphinate also binds at the regulatory site and its binding is reflected by catalytic events at the active site 20 Å away. (4) YPDC stabilizes an electrostatic model for the 4'-aminopyrimidinium ionization state, an important contribution of the protein to catalysis. The combination of tools used provides time-resolved details about individual events during ThDP catalysis; the methods are transferable to other ThDP superfamily members. © 2012 American Chemical Society

  12. Activation of thiamin diphosphate in enzymes.

    PubMed

    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.

  13. Mevalonate Biosynthesis Intermediates Are Key Regulators of Innate Immunity in Bovine Endometritis.

    PubMed

    Healey, Gareth D; Collier, Christine; Griffin, Sholeem; Schuberth, Hans-Joachim; Sandra, Olivier; Smith, David G; Mahan, Suman; Dieuzy-Labaye, Isabelle; Sheldon, I Martin

    2016-01-15

    Metabolic changes can influence inflammatory responses to bacteria. To examine whether localized manipulation of the mevalonate pathway impacts innate immunity, we exploited a unique mucosal disease model, endometritis, where inflammation is a consequence of innate immunity. IL responses to pathogenic bacteria and LPS were modulated in bovine endometrial cell and organ cultures by small molecules that target the mevalonate pathway. Treatment with multiple statins, bisphosphonates, squalene synthase inhibitors, and small interfering RNA showed that inhibition of farnesyl-diphosphate farnesyl transferase (squalene synthase), but not 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyl diphosphate synthase, reduced endometrial organ and cellular inflammatory responses to pathogenic bacteria and LPS. Although manipulation of the mevalonate pathway reduced cellular cholesterol, impacts on inflammation were independent of cholesterol concentration as cholesterol depletion using cyclodextrins did not alter inflammatory responses. Treatment with the isoprenoid mevalonate pathway-intermediates, farnesyl diphosphate and geranylgeranyl diphosphate, also reduced endometrial cellular inflammatory responses to LPS. These data imply that manipulating the mevalonate pathway regulates innate immunity within the endometrium, and that isoprenoids are regulatory molecules in this process, knowledge that could be exploited for novel therapeutic strategies.

  14. Mevalonate Biosynthesis Intermediates Are Key Regulators of Innate Immunity in Bovine Endometritis

    PubMed Central

    Collier, Christine; Griffin, Sholeem; Schuberth, Hans-Joachim; Sandra, Olivier; Smith, David G.; Mahan, Suman; Dieuzy-Labaye, Isabelle; Sheldon, I. Martin

    2016-01-01

    Metabolic changes can influence inflammatory responses to bacteria. To examine whether localized manipulation of the mevalonate pathway impacts innate immunity, we exploited a unique mucosal disease model, endometritis, where inflammation is a consequence of innate immunity. IL responses to pathogenic bacteria and LPS were modulated in bovine endometrial cell and organ cultures by small molecules that target the mevalonate pathway. Treatment with multiple statins, bisphosphonates, squalene synthase inhibitors, and small interfering RNA showed that inhibition of farnesyl-diphosphate farnesyl transferase (squalene synthase), but not 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyl diphosphate synthase, reduced endometrial organ and cellular inflammatory responses to pathogenic bacteria and LPS. Although manipulation of the mevalonate pathway reduced cellular cholesterol, impacts on inflammation were independent of cholesterol concentration as cholesterol depletion using cyclodextrins did not alter inflammatory responses. Treatment with the isoprenoid mevalonate pathway-intermediates, farnesyl diphosphate and geranylgeranyl diphosphate, also reduced endometrial cellular inflammatory responses to LPS. These data imply that manipulating the mevalonate pathway regulates innate immunity within the endometrium, and that isoprenoids are regulatory molecules in this process, knowledge that could be exploited for novel therapeutic strategies. PMID:26673142

  15. Staphylococcus aureus mevalonate kinase: isolation and characterization of an enzyme of the isoprenoid biosynthetic pathway.

    PubMed

    Voynova, Natalya E; Rios, Sandra E; Miziorko, Henry M

    2004-01-01

    It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent K(m) values for R,S-mevalonate and ATP were 41 and 339 micro M, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the K(i) values were 46 and 45 micro M for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (K(i) difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.

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

    PubMed

    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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  18. A critical role of mevalonate for peptidoglycan synthesis in Staphylococcus aureus.

    PubMed

    Matsumoto, Yasuhiko; Yasukawa, Jyunichiro; Ishii, Masaki; Hayashi, Yohei; Miyazaki, Shinya; Sekimizu, Kazuhisa

    2016-03-10

    3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, a mevalonate synthetase, is required for the growth of Staphylococcus aureus. However, the essential role of the enzyme in cell growth has remained unclear. Here we show that three mutants possessed single-base substitutions in the mvaA gene, which encodes HMG-CoA reductase, show a temperature-sensitive phenotype. The phenotype was suppressed by the addition of mevalonate or farnesyl diphosphate, which is a product synthesized from mevalonate. Farnesyl diphosphate is a precursor of undecaprenyl phosphate that is required for peptidoglycan synthesis. The rate of peptidoglycan synthesis was decreased in the mvaA mutants under the non-permissive conditions and the phenotype was suppressed by the addition of mevalonate. HMG-CoA reductase activities of mutant MvaA proteins in the temperature sensitive mutants were lower than that of wild-type MvaA protein. Our findings from genetic and biochemical analyses suggest that mevalonate produced by HMG-CoA reductase is required for peptidoglycan synthesis for S. aureus cell growth.

  19. Mevalonate kinase deficiencies: from mevalonic aciduria to hyperimmunoglobulinemia D syndrome

    PubMed Central

    Haas, Dorothea; Hoffmann, Georg F

    2006-01-01

    Mevalonic aciduria (MVA) and hyperimmunoglobulinemia D syndrome (HIDS) represent the two ends of a clinical spectrum of disease caused by deficiency of mevalonate kinase (MVK), the first committed enzyme of cholesterol biosynthesis. At least 30 patients with MVA and 180 patients with HIDS have been reported worldwide. MVA is characterized by psychomotor retardation, failure to thrive, progressive cerebellar ataxia, dysmorphic features, progressive visual impairment and recurrent febrile crises. The febrile episodes are commonly accompanied by hepatosplenomegaly, lymphadenopathy, abdominal symptoms, arthralgia and skin rashes. Life expectancy is often compromised. In HIDS, only febrile attacks are present, but a subgroup of patients may also develop neurological abnormalities of varying degree such as mental retardation, ataxia, ocular symptoms and epilepsy. A reduced activity of MVK and pathogenic mutations in the MVK gene have been demonstrated as the common genetic basis in both disorders. In MVA, the diagnosis is established by detection of highly elevated levels of mevalonic acid excreted in urine. Increased levels of immunoglobulin D (IgD) and, in most patients of immunoglobulin A (IgA), in combination with enhanced excretion of mevalonic acid provide strong evidence for HIDS. The diagnosis is confirmed by low activity of mevalonate kinase or by demonstration of disease-causing mutations. Genetic counseling should be offered to families at risk. There is no established successful treatment for MVA. Simvastatin, an inhibitor of HMG-CoA reductase, and anakinra have been shown to have beneficial effect in HIDS. PMID:16722536

  20. Genetics Home Reference: mevalonate kinase deficiency

    MedlinePlus

    ... shape, leading to a reduction of mevalonate kinase enzyme activity. Despite this shortage (deficiency) of mevalonate kinase activity, ... who have less than 1 percent of normal enzyme activity usually develop MVA. Learn more about the gene ...

  1. Bacterial Metabolism of Mevalonic Acid1

    PubMed Central

    Siddiq, Majid A.; Rodwell, Victor W.

    1967-01-01

    Soluble cell-free extracts of actinomycete S4 grown on media containing mevalonate catalyze acetoacetate formation from mevalonate, mevaldate, and β-hydroxy-β-methylglutaryl-coenzyme A (CoA). Conversion of mevalonate to acetoacetate involves formation of free β-hydroxy-β-methylglutaryl-CoA, but not free mevaldate. The reaction favors mevalonate oxidation, and nicotinamide adenine dinucleotide, rather than nicotinamide adenine dinucleotide phosphate, acts as oxidant. PMID:4289807

  2. A new alternative non-mevalonate pathway for isoprenoid biosynthesis in eubacteria and plants.

    PubMed

    Paseshnichenko, V A

    1998-02-01

    Data concerning the discovery of an alternative non-mevalonate pathway for isoprenoid biosynthesis leading to isopentenyl diphosphate formation are reviewed. This pathway has been discovered in experiments with several eubacteria producing triterpenoids of the hopane series. 13C-labeled acetate, glucose, and triose phosphates were used as precursors. The 13C-labeling patterns in isoprenoids were studied by 13C-NMR spectrometry. In eubacteria the universal C5 precursor--isopentenyl diphosphate--did not appear to form via the classical acetate/mevalonate pathway, but via a novel glyceraldehyde 3-phosphate/pyruvate pathway. It is postulated that the condensation of the C2 unit formed as a result of pyruvate decarboxylation with the C3 unit (glyceraldehyde 3-phosphate) and the next transposition leads to the formation of the branched C5 precursor--isopentenyl diphosphate. In Scenedesmus obliquus not only all plastid isoprenoids (carotenoids and prenyl side chains of chlorophylls and plastoquinone-9) were formed via this novel pathway, but also the non-plastid cytoplasmic sterols. In higher plants the plastid isoprenoids were formed via the glyceraldehyde 3-phosphate/pyruvate pathway, while the cytoplasmic sterols were formed via the acetate/mevalonate pathway.

  3. Mevalonate kinase deficiency: current perspectives

    PubMed Central

    Favier, Leslie A; Schulert, Grant S

    2016-01-01

    Mevalonate kinase deficiency (MKD) is a recessively inherited autoinflammatory disorder with a spectrum of manifestations, including the well-defined clinical phenotypes of hyperimmunoglobulinemia D and periodic fever syndrome and mevalonic aciduria. Patients with MKD have recurrent attacks of hyperinflammation associated with fever, abdominal pain, arthralgias, and mucocutaneous lesions, and more severely affected patients also have dysmorphisms and central nervous system anomalies. MKD is caused by mutations in the gene encoding mevalonate kinase, with the degree of residual enzyme activity largely determining disease severity. Mevalonate kinase is essential for the biosynthesis of nonsterol isoprenoids, which mediate protein prenylation. Although the precise pathogenesis of MKD remains unclear, increasing evidence suggests that deficiency in protein prenylation leads to innate immune activation and systemic hyperinflammation. Given the emerging understanding of MKD as an autoinflammatory disorder, recent treatment approaches have largely focused on cytokine-directed biologic therapy. Herein, we review the current genetic and pathologic understanding of MKD, its various clinical phenotypes, and the evolving treatment approach for this multifaceted disorder. PMID:27499643

  4. How thiamine diphosphate is activated in enzymes.

    PubMed

    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.

  5. Negative Feedbacks by Isoprenoids on a Mevalonate Kinase Expressed in the Corpora Allata of Mosquitoes

    PubMed Central

    Noriega, Fernando G.

    2015-01-01

    Background Juvenile hormones (JH) regulate development and reproductive maturation in insects. JHs are synthesized through the mevalonate pathway (MVAP), an ancient metabolic pathway present in the three domains of life. Mevalonate kinase (MVK) is a key enzyme in the MVAP. MVK catalyzes the synthesis of phosphomevalonate (PM) by transferring the γ-phosphoryl group from ATP to the C5 hydroxyl oxygen of mevalonic acid (MA). Despite the importance of MVKs, these enzymes have been poorly characterized in insects. Results We functionally characterized an Aedes aegypti MVK (AaMVK) expressed in the corpora allata (CA) of the mosquito. AaMVK displayed its activity in the presence of metal cofactors. Different nucleotides were used by AaMVK as phosphoryl donors. In the presence of Mg2+, the enzyme has higher affinity for MA than ATP. The activity of AaMVK was regulated by feedback inhibition from long-chain isoprenoids, such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Conclusions AaMVK exhibited efficient inhibition by GPP and FPP (Ki less than 1 μM), and none by isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DPPM). These results suggest that GPP and FPP might act as physiological inhibitors in the synthesis of isoprenoids in the CA of mosquitoes. Changing MVK activity can alter the flux of precursors and therefore regulate juvenile hormone biosynthesis. PMID:26566274

  6. p53 regulates the mevalonate pathway in human glioblastoma multiforme

    PubMed Central

    Laezza, C; D'Alessandro, A; Di Croce, L; Picardi, P; Ciaglia, E; Pisanti, S; Malfitano, A M; Comegna, M; Faraonio, R; Gazzerro, P; Bifulco, M

    2015-01-01

    The mevalonate (MVA) pathway is an important metabolic pathway implicated in multiple aspects of tumorigenesis. In this study, we provided evidence that p53 induces the expression of a group of enzymes of the MVA pathway including 3′-hydroxy-3′-methylglutaryl-coenzyme A reductase, MVA kinase, farnesyl diphosphate synthase and farnesyl diphosphate farnesyl transferase 1, in the human glioblastoma multiforme cell line, U343 cells, and in normal human astrocytes, NHAs. Genetic and pharmacologic perturbation of p53 directly influences the expression of these genes. Furthermore, p53 is recruited to the gene promoters in designated p53-responsive elements, thereby increasing their transcription. Such effect was abolished by site-directed mutagenesis in the p53-responsive element of promoter of the genes. These findings highlight another aspect of p53 functions unrelated to tumor suppression and suggest p53 as a novel regulator of the MVA pathway providing insight into the role of this pathway in cancer progression. PMID:26469958

  7. Perspectives in anti-infective drug design. The late steps in the biosynthesis of the universal terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate.

    PubMed

    Rohdich, Felix; Bacher, Adelbert; Eisenreich, Wolfgang

    2004-10-01

    A mevalonate-independent pathway for the biosynthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that has been elucidated during the last decade is essential in plants, many eubacteria and apicomplexan parasites, but is absent in Archaea and animals. The enzymes of the pathway are potential targets for the development of novel antibiotic, antimalarial and herbicidal agents. This review is focused on the late steps of this pathway. The intermediate 2C-methyl-D-erythritol 2,4-cyclodiphosphate is converted into IPP and DMAPP via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of the iron-sulfur proteins IspG and IspH. IPP and DMAPP can be interconverted by IPP isomerase which is essential in microorganisms using the mevalonate pathway, whereas its presence is optional in microorganisms using the non-mevalonate pathway. A hitherto unknown family of IPP isomerases using FMN as coenzyme has been discovered recently in Archaea and certain eubacteria.

  8. Formation of isobutene from 3-hydroxy-3-methylbutyrate by diphosphomevalonate decarboxylase.

    PubMed

    Gogerty, David S; Bobik, Thomas A

    2010-12-01

    Isobutene is an important commercial chemical used for the synthesis of butyl rubber, terephthalic acid, specialty chemicals, and a gasoline performance additive known as alkylate. Currently, isobutene is produced from petroleum and hence is nonrenewable. Here, we report that the Saccharomyces cerevisiae mevalonate diphosphate decarboxylase (ScMDD) can convert 3-hydroxy-3-methylbutyrate (3-HMB) to isobutene. Whole cells of Escherichia coli producing ScMDD with an N-terminal 6×His tag (His(6)-ScMDD) formed isobutene from 3-HMB at a rate of 154 pmol h(-1) g cells(-1). In contrast, no isobutene was detected from control cells lacking ScMDD. His(6)-ScMDD was purified by nickel affinity chromatography and shown to produce isobutene from 3-HMB at a rate of 1.33 pmol min(-1) mg(-1) protein. Controls showed that both His(6)-ScMDD and 3-HMB were required for detectable isobutene formation. Isobutene was identified by gas chromatography (GC) with flame ionization detection as well as by GC-mass spectrometry (MS). ScMDD was subjected to error-prone PCR, and two improved variants were characterized, ScMDD1 (I145F) and ScMDD2 (R74H). Whole cells of E. coli producing ScMDD1 and ScMDD2 produced isobutene from 3-HMB at rates of 3,000 and 5,888 pmol h(-1) g cells(-1), which are 19- and 38-fold increases compared to rates for cells producing His(6)-ScMDD. This showed that genetic modifications can be used to increase the rate at which ScMDD converts 3-HMB to isobutene. Because 3-HMB can be produced from l-leucine, ScMDD has a potential application for the production of renewable isobutene. Moreover, isobutene is a gas, which might simplify its purification from a fermentation medium, substantially reducing production costs.

  9. Geranyl diphosphate synthase from mint

    DOEpatents

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    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.

  10. Geranyl diphosphate synthase from mint

    DOEpatents

    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.

  11. Studies on thiamine diphosphate-dependent enzymes.

    PubMed

    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.

  12. Efficient Use of Exogenous Isoprenols for Protein Isoprenylation by MDA-MB-231 Cells Is Regulated Independently of the Mevalonate Pathway*

    PubMed Central

    Onono, Fredrick; Subramanian, Thangaiah; Sunkara, Manjula; Subramanian, Karunai Leela; Spielmann, H. Peter; Morris, Andrew J.

    2013-01-01

    Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations >10 μm, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition. PMID:23908355

  13. Evolutionary hypothesis of the Mevalonate Kinase Deficiency.

    PubMed

    Vuch, J; Marcuzzi, A; Bianco, A M; Tommasini, A; Zanin, V; Crovella, S

    2013-01-01

    Mevalonate Kinase Deficiency (MKD) is an autosomal-recessively inherited disorder of cholesterol biosynthesis with higher prevalence in the Netherlands and other North European countries. MKD is due to mutations in the second enzyme of mevalonate pathway (mevalonate kinase, MK/MVK) which results in reduced enzymatic activity and in the consequent shortage of downstream compounds. In most severe cases the deregulation of mevalonate pathway is associated with a decrease in serum cholesterol. More than 100 pathological mutations have been described in the MVK gene so far, and a founder effect has been hypothesized as responsible for the diffusion of the most frequent disease-associated mutations. In the acute phase of disease, patients affected with MKD present low cholesterol levels comparable to their basal physiologic conditions, already characterized by lower cholesterol levels when compared to healthy individuals. Low cholesterol levels are widely known to correlate with the reduction of cardiovascular events. We hypothesize a selective advantage for heterozygote carriers of the most frequent MVK mutations in those countries where the diet is characterized by high consumption of saturated animal fats rich in cholesterol. This could explain the maintenance in North European population of the main mutations leading to MKD and the distribution world-wide of these mutations that followed the migrations of North European populations.

  14. Weekly oral alendronate in mevalonate kinase deficiency

    PubMed Central

    2013-01-01

    Background Mevalonate kinase deficiency (MKD) is caused by mutations in the MVK gene, encoding the second enzyme of mevalonate pathway, which results in subsequent shortage of downstream compounds, and starts in childhood with febrile attacks, skin, joint, and gastrointestinal symptoms, sometimes induced by vaccinations. Methods For a history of early-onset corticosteroid-induced reduction of bone mineral density in a 14-year-old boy with MKD, who also had presented three bone fractures, we administered weekly oral alendronate, a drug widely used in the management of osteoporosis and other high bone turnover diseases, which blocks mevalonate and halts the prenylation process. Results All of the patient’s MKD clinical and laboratory abnormalities were resolved after starting alendronate treatment. Conclusions This observation appears enigmatic, since alendronate should reinforce the metabolic block characterizing MKD, but is crucial because of the ultimate improvement shown by this patient. The anti-inflammatory properties of bisphosphonates are a new question for debate among physicians across various specialties, and requires further biochemical and clinical investigation. PMID:24360083

  15. Putative modifier genes in mevalonate kinase deficiency.

    PubMed

    Marcuzzi, Annalisa; Vozzi, Diego; Girardelli, Martina; Tricarico, Paola Maura; Knowles, Alessandra; Crovella, Sergio; Vuch, Josef; Tommasini, Alberto; Piscianz, Elisa; Bianco, Anna Monica

    2016-04-01

    Mevalonate kinase deficiency (MKD) is an autosomal recessive auto‑inflammatory disease, caused by impairment of the mevalonate pathway. Although the molecular mechanism remains to be elucidated, there is clinical evidence suggesting that other regulatory genes may be involved in determining the phenotype. The identification of novel target genes may explain non‑homogeneous genotype‑phenotype correlations, and provide evidence in support of the hypothesis that novel regulatory genes predispose or amplify deregulation of the mevalonate pathway in this orphan disease. In the present study, DNA samples were obtained from five patients with MKD, which were then analyzed using whole exome sequencing. A missense variation in the PEX11γ gene was observed in homozygosis in P2, possibly correlating with visual blurring. The UNG rare gene variant was detected in homozygosis in P5, without correlating with a specific clinical phenotype. A number of other variants were found in the five analyzed DNA samples from the MKD patients, however no correlation with the phenotype was established. The results of the presents study suggested that further analysis, using next generation sequencing approaches, is required on a larger sample size of patients with MKD, who share the same MVK mutations and exhibit 'extreme' clinical phenotypes. As MVK mutations may be associated with MKD, the identification of specific modifier genes may assist in providing an earlier diagnosis.

  16. Dimethylallyl diphosphate and geranyl diphosphate pools of plant species characterized by different isoprenoid emissions.

    PubMed

    Nogués, Isabel; Brilli, Federico; Loreto, Francesco

    2006-06-01

    Dimethylallyl diphosphate (DMADP) and geranyl diphosphate (GDP) are the last precursors of isoprene and monoterpenes emitted by leaves, respectively. DMADP and GDP pools were measured in leaves of plants emitting isoprene (Populus alba), monoterpenes (Quercus ilex and Mentha piperita), or nonemitting isoprenoids (Prunus persica). Detectable pools were found in all plant species, but P. persica showed the lowest pool size, which indicates a limitation of the whole pathway leading to isoprenoid biosynthesis in nonemitting species. The pools of DMADP and GDP of nonemitting, isoprene-emitting, and monoterpene-emitting species were partially labeled (generally 40%-60% of total carbon-incorporated (13)C) within the same time by which volatile isoprenoids are fully labeled (15 min). This indicates the coexistence of two pools for both precursors, the rapidly labeled pool presumably occurring in chloroplasts and thereby synthesized by the methylerythritol phosphate pathway and the nonlabeled pool presumably located in the cytosol and synthesized by the mevalonic pathway. In M. piperita storing monoterpenes in specialized leaf structures, the GDP pool remained totally unlabeled, indicating either that monoterpenes are totally formed by the mevalonic pathway or that labeling occurs slowly in comparison to the large pool of stored monoterpenes in this plant. The pools of DMADP and GDP increased during the season (from May to July) but decreased when the leaf was darkened or exposed to very high temperature. In the dark, the pool of DMADP of the isoprene-emitting species decreased faster than the pool of GDP. However, after 6 h of darkness, both pools were depleted to about 10% of the pool size in illuminated leaves. This indicates that both the chloroplastic and the cytosolic pools of precursors are depleted in the dark. When comparing measurements over the season and at different temperatures, an inverse correlation was observed between isoprene emission by P. alba and the

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

    PubMed Central

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

    2013-01-01

    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. PMID:24167290

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

    PubMed

    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.

  19. Isoprenoid biosynthesis in higher plants and in Escherichia coli: on the branching in the methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate.

    PubMed Central

    Hoeffler, Jean-François; Hemmerlin, Andréa; Grosdemange-Billiard, Catherine; Bach, Thomas J; Rohmer, Michel

    2002-01-01

    In the bacterium Escherichia coli, the mevalonic-acid (MVA)-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is characterized by two branches leading separately to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The signature of this branching is the retention of deuterium in DMAPP and the deuterium loss in IPP after incorporation of 1-[4-(2)H]deoxy-d-xylulose ([4-(2)H]DX). Feeding tobacco BY-2 cell-suspension cultures with [4-(2)H]DX resulted in deuterium retention in the isoprene units derived from DMAPP, as well as from IPP in the plastidial isoprenoids, phytoene and plastoquinone, synthesized via the MEP pathway. This labelling pattern represents direct evidence for the presence of the DMAPP branch of the MEP pathway in a higher plant, and shows that IPP can be synthesized from DMAPP in plant plastids, most probably via a plastidial IPP isomerase. PMID:12010124

  20. Probing Ligand-binding Pockets of the Mevalonate Pathway Enzymes from Streptococcus pneumoniae*

    PubMed Central

    Lefurgy, Scott T.; Rodriguez, Sofia B.; Park, Chan Sun; Cahill, Sean; Silverman, Richard B.; Leyh, Thomas S.

    2010-01-01

    Diphosphomevalonate (Mev·pp) is the founding member of a new class of potential antibiotics targeting the Streptococcus pneumoniae mevalonate (Mev) pathway. We have synthesized a series of Mev·pp analogues designed to simultaneously block two steps in this pathway, through allosteric inhibition of mevalonate kinase (MK) and, for five of the analogues, by mechanism-based inactivation of diphosphomevalonate decarboxylase (DPM-DC). The analogue series expands the C3-methyl group of Mev·pp with hydrocarbons of varying size, shape, and chemical and physical properties. Previously, we established the feasibility of a prodrug strategy in which unphosphorylated Mev analogues could be enzymatically converted to the active Mev·pp forms by the endogenous MK and phosphomevalonate kinase. We now report the kinetic parameters for the turnover of non-, mono-, and diphosphorylated analogues as substrates and inhibitors of the three mevalonate pathway enzymes. The inhibition of MK by Mev·pp analogues revealed that the allosteric site is selective for compact, electron-rich C3-subsitutents. The lack of reactivity of analogues with DPM-DC provided evidence, counter to the existing model, for a decarboxylation transition state that is concerted rather than dissociative. The Mev pathway is composed of three structurally and functionally conserved enzymes that catalyze consecutive steps in a metabolic pathway. The current work reveals that these enzymes exhibit significant differences in specificity toward R-group substitution at C3 and that these patterns are explained well by changes in the volume of the C3 R-group-binding pockets of the enzymes. PMID:20404339

  1. Mutations in the mevalonate pathway genes in Chinese patients with porokeratosis.

    PubMed

    Li, M; Li, Z; Wang, J; Ni, C; Sun, Z; Wilson, N J; Zhang, J; Chen, F; Li, X; Du, X; Yu, H; Zhang, L; Smith, F J D; Zhang, G; Yao, Z

    2016-09-01

    Porokeratosis (PK, MIM 175800) is a chronic autosomal dominant cutaneous keratinization disorder, which has a wide variety of clinical manifestations. We analysed the molecular basis of 10 families and 12 sporadic cases with different subtypes of porokeratosis in the Chinese population. Genomic DNA was extracted from peripheral blood samples. Mutation screening was performed by direct sequencing of exons and flanking intron-exon boundaries for the entire coding region of four mevalonate pathway genes and SLC17A9 gene. We detected three novel mutations and seven previously described mutations by direct sequence analysis of the PCR products. Mutations p.Phe249Ser and p.Asn292Ser in mevalonate decarboxylase (MVD) were the most common mutations in this PK cohort; their presence was 27.3% and 13.6% respectively. This study extended the mutation spectrum of PK in the Chinese Han population and provided further evidence for the genetic basis of PK. We first identified MVD simultaneously responsible for porokeratosis palmaris et plantaris disseminate development and confirmed the genotype-phenotype correlations. © 2016 European Academy of Dermatology and Venereology.

  2. Establishment of a novel anabolism-based addiction system with an artificially introduced mevalonate pathway: complete stabilization of plasmids as universal application in white biotechnology.

    PubMed

    Kroll, Jens; Steinle, Anna; Reichelt, Rudolf; Ewering, Christian; Steinbüchel, Alexander

    2009-05-01

    Plasmid stability in recombinant microorganisms is a very important requirement for highly efficient plasmid-based production processes in biotechnology. To stably maintain plasmids, we developed in this study an efficient and stringent novel anabolism-based addiction system, which can be widely used. This novel addiction system is based on two components: (i) an Escherichia coli HMS174(DE3) knockout mutant of the ispH gene coding for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2) of the deoxyxylulose 5-phosphate (DXP) pathway, impairing the synthesis of isopentenyl pyrophosphate (IPP) and (ii) a completely synthetic and episomal mevalonate (MVA) pathway as an alternative supplier of essential IPP. The latter is encoded by a plasmid that contains the genes for HMG-CoA reductases from Lactococcus lactis and Staphylococcus aureus plus HMG-CoA-synthase, MVA kinase, MVP kinase and MVPP decarboxylase from S. aureus. This plasmid should then also harbor the genes for the protein or for the pathway that will be produced or that will be utilized for production of a chemical. To demonstrate the functionality of this addiction system, a mutated cyanophycin synthetase gene (cphA(6308)C595S) was used. To determine plasmid stabilities, flasks experiments in media supplied or not supplied with antibiotics were carried out with the knockout mutant and two control strains, one harboring plasmid pCOLADuet-1::MVA1-5::cphA(6308) and the other harboring a conventional expression plasmid pET-23a::cphA(6308). As revealed by measuring the colony-forming units of aliquots spread on solid media with or without antibiotics, the knockout mutant revealed a plasmid stability of 100% whereas the control strains exhibited plasmid stabilities of only 64% and 2%, respectively. Radiometric enzyme activity measurements for CphA revealed only 95% and 12.5% of the activity in the control strains harboring pCOLADuet-1::MVA1-5::cphA(6308) and pET-23a::cphA(6308), respectively, in

  3. Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraldehyde 3-phosphate non-mevalonate pathway in the green alga Scenedesmus obliquus.

    PubMed

    Schwender, J; Seemann, M; Lichtenthaler, H K; Rohmer, M

    1996-05-15

    Isoprenoid biosynthesis was investigated in the green alga Scenedesmus obliquus grown heterotrophically on 13C-labelled glucose and acetate. Several isoprenoid compounds were isolated and investigated by 13C-NMR spectroscopy. According to the 13C-labelling pattern indicated by the 13C-NMR spectra, the biosynthesis of all plastidic isoprenoids investigated (prenyl side-chains of chlorophylls and plastoquinone-9, and the carotenoids beta-carotene and lutein), as well as of the non-plastidic cytoplasmic sterols, does not proceed via the classical acetate/mevalonate pathway (which leads from acetyl-CoA via mevalonate to isopentenyl diphosphate), but via the novel glyceraldehyde 3-phosphate/pyruvate route recently detected in eubacteria. Formation of isopentenyl diphosphate involves the condensation of a C2 unit derived from pyruvate decarboxylation with glyceraldehyde 3-phosphate and a transposition yielding the branched C5 skeleton of isoprenic units.

  4. Diagnostic Value of Urinary Mevalonic Acid Excretion in Patients with a Clinical Suspicion of Mevalonate Kinase Deficiency (MKD).

    PubMed

    Jeyaratnam, Jerold; Ter Haar, Nienke M; de Sain-van der Velden, Monique G M; Waterham, Hans R; van Gijn, Mariëlle E; Frenkel, Joost

    2016-01-01

    In patients suffering from mevalonate kinase deficiency (MKD), the reduced enzyme activity leads to an accumulation of mevalonic acid which is excreted in the urine. This study aims to evaluate the diagnostic value of urinary mevalonic acid measurement in patients with a clinical suspicion of mevalonate kinase deficiency. In this single-center, retrospective analysis, all patients in whom both measurement of mevalonic acid and genetic testing had been performed in the preceding 17 years have been included. The presence of two pathogenic MVK mutations or demonstration of decreased enzyme activity was considered to be the gold standard for the diagnosis of MKD. Sixty-one patients were included in this study. Thirteen of them harbored two MVK mutations; twelve of them showed elevated levels of mevalonic acid. Forty-eight patients did not harbor any MVK mutations, yet five of them excreted increased amounts of mevalonic acid. This corresponds to a sensitivity of 92%, a specificity of 90%, a positive predictive value of 71%, and a negative predictive value of 98%. The positive likelihood ratio is 10 and the negative likelihood ratio is 0.09. MKD seems very unlikely in patients with a normal mevalonic acid excretion, but it cannot be excluded completely. Further, a positive urinary mevalonic acid excretion still requires MVK analysis to confirm the diagnosis of MKD. Therefore, detection of urinary mevalonic acid should not be mandatory before genetic testing. However, as long as genetic testing is not widely available and affordable, measurement of urinary mevalonic acid is a fair way to select patients for MVK gene analysis or enzyme assay.

  5. Methylerythritol and mevalonate pathway contributions to biosynthesis of mono-, sesqui-, and diterpenes in glandular trichomes and leaves of Stevia rebaudiana Bertoni.

    PubMed

    Wölwer-Rieck, Ursula; May, Bianca; Lankes, Christa; Wüst, Matthias

    2014-03-19

    The biosynthesis of the diterpenoid steviol glycosides rebaudioside A and stevioside in nonrooted cuttings of Stevia rebaudiana was investigated by feeding experiments using the labeled key precursors [5,5-(2)H2]-mevalonic acid lactone (d2-MVL) and [5,5-(2)H2]-1-deoxy-d-xylulose (d2-DOX). Labeled glycosides were extracted from the leaves and stems and were directly analyzed by LC-(-ESI)-MS/MS and by GC-MS after hydrolysis and derivatization of the resulting isosteviol to the corresponding TMS-ester. Additionally, the incorporation of the proffered d2-MVL and d2-DOX into volatile monoterpenes, sesquiterpenes, and diterpenes in glandular trichomes on leaves and stems was investigated by headspace-solid phase microextraction-GC-MS (HS-SPME-GC-MS). Incorporation of the labeled precursors indicated that diterpenes in leaves and monoterpenes and diterpenes in glandular trichomes are predominately biosynthesized via the methylerythritol phosphate (MEP) pathway, whereas both the MEP and mevalonate (MVA) pathways contribute to the biosynthesis of sesquiterpenes at equal rates in glandular trichomes. These findings give evidence for a transport of MEP pathway derived farnesyl diphosphate precursors from plastids to the cytosol. Contrarily, the transport of MVA pathway derived geranyl diphosphate and geranylgeranyl diphosphate precursors from the cytosol to the plastid is limited.

  6. Mevalonate kinase deficiency and neuroinflammation: balance between apoptosis and pyroptosis.

    PubMed

    Tricarico, Paola Maura; Marcuzzi, Annalisa; Piscianz, Elisa; Monasta, Lorenzo; Crovella, Sergio; Kleiner, Giulio

    2013-11-26

    Mevalonic aciduria, a rare autosomal recessive disease, represents the most severe form of the periodic fever, known as Mevalonate Kinase Deficiency. This disease is caused by the mutation of the MVK gene, which codes for the enzyme mevalonate kinase, along the cholesterol pathway. Mevalonic aciduria patients show recurrent fever episodes with associated inflammatory symptoms, severe neurologic impairments, or death, in early childhood. The typical neurodegeneration occurring in mevalonic aciduria is linked both to the intrinsic apoptosis pathway (caspase-3 and -9), which is triggered by mitochondrial damage, and to pyroptosis (caspase-1). These cell death mechanisms seem to be also related to the assembly of the inflammasome, which may, in turn, activate pro-inflammatory cytokines and chemokines. Thus, this particular molecular platform may play a crucial role in neuroinflammation mechanisms. Nowadays, a specific therapy is still lacking and the pathogenic mechanisms involving neuroinflammation and neuronal dysfunction have not yet been completely understood, making mevalonic aciduria an orphan drug disease. This review aims to analyze the relationship among neuroinflammation, mitochondrial damage, programmed cell death, and neurodegeneration. Targeting inflammation and degeneration in the central nervous system might help identify promising treatment approaches for mevalonic aciduria or other diseases in which these mechanisms are involved.

  7. Mevalonate Kinase Deficiency and Neuroinflammation: Balance between Apoptosis and Pyroptosis

    PubMed Central

    Tricarico, Paola Maura; Marcuzzi, Annalisa; Piscianz, Elisa; Monasta, Lorenzo; Crovella, Sergio; Kleiner, Giulio

    2013-01-01

    Mevalonic aciduria, a rare autosomal recessive disease, represents the most severe form of the periodic fever, known as Mevalonate Kinase Deficiency. This disease is caused by the mutation of the MVK gene, which codes for the enzyme mevalonate kinase, along the cholesterol pathway. Mevalonic aciduria patients show recurrent fever episodes with associated inflammatory symptoms, severe neurologic impairments, or death, in early childhood. The typical neurodegeneration occurring in mevalonic aciduria is linked both to the intrinsic apoptosis pathway (caspase-3 and -9), which is triggered by mitochondrial damage, and to pyroptosis (caspase-1). These cell death mechanisms seem to be also related to the assembly of the inflammasome, which may, in turn, activate pro-inflammatory cytokines and chemokines. Thus, this particular molecular platform may play a crucial role in neuroinflammation mechanisms. Nowadays, a specific therapy is still lacking and the pathogenic mechanisms involving neuroinflammation and neuronal dysfunction have not yet been completely understood, making mevalonic aciduria an orphan drug disease. This review aims to analyze the relationship among neuroinflammation, mitochondrial damage, programmed cell death, and neurodegeneration. Targeting inflammation and degeneration in the central nervous system might help identify promising treatment approaches for mevalonic aciduria or other diseases in which these mechanisms are involved. PMID:24287904

  8. Mevalonate kinase deficiency: disclosing the role of mevalonate pathway modulation in inflammation.

    PubMed

    Marcuzzi, Annalisa; Crovella, Sergio; Monasta, Lorenzo; Vecchi Brumatti, Liza; Gattorno, Marco; Frenkel, Joost

    2012-01-01

    Inflammation is a highly regulated process involved both in the response to pathogens as well as in tissue homeostasis. In recent years, a complex network of proteins in charge of inflammation control has been revealed by the study of hereditary periodic fever syndromes. Most of these proteins belong to a few families and share the capability of sensing pathogen-associated and damageassociated molecular patterns. By interacting with each other, these proteins participate in the assembly of molecular platforms, called inflammasomes, which ultimately lead to the activation of cytokines, to the transcription of inflammatory genes or to the induction of cell apoptosis. Among hereditary periodic fever syndromes, mevalonate kinase deficiency (MKD) is the sole in which the phenotype did not directly associate with a deficiency of these proteins, but with a metabolic defect of the mevalonate pathway, highlighting the importance of this metabolic pathway in the inflammation control. Noteworthy, drugs acting on this pathway can greatly influence the inflammatory response. The modulation of inflammation by mevalonate pathway is of interest, since it may involve mechanisms not directly referable to inflammasomes. MKD provides a model to study these mechanisms and possibly to develop new classes of anti-inflammatory drugs.

  9. Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway.

    PubMed

    Zhu, Wen-Liang; Cui, Jin-Yu; Cui, Lan-Yu; Liang, Wei-Fan; Yang, Song; Zhang, Chong; Xing, Xin-Hui

    2016-03-01

    Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

  10. Feasibility of gas/solid carboligation: conversion of benzaldehyde to benzoin using thiamine diphosphate-dependent enzymes.

    PubMed

    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.

  11. Mevalonate Pathway Regulates Cell Size Homeostasis and Proteostasis through Autophagy

    PubMed Central

    Miettinen, Teemu P.; Björklund, Mikael

    2015-01-01

    Summary Balance between cell growth and proliferation determines cell size homeostasis, but little is known about how metabolic pathways are involved in the maintenance of this balance. Here, we perform a screen with a library of clinically used drug molecules for their effects on cell size. We find that statins, inhibitors of the mevalonate pathway, reduce cell proliferation and increase cell size and cellular protein density in various cell types, including primary human cells. Mevalonate pathway effects on cell size and protein density are mediated through geranylgeranylation of the small GTPase RAB11, which is required for basal autophagic flux. Our results identify the mevalonate pathway as a metabolic regulator of autophagy and expose a paradox in the regulation of cell size and proteostasis, where inhibition of an anabolic pathway can cause an increase in cell size and cellular protein density. PMID:26686643

  12. The mevalonate pathway regulates primitive streak formation via protein farnesylation

    PubMed Central

    Okamoto-Uchida, Yoshimi; Yu, Ruoxing; Miyamura, Norio; Arima, Norie; Ishigami-Yuasa, Mari; Kagechika, Hiroyuki; Yoshida, Suguru; Hosoya, Takamitsu; Nawa, Makiko; Kasama, Takeshi; Asaoka, Yoichi; Alois, Reiner Wimmer; Elling, Ulrich; Penninger, Josef M.; Nishina, Sachiko; Azuma, Noriyuki; Nishina, Hiroshi

    2016-01-01

    The primitive streak in peri-implantation embryos forms the mesoderm and endoderm and controls cell differentiation. The metabolic cues regulating primitive streak formation remain largely unknown. Here we utilised a mouse embryonic stem (ES) cell differentiation system and a library of well-characterised drugs to identify these metabolic factors. We found that statins, which inhibit the mevalonate metabolic pathway, suppressed primitive streak formation in vitro and in vivo. Using metabolomics and pharmacologic approaches we identified the downstream signalling pathway of mevalonate and revealed that primitive streak formation requires protein farnesylation but not cholesterol synthesis. A tagging-via-substrate approach revealed that nuclear lamin B1 and small G proteins were farnesylated in embryoid bodies and important for primitive streak gene expression. In conclusion, protein farnesylation driven by the mevalonate pathway is a metabolic cue essential for primitive streak formation. PMID:27883036

  13. Biochemical characterization of recombinant mevalonate kinase from Bacopa monniera.

    PubMed

    Kumari, Uma; Vishwakarma, Rishi K; Sonawane, Prashant; Abbassi, Shakeel; Khan, Bashir M

    2015-01-01

    Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.36) plays a key role in isoprenoid biosynthetic pathway in plants. MK catalyzes the phosphorylation of mevalonate to form mevalonate-5-phosphate. The recombinant BmMK was cloned and over-expressed in E. coli BL21 (DE3), and purified to homogeneity by affinity chromatography followed by gel filtration. Optimum pH and temperature for forward reaction was found to be 7.0 and 30 °C, respectively. The enzyme was most stable at pH 8 at 25 °C with deactivation rate constant (Kd*) 1.398 × 10(-4) and half life (t1/2) 49 h. pH activity profile of BmMK indicates the involvement of carboxylate ion, histidine, lysine, arginine or aspartic acid at the active site of enzyme. Activity of recombinant BmMK was confirmed by phosphorylation of RS-mevalonate in the presence of Mg(2+), having Km and Vmax 331.9 μM and 719.1 pKat μg(-1), respectively. The values of kcat and kcat/Km for RS-mevalonate were determined to be 143.82 s(-1) and 0.43332 M(-1) s(-1) and kcat and kcat/Km values for ATP were found 150.9 s(-1) and 1.023 M(-1) s(-1). The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2.

  14. Mevalonate kinase deficiency and Dutch type periodic fever.

    PubMed

    Frenkel, J; Houten, S M; Waterham, H R; Wanders, R J; Rijkers, G T; Kimpen, J L; Duran, R; Poll-The, B T; Kuis, W

    2000-01-01

    Dutch type periodic fever (DPF) is an autosomal recessive hereditary fever syndrome. Cases have been reported worldwide, the majority from France and The Netherlands. From infancy the patients suffer fever attacks that recur every 2-8 weeks, often precipitated by immunizations, infections or emotional stress. Fever lasts 2-7 days and can be accompanied by malaise, headache, diarrhea, abdominal pain, vomiting, skin rashes, arthralgia, arthritis, tender lymphadenopathy, hepatosplenomegaly, and oral and genital ulcers. Laboratory evaluation during fever shows granulocytosis and elevated acute phase reactants. DPF is caused by a deficiency of the enzyme mevalonate kinase (MK). Besides DPF, the spectrum of MK deficiency includes a severe phenotype, mevalonic aciduria (MA). MA patients have less residual MK activity, leading to substantially higher urinary mevalonic acid excretion than in DPF. Mevalonic aciduria is characterized by mental retardation and dysmorphic features in addition to the clinical features of DPF. At the genomic level, several mutations of varying severity have been identified. The DPF phenotype is caused by one particular mild missense mutation. Most patients are compound heterozygotes for this mutation and a more severe mutation. The mechanism by which MK deficiency leads to fever is not understood. The vast majority of DPF patients have persistently elevated serum IgD and can be classified as having hyperimmunoglobulinemia D and periodic fever syndrome (HIDS). Conversely, most HIDS patients have MK deficiency and hence DPF, but the two disorders do not overlap entirely.

  15. Genomic variations of the mevalonate pathway in porokeratosis

    PubMed Central

    Zhang, Zhenghua; Li, Caihua; Wu, Fei; Ma, Ruixiao; Luan, Jing; Yang, Feng; Liu, Weida; Wang, Li; Zhang, Shoumin; Liu, Yan; Gu, Jun; Hua, Wenlian; Fan, Min; Peng, Hua; Meng, Xuemei; Song, Ningjing; Bi, Xinling; Gu, Chaoying; Zhang, Zhen; Huang, Qiong; Chen, Lianjun; Xiang, Leihong; Xu, Jinhua; Zheng, Zhizhong; Jiang, Zhengwen

    2015-01-01

    Porokeratosis (PK) is a heterogeneous group of keratinization disorders. No causal genes except MVK have been identified, even though the disease was linked to several genomic loci. Here, we performed massively parallel sequencing and exonic CNV screening of 12 isoprenoid genes in 134 index PK patients (61 familial and 73 sporadic) and identified causal mutations in three novel genes (PMVK, MVD, and FDPS) in addition to MVK in the mevalonate pathway. Allelic expression imbalance (AEI) assays were performed in 13 lesional tissues. At least one mutation in one of the four genes in the mevalonate pathway was found in 60 (98%) familial and 53 (73%) sporadic patients, which suggests that isoprenoid biosynthesis via the mevalonate pathway may play a role in the pathogenesis of PK. Significantly reduced expression of the wild allele was common in lesional tissues due to gene conversion or some other unknown mechanism. A G-to-A RNA editing was observed in one lesional tissue without AEI. In addition, we observed correlations between the mutations in the four mevalonate pathway genes and clinical manifestations in the PK patients, which might support a new and simplified classification of PK under the guidance of genetic testing. DOI: http://dx.doi.org/10.7554/eLife.06322.001 PMID:26202976

  16. Genomic variations of the mevalonate pathway in porokeratosis.

    PubMed

    Zhang, Zhenghua; Li, Caihua; Wu, Fei; Ma, Ruixiao; Luan, Jing; Yang, Feng; Liu, Weida; Wang, Li; Zhang, Shoumin; Liu, Yan; Gu, Jun; Hua, Wenlian; Fan, Min; Peng, Hua; Meng, Xuemei; Song, Ningjing; Bi, Xinling; Gu, Chaoying; Zhang, Zhen; Huang, Qiong; Chen, Lianjun; Xiang, Leihong; Xu, Jinhua; Zheng, Zhizhong; Jiang, Zhengwen

    2015-07-23

    Porokeratosis (PK) is a heterogeneous group of keratinization disorders. No causal genes except MVK have been identified, even though the disease was linked to several genomic loci. Here, we performed massively parallel sequencing and exonic CNV screening of 12 isoprenoid genes in 134 index PK patients (61 familial and 73 sporadic) and identified causal mutations in three novel genes (PMVK, MVD, and FDPS) in addition to MVK in the mevalonate pathway. Allelic expression imbalance (AEI) assays were performed in 13 lesional tissues. At least one mutation in one of the four genes in the mevalonate pathway was found in 60 (98%) familial and 53 (73%) sporadic patients, which suggests that isoprenoid biosynthesis via the mevalonate pathway may play a role in the pathogenesis of PK. Significantly reduced expression of the wild allele was common in lesional tissues due to gene conversion or some other unknown mechanism. A G-to-A RNA editing was observed in one lesional tissue without AEI. In addition, we observed correlations between the mutations in the four mevalonate pathway genes and clinical manifestations in the PK patients, which might support a new and simplified classification of PK under the guidance of genetic testing.

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

    PubMed

    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.

  18. Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase.

    PubMed

    Zhou, Changfang; Li, Ziru; Wiberley-Bradford, Amy E; Weise, Sean E; Sharkey, Thomas D

    2013-09-15

    Isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) are building units for all isoprenoids; thus, intracellular pool sizes of IDP and DMADP play important roles in living organisms. Several methods have been used to quantify the amount of DMADP or the combined amount of IDP plus DMADP, but measuring the DMADP/IDP ratio has been difficult. In this study, a method was developed to measure the ratio of DMADP/IDP. Catalyzed by a recombinant IDP isomerase (IDI) together with a recombinant isoprene synthase (IspS), IDP was converted to isoprene, which was then detected by chemiluminescence. With this method, the in vitro equilibrium ratio of DMADP/IDP was found to be 2.11:1. IDP and DMADP pools were significantly increased in Escherichia coli transformed with methylerythritol 4-phosphate pathway genes; the ratio of DMADP/IDP was 3.85. An E. coli strain transformed with IspS but no additional IDI had a lower DMADP level and a DMADP/IDP ratio of 1.05. Approximately 90% of the IDP and DMADP pools in light-adapted kudzu leaves were light dependent and so presumably were located in the chloroplasts; the DMADP/IDP ratios in chloroplasts and cytosol were the same as the in vitro ratio (2.04 in the light and 2.32 in the dark).

  19. Mevalonate kinase deficiency leads to decreased prenylation of Rab GTPases.

    PubMed

    Jurczyluk, Julie; Munoz, Marcia A; Skinner, Oliver P; Chai, Ryan C; Ali, Naveid; Palendira, Umaimainthan; Quinn, Julian Mw; Preston, Alexandra; Tangye, Stuart G; Brown, Andrew J; Argent, Elizabeth; Ziegler, John B; Mehr, Sam; Rogers, Michael J

    2016-11-01

    Mevalonate kinase deficiency (MKD) is caused by mutations in a key enzyme of the mevalonate-cholesterol biosynthesis pathway, leading to recurrent autoinflammatory disease characterised by enhanced release of interleukin-1β (IL-1β). It is currently believed that the inflammatory phenotype of MKD is triggered by temperature-sensitive loss of mevalonate kinase activity and reduced biosynthesis of isoprenoid lipids required for the prenylation of small GTPase proteins. However, previous studies have not clearly shown any change in protein prenylation in patient cells under normal conditions. With lymphoblast cell lines from two compound heterozygous MKD patients, we used a highly sensitive in vitro prenylation assay, together with quantitative mass spectrometry, to reveal a subtle accumulation of unprenylated Rab GTPases in cells cultured for 3 days or more at 40 °C compared with 37 °C. This included a 200% increase in unprenylated Rab7A, Rab14 and Rab1A. Inhibition of sterol regulatory element-binding protein (SREBP) activation by fatostatin led to more pronounced accumulation of unprenylated Rab proteins in MKD cells but not parent cells, suggesting that cultured MKD cells may partially overcome the loss of isoprenoid lipids by SREBP-mediated upregulation of enzymes required for isoprenoid biosynthesis. Furthermore, while inhibition of Rho/Rac/Rap prenylation promoted the release of IL-1β, specific inhibition of Rab prenylation by NE10790 had no effect in human peripheral blood mononuclear cells or human THP-1 monocytic cells. These studies demonstrate for the first time that mutations in mevalonate kinase can lead to a mild, temperature-induced defect in the prenylation of small GTPases, but that loss of prenylated Rab GTPases is not the cause of enhanced IL-1β release in MKD.

  20. Control of the innate immune response by the mevalonate pathway

    PubMed Central

    Akula, Murali K.; Shi, Man; Jiang, Zhaozhao; Foster, Celia E.; Miao, David; Li, Annie S.; Zhang, Xiaoman; Gavin, Ruth M.; Forde, Sorcha D.; Germain, Gail; Carpenter, Susan; Rosadini, Charles V.; Gritsman, Kira; Chae, Jae Jin; Hampton, Randolph; Silverman, Neal; Gravallese, Ellen M.; Kagan, Jonathan C.; Fitzgerald, Katherine A.; Kastner, Daniel L.; Golenbock, Douglas T.; Bergo, Martin O.; Wang, Donghai

    2016-01-01

    Deficiency of mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate (GGPP), a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, protein post-translational modification catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) cause autoinflammatory Familial Mediterranean Fever (FMF) syndrome. Here, we show that protein geranylgeranylation enables Toll-like receptor (TLR)-induced phosphatidylinositol-3-OH kinase PI(3)K) activation by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages deficient for GGTase I or p110δ exhibited constitutive interleukin-1β release that was MEFV-dependent, but NLRP3-, AIM2- and NLRC4- inflammasome independent. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows for an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome. PMID:27270400

  1. Enzymatic synthesis of isotopically labeled isoprenoid diphosphates.

    PubMed

    Christensen, D J; Poulter, C D

    1994-07-01

    Recombinant yeast isopentenyl diphosphate (IPP) isomerase and avian farnesyl diphosphate (FPP) synthase from overproducing strains of Escherichia coli were used to synthesize FPP from IPP and dimethylallyl diphosphate (DMAPP). [2,4,5-13C3]IPP and [2,4,5-13C3]DMAPP were synthesized from ethyl [2-13C]bromoacetate and [1,3-13C2]acetone. Thes compounds were used as substrates for enzymatic synthesis of FPP selectivity labeled at the first or third isoprene residue or at all three.

  2. Propioin synthesis using thiamine diphosphate-dependent enzymes.

    PubMed

    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%).

  3. Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    van Deenen, Nicole; Bachmann, Anne-Lena; Schmidt, Thomas; Schaller, Hubert; Sand, Jennifer; Prüfer, Dirk; Schulze Gronover, Christian

    2012-04-01

    Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products.

  4. Identification of an active site alanine in mevalonate kinase through characterization of a novel mutation in mevalonate kinase deficiency.

    PubMed

    Hinson, D D; Chambliss, K L; Hoffmann, G F; Krisans, S; Keller, R K; Gibson, K M

    1997-10-17

    Sequencing of polymerase chain reaction-amplified cDNAs from cultured cells of three patients with mevalonate kinase deficiency revealed a G --> A transversion at nucleotide 1000 of the coding region, converting alanine to threonine at position 334 (A334T). To characterize this defect, we expressed wild-type and mutant cDNAs in Escherichia coli as the glutathione S-transferase fusion proteins, with purification by affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis for wild-type and mutant fusion proteins indicated an expected molecular mass of 42-43 kDa. Kinetic characterization of the wild-type fusion protein yielded Km values of 150 +/- 23 and 440 +/- 190 microM (mean +/- S.E.) for substrates (RS)-mevalonate and ATP, respectively. Expressed wild-type mevalonate kinase (MKase) had a maximum velocity of 13.6 +/- 1.4 units/mg of protein (n = 22, +/-S.E.), whereas the A334T mutation yielded an enzyme with average Vmax of 0.26 +/- 0.02 unit/mg of protein (n = 6, +/-S.E.), representing a decrease to 1.4% of control Vmax. Restriction digestion with HhaI, in conjunction with direct sequencing of cDNAs, revealed that two patients were homozygous and one heterozygous for the A334T allele, establishing autosomal recessive inheritance within families. Although the A334T enzyme had a normal Km for ATP of 680 +/- 226 microM (n = 3, +/-S.E.), the Michaelis constant for (RS)-mevalonate was increased >30-fold to 4623 +/- 1167 microM (n = 4, +/-S.E.) under standard assay conditions. Comparable kinetic results were obtained using extracts of lymphoblasts, which were homozygous for the A334T allele. Alanine 334 is invariant in MKase from bacteria to man and located in a glycine-rich region postulated to have homology with ATP-binding sequences. Our results indicate that the bacterial expression system for human MKase will provide a useful model system in which to analyze inherited mutations and identify the first active site residue in MKase associated with

  5. Mevalonate kinase deficiency leads to decreased prenylation of Rab GTPases

    PubMed Central

    Jurczyluk, Julie; Munoz, Marcia A; Skinner, Oliver P; Chai, Ryan C; Ali, Naveid; Palendira, Umaimainthan; Quinn, Julian MW; Preston, Alexandra; Tangye, Stuart G; Brown, Andrew J; Argent, Elizabeth; Ziegler, John B; Mehr, Sam; Rogers, Michael J

    2016-01-01

    Mevalonate kinase deficiency (MKD) is caused by mutations in a key enzyme of the mevalonate–cholesterol biosynthesis pathway, leading to recurrent autoinflammatory disease characterised by enhanced release of interleukin-1β (IL-1β). It is currently believed that the inflammatory phenotype of MKD is triggered by temperature-sensitive loss of mevalonate kinase activity and reduced biosynthesis of isoprenoid lipids required for the prenylation of small GTPase proteins. However, previous studies have not clearly shown any change in protein prenylation in patient cells under normal conditions. With lymphoblast cell lines from two compound heterozygous MKD patients, we used a highly sensitive in vitro prenylation assay, together with quantitative mass spectrometry, to reveal a subtle accumulation of unprenylated Rab GTPases in cells cultured for 3 days or more at 40 °C compared with 37 °C. This included a 200% increase in unprenylated Rab7A, Rab14 and Rab1A. Inhibition of sterol regulatory element-binding protein (SREBP) activation by fatostatin led to more pronounced accumulation of unprenylated Rab proteins in MKD cells but not parent cells, suggesting that cultured MKD cells may partially overcome the loss of isoprenoid lipids by SREBP-mediated upregulation of enzymes required for isoprenoid biosynthesis. Furthermore, while inhibition of Rho/Rac/Rap prenylation promoted the release of IL-1β, specific inhibition of Rab prenylation by NE10790 had no effect in human peripheral blood mononuclear cells or human THP-1 monocytic cells. These studies demonstrate for the first time that mutations in mevalonate kinase can lead to a mild, temperature-induced defect in the prenylation of small GTPases, but that loss of prenylated Rab GTPases is not the cause of enhanced IL-1β release in MKD. PMID:27377765

  6. Genetics Home Reference: aromatic l-amino acid decarboxylase deficiency

    MedlinePlus

    ... aromatic l-amino acid decarboxylase deficiency aromatic l-amino acid decarboxylase deficiency Printable PDF Open All Close All ... view the expand/collapse boxes. Description Aromatic l-amino acid decarboxylase (AADC) deficiency is an inherited disorder that ...

  7. Production of taxadiene by engineering of mevalonate pathway in Escherichia coli and endophytic fungus Alternaria alternata TPF6.

    PubMed

    Bian, Guangkai; Yuan, Yujie; Tao, Hui; Shi, Xiaofei; Zhong, Xiaofang; Han, Yichao; Fu, Shuai; Fang, Chengxiang; Deng, Zixin; Liu, Tiangang

    2017-04-01

    Taxol (paclitaxel) is a diterpenoid compound with significant and extensive applications in the treatment of cancer. The production of Taxol and relevant intermediates by engineered microbes is an attractive alternative to the semichemical synthesis of Taxol. In this study, based on a previously developed platform, the authors first established taxadiene production in mutant E. coli T2 and T4 by engineering of the mevalonate (MVA) pathway. The authors then developed an Agrobacterium tumefaciens-mediated transformation (ATMT) method and verified the strength of heterologous promoters in Alternaria alternata TPF6. The authors next transformed the taxadiene-producing platform into A. alternata TPF6, and the MVA pathway was engineered, with introduction of the plant taxadiene-forming gene. Notably, by co-overexpression of isopentenyl diphosphate isomerase (Idi), a truncated version of 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1), and taxadiene synthase (TS), the authors could detect 61.9 ± 6.3 μg/L taxadiene in the engineered strain GB127. This is the first demonstration of taxadiene production in filamentous fungi, and the approach presented in this study provides a new method for microbial production of Taxol. The well-established ATMT method and the known promoter strengths facilitated further engineering of taxaenes in this fungus. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells.

    PubMed

    Hemmerlin, Andréa; Hoeffler, Jean-François; Meyer, Odile; Tritsch, Denis; Kagan, Isabelle A; Grosdemange-Billiard, Catherine; Rohmer, Michel; Bach, Thomas J

    2003-07-18

    In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.

  9. Genome-wide RNAi analysis reveals that simultaneous inhibition of specific mevalonate pathway genes potentiates tumor cell death.

    PubMed

    Pandyra, Aleksandra A; Mullen, Peter J; Goard, Carolyn A; Ericson, Elke; Sharma, Piyush; Kalkat, Manpreet; Yu, Rosemary; Pong, Janice T; Brown, Kevin R; Hart, Traver; Gebbia, Marinella; Lang, Karl S; Giaever, Guri; Nislow, Corey; Moffat, Jason; Penn, Linda Z

    2015-09-29

    The mevalonate (MVA) pathway is often dysregulated or overexpressed in many cancers suggesting tumor dependency on this classic metabolic pathway. Statins, which target the rate-limiting enzyme of this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), are promising agents currently being evaluated in clinical trials for anti-cancer efficacy. To uncover novel targets that potentiate statin-induced apoptosis when knocked down, we carried out a pooled genome-wide short hairpin RNA (shRNA) screen. Genes of the MVA pathway were amongst the top-scoring targets, including sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) and geranylgeranyl diphosphate synthase 1 (GGPS1). Each gene was independently validated and shown to significantly sensitize A549 cells to statin-induced apoptosis when knocked down. SREBP2 knockdown in lung and breast cancer cells completely abrogated the fluvastatin-induced upregulation of sterol-responsive genes HMGCR and HMGCS1. Knockdown of SREBP2 alone did not affect three-dimensional growth of lung and breast cancer cells, yet in combination with fluvastatin cell growth was disrupted. Taken together, these results show that directly targeting multiple levels of the MVA pathway, including blocking the sterol-feedback loop initiated by statin treatment, is an effective and targetable anti-tumor strategy.

  10. Genome-wide RNAi analysis reveals that simultaneous inhibition of specific mevalonate pathway genes potentiates tumor cell death

    PubMed Central

    Pandyra, Aleksandra A.; Mullen, Peter J.; Goard, Carolyn A.; Ericson, Elke; Sharma, Piyush; Kalkat, Manpreet; Yu, Rosemary; Pong, Janice T.; Brown, Kevin R.; Hart, Traver; Gebbia, Marinella; Lang, Karl S.; Giaever, Guri; Nislow, Corey; Moffat, Jason; Penn, Linda Z.

    2015-01-01

    The mevalonate (MVA) pathway is often dysregulated or overexpressed in many cancers suggesting tumor dependency on this classic metabolic pathway. Statins, which target the rate-limiting enzyme of this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), are promising agents currently being evaluated in clinical trials for anti-cancer efficacy. To uncover novel targets that potentiate statin-induced apoptosis when knocked down, we carried out a pooled genome-wide short hairpin RNA (shRNA) screen. Genes of the MVA pathway were amongst the top-scoring targets, including sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) and geranylgeranyl diphosphate synthase 1 (GGPS1). Each gene was independently validated and shown to significantly sensitize A549 cells to statin-induced apoptosis when knocked down. SREBP2 knockdown in lung and breast cancer cells completely abrogated the fluvastatin-induced upregulation of sterol-responsive genes HMGCR and HMGCS1. Knockdown of SREBP2 alone did not affect three-dimensional growth of lung and breast cancer cells, yet in combination with fluvastatin cell growth was disrupted. Taken together, these results show that directly targeting multiple levels of the MVA pathway, including blocking the sterol-feedback loop initiated by statin treatment, is an effective and targetable anti-tumor strategy. PMID:26353928

  11. Hyper-IgD syndrome/mevalonate kinase deficiency: what is new?

    PubMed

    Mulders-Manders, C M; Simon, A

    2015-07-01

    Mevalonate kinase deficiency or hyper-IgD syndrome is a hereditary autoinflammatory syndrome caused by mutations in the mevalonate kinase gene. In this review, we will discuss new findings in this disorder that have been published in the last 2 years. This includes new insights into pathophysiology, treatment, and the clinical phenotype linked to the genetic defect.

  12. Regulation of the Mevalonate Pathway for the Prevention of Breast Cancer

    DTIC Science & Technology

    2002-08-01

    cholesterol biosynthesis. Cancer Res 1990;50(11):3270-3. 6. Wejde J, Blegen H, Larsson 0. Requirement for mevalonate in the control of proliferation...and normal cells from GI to multiple cell cycles by lovastatin. Cancer Res. 1991;51:3602-3609. 11. Larsson 0, Blegen H. Regulatory role of mevalonate

  13. Diphosphates and diphosphonates in polyoxometalate chemistry.

    PubMed

    Banerjee, Abhishek; Bassil, Bassem S; Röschenthaler, Gerd-Volker; Kortz, Ulrich

    2012-11-21

    In the wide area of polyoxometalate (POM) chemistry, diphosphate/diphosphonate-based POMs represent a more recent area of study. However, in this short time it has emerged to become very dynamic, as shown by the wide variety of compounds reported. Ever since the discovery of the first polyoxotungstate framework constructed from diphosph(on)ate ligands, a widespread investigation on the preparative chemistry and properties of such compounds has followed. The main focus of such a study is based on factors such as the oxidation state of the metal, the effect of pH and temperature during synthesis, and the presence of different functional groups on the diphosphonate. In this review we discuss in detail all diphosphate/diphosphonate-based POMs, beginning with early developments, subsequent growth in interest, and finally focusing on the very latest developments.

  14. Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

    PubMed Central

    2013-01-01

    Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic

  15. The effect of decreased plasma cholesterol concentration on circulatinga mevalonate metabolism in rats.

    PubMed

    Feingold, K R; Wiley, M H; MacRae, G; Siperstein, M D

    1981-08-01

    Circulating mevalonate is metabolized by two mechanisms: the sterol pathways leading to cholesterol and the shunt pathway resulting in CO2 production. The kidney is the chief site of circulating mevalonate metabolism by both pathways. The present study investigated the effect of plasma cholesterol concentration on circulating mevalonate metabolism. 3-Aminopyrazolo(3,4-d)pyrimidine and Triton WR 1339 were utilized to induce "functional hypocholesterolemia". An enhancement of both renal total nonsaponifiable lipid synthesis (36-43%) and cholesterol synthesis (42%) from circulatinga mevalonate was observed when "functional hypocholesterolemia" was induced by either compound. Hepatic total nonsaponifiable lipid synthesis from circulating mevalonate was not enhanced in the Triton-treated animals, but 4-aminopyrazolo(3,4-d)pyrimidine treatment increased accumulation of total labeled nonsaponifiable lipids and cholesterol. No increase in labeled total nonsaponifiable lipids or cholesterol in the carcass was observed after treatment with wither compound. "Functional hypocholesterolemia" reduced the shunt pathway of circulating mevalonate metabolism by approximately 30%. This reduction occurred in both the renal and extrarenal shunt pathways. These data indicate that plasma cholesterol concentration regulates the in vivo metabolism of circulating mevalonate in that hypocholesterolemia reduces the shunt pathway and stimulates sterologenesis, and effect chiefly localized to athe kidneys.

  16. The mevalonate pathway and the synthesis of juvenile hormone in insects.

    PubMed

    Bellés, Xavier; Martín, David; Piulachs, Maria-Dolors

    2005-01-01

    The mevalonate pathway in insects has two important peculiarities, the absence of the sterol branch and the synthesis of juvenile hormone (JH), that may have influenced the mechanisms of regulation. The data available on these mechanisms indicate that cholesterol does not play a regulatory role and that JH modulates transcript levels of a number of genes of the mevalonate pathway or can influence the translatability and/or stability of the transcripts themselves. These data suggest that the mevalonate pathway in insects can best be interpreted in terms of coordinated regulation, in which regulators act in parallel to a number of enzymes, as occurs in the cholesterol-driven pathway in vertebrates.

  17. Perturbation of the Monomer-Monomer Interfaces of the Benzoylformate Decarboxylase Tetramer

    SciTech Connect

    Andrews, Forest H.; Rogers, Megan P.; Paul, Lake N.; McLeish, Michael J.

    2014-08-14

    The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer–monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer–tetramer equilibrium and to provide an ~450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

  18. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, 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 large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  19. Zymographic detection of cinnamic acid decarboxylase activity.

    PubMed

    Prim, Núria; Pastor, F I Javier; Diaz, Pilar

    2002-11-01

    The manuscript includes a concise description of a new, fast and simple method for detection of cinnamic acid decarboxylase activity. The method is based on a color shift caused a by pH change and may be an excellent procedure for large screenings of samples from natural sources, as it involves no complex sample processing or purification. The method developed can be used in preliminary approaches to biotransformation processes involving detection of hydroxycinnamic acid decarboxylase activity.

  20. Isolation and characterization of a benzoylformate decarboxylase and a NAD+/NADP+-dependent benzaldehyde dehydrogenase involved in D-phenylglycine metabolism in Pseudomonas stutzeri ST-201.

    PubMed

    Saehuan, Choedchai; Rojanarata, Theerasak; Wiyakrutta, Suthep; McLeish, Michael J; Meevootisom, Vithaya

    2007-11-01

    Following induction with D-phenylglycine both d-phenylglycine aminotransferase activity and benzoylformate decarboxylase activity were observed in cultures of Pseudomonas stutzeri ST-201. Induction with benzoylformate, on the other hand, induced only benzoylformate decarboxylase activity. Purification of the benzoylformate decarboxylase, followed by N-terminal sequencing, enabled the design of probes for hybridization with P. stutzeri ST-201 genomic DNA libraries. Sequencing of two overlapping genomic DNA restriction fragments revealed two open reading frames which were denoted dpgB and dpgC. Sequence alignments suggested that the genes encoded a thiamin-diphosphate-dependent decarboxylase and an aldehyde dehydrogenase, respectively. Both genes were isolated and expressed in Escherichia coli. The dpgB gene product was confirmed as a benzoylformate decarboxylase while the dpgC gene product was characterized as a NAD+/NADP+-dependent benzaldehyde dehydrogenase. In keeping with their high sequence identities (both greater than 85%) the kinetic properties of the two enzymes were similar to those of the homologous enzymes in the mandelate pathway of Pseudomonas putida ATCC 12633. However, Pseudomonas stutzeri ST-201 was unable to grow on either isomer of mandelate, and sequencing indicated that the dpgB gene did not form part of an operon. Thus it appears that the two enzymes form part of a d-phenylglycine, rather than mandelate, degrading pathway.

  1. Mevalonate production by engineered acetogen biocatalyst during continuous fermentation of syngas or CO₂/H₂ blend.

    PubMed

    Kiriukhin, Michael; Tyurin, Michael

    2014-02-01

    Naturally mevalonate-resistant acetogen Clostridium sp. MT1243 produced only 425 mM acetate during syngas fermentation. Using Clostridium sp. MT1243 we engineered biocatalyst selectively producing mevalonate from synthesis gas or CO₂/H₂ blend. Acetate production and spore formation were eliminated from Clostridium sp. MT1243 using Cre-lox66/lox71-system. Cell energy released via elimination of phosphotransacetylase, acetate kinase and early stage sporulation genes powered mevalonate accumulation in fermentation broth due to expression of synthetic thiolase, HMG-synthase, and HMG-reductase, three copies of each, integrated using Tn7-approach. Recombinants produced 145 mM mevalonate in five independent single-step fermentation runs 25 days each in five repeats using syngas blend 60% CO and 40% H₂ (v/v) (p < 0.005). Mevalonate production was 97 mM if only CO₂/H₂ blend was fed instead of syngas (p < 0.005). Mevalonate from CO₂/H₂ blend might serve as a commercial route to mitigate global warming in proportion to CO₂ fermentation scale worldwide.

  2. Biosynthesis of Taxadiene in Saccharomyces cerevisiae : selection of geranylgeranyl diphosphate synthase directed by a computer-aided docking strategy.

    PubMed

    Ding, Ming-Zhu; Yan, Hui-Fang; Li, Lin-Feng; Zhai, Fang; Shang, Lu-Qing; Yin, Zheng; Yuan, Ying-Jin

    2014-01-01

    Identification of efficient key enzymes in biosynthesis pathway and optimization of the fitness between functional modules and chassis are important for improving the production of target compounds. In this study, the taxadiene biosynthesis pathway was firstly constructed in yeast by transforming ts gene and overexpressing erg20 and thmgr. Then, the catalytic capabilities of six different geranylgeranyl diphosphate synthases (GGPPS), the key enzyme in mevalonic acid (MVA) pathway catalyzing famesyl diphosphate (FPP) to geranylgeranyl diphosphate (GGPP), were predicted using enzyme-substrate docking strategy. GGPPSs from Taxus baccata x Taxus cuspidate (GGPPSbc), Erwinia herbicola (GGPPSeh), and S. cerevisiae (GGPPSsc) which ranked 1st, 4th and 6th in docking with FPP were selected for construction. The experimental results were consistent with the computer prediction that the engineered yeast with GGPPSbc exhibited the highest production. In addition, two chassis YSG50 and W303-1A were chosen, and the titer of taxadiene reached 72.8 mg/L in chassis YSG50 with GGPPSbc. Metabolomic study revealed that the contents of tricarboxylic acid cycle (TCA) intermediates and their precursor amino acids in chassis YSG50 was lower than those in W303-1A, indicating less carbon flux was divided into TCA cycle. Furthermore, the levels of TCA intermediates in the taxadiene producing yeasts were lower than those in chassis YSG50. Thus, it may result in more carbon flux in MVA pathway in chassis YSG50, which suggested that YSG50 was more suitable for engineering the taxadiene producing yeast. These results indicated that computer-aided protein modeling directed isoenzyme selection strategy and metabolomic study could guide the rational design of terpenes biosynthetic cells.

  3. Biosynthesis of Taxadiene in Saccharomyces cerevisiae : Selection of Geranylgeranyl Diphosphate Synthase Directed by a Computer-Aided Docking Strategy

    PubMed Central

    Li, Lin-feng; Zhai, Fang; Shang, Lu-qing; Yin, Zheng; Yuan, Ying-jin

    2014-01-01

    Identification of efficient key enzymes in biosynthesis pathway and optimization of the fitness between functional modules and chassis are important for improving the production of target compounds. In this study, the taxadiene biosynthesis pathway was firstly constructed in yeast by transforming ts gene and overexpressing erg20 and thmgr. Then, the catalytic capabilities of six different geranylgeranyl diphosphate synthases (GGPPS), the key enzyme in mevalonic acid (MVA) pathway catalyzing famesyl diphosphate (FPP) to geranylgeranyl diphosphate (GGPP), were predicted using enzyme-substrate docking strategy. GGPPSs from Taxus baccata x Taxus cuspidate (GGPPSbc), Erwinia herbicola (GGPPSeh), and S. cerevisiae (GGPPSsc) which ranked 1st, 4th and 6th in docking with FPP were selected for construction. The experimental results were consistent with the computer prediction that the engineered yeast with GGPPSbc exhibited the highest production. In addition, two chassis YSG50 and W303-1A were chosen, and the titer of taxadiene reached 72.8 mg/L in chassis YSG50 with GGPPSbc. Metabolomic study revealed that the contents of tricarboxylic acid cycle (TCA) intermediates and their precursor amino acids in chassis YSG50 was lower than those in W303-1A, indicating less carbon flux was divided into TCA cycle. Furthermore, the levels of TCA intermediates in the taxadiene producing yeasts were lower than those in chassis YSG50. Thus, it may result in more carbon flux in MVA pathway in chassis YSG50, which suggested that YSG50 was more suitable for engineering the taxadiene producing yeast. These results indicated that computer-aided protein modeling directed isoenzyme selection strategy and metabolomic study could guide the rational design of terpenes biosynthetic cells. PMID:25295588

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

    SciTech Connect

    Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric; Kaiser, Marcel; Amzel, L. Mario; Gabelli, Sandra B.

    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.

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

    PubMed

    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. X-ray analysis of azido-thymidine diphosphate binding to nucleoside diphosphate kinase.

    PubMed

    Xu, Y; Sellam, O; Moréra, S; Sarfati, S; Biondi, R; Véron, M; Janin, J

    1997-07-08

    To be effective as antiviral agent, AZT (3'-azido-3'-deoxythymidine) must be converted to a triphosphate derivative by cellular kinases. The conversion is inefficient and, to understand why AZT diphosphate is a poor substrate of nucleoside diphosphate (NDP) kinase, we determined a 2.3-A x-ray structure of a complex with the N119A point mutant of Dictyostelium NDP kinase. It shows that the analog binds at the same site and, except for the sugar ring pucker which is different, binds in the same way as the natural substrate thymidine diphosphate. However, the azido group that replaces the 3'OH of the deoxyribose in AZT displaces a lysine side chain involved in catalysis. Moreover, it is unable to make an internal hydrogen bond to the oxygen bridging the beta- and gamma-phosphate, which plays an important part in phosphate transfer.

  7. Microglia activation and interaction with neuronal cells in a biochemical model of mevalonate kinase deficiency.

    PubMed

    Tricarico, Paola Maura; Piscianz, Elisa; Monasta, Lorenzo; Kleiner, Giulio; Crovella, Sergio; Marcuzzi, Annalisa

    2015-08-01

    Mevalonate kinase deficiency is a rare disease whose worst manifestation, characterised by severe neurologic impairment, is called mevalonic aciduria. The progressive neuronal loss associated to cell death can be studied in vitro with a simplified model based on a biochemical block of the mevalonate pathway and a subsequent inflammatory trigger. The aim of this study was to evaluate the effect of the mevalonate blocking on glial cells (BV-2) and the following effects on neuronal cells (SH-SY5Y) when the two populations were cultured together. To better understand the cross-talk between glial and neuronal cells, as it happens in vivo, BV-2 and SH-SY5Y were co-cultured in different experimental settings (alone, transwell, direct contact); the effect of mevalonate pathway biochemical block by Lovastatin, followed by LPS inflammatory trigger, were evaluated by analysing programmed cell death and mitochondrial membrane potential, cytokines' release and cells' morphology modifications. In this experimental condition, glial cells underwent an evident activation, confirmed by elevated pro-inflammatory cytokines release, typical of these disorders, and a modification in morphology. Moreover, the activation induced an increase in apoptosis. When glial cells were co-cultured with neurons, their activation caused an increase of programmed cell death also in neuronal cells, but only if the two populations were cultured in direct contact. Our findings, being aware of the limitations related to the cell models used, represent a preliminary step towards understanding the pathological and neuroinflammatory mechanisms occurring in mevalonate kinase diseases. Contact co-culture between neuronal and microglial cells seems to be a good model to study mevalonic aciduria in vitro, and to contribute to the identification of potential drugs able to block microglial activation for this orphan disease. In fact, in such a pathological condition, we demonstrated that microglial cells are

  8. Dopa Decarboxylase Modulates Tau Toxicity.

    PubMed

    Kow, Rebecca L; Sikkema, Carl; Wheeler, Jeanna M; Wilkinson, Charles W; Kraemer, Brian C

    2017-06-15

    The microtubule-associated protein tau accumulates into toxic aggregates in multiple neurodegenerative diseases. We found previously that loss of D2-family dopamine receptors ameliorated tauopathy in multiple models including a Caenorhabditis elegans model of tauopathy. To better understand how loss of D2-family dopamine receptors can ameliorate tau toxicity, we screened a collection of C. elegans mutations in dopamine-related genes (n = 45) for changes in tau transgene-induced behavioral defects. These included many genes responsible for dopamine synthesis, metabolism, and signaling downstream of the D2 receptors. We identified one dopamine synthesis gene, dopa decarboxylase (DDC), as a suppressor of tau toxicity in tau transgenic worms. Loss of the C. elegans DDC gene, bas-1, ameliorated the behavioral deficits of tau transgenic worms, reduced phosphorylated and detergent-insoluble tau accumulation, and reduced tau-mediated neuron loss. Loss of function in other genes in the dopamine and serotonin synthesis pathways did not alter tau-induced toxicity; however, their function is required for the suppression of tau toxicity by bas-1. Additional loss of D2-family dopamine receptors did not synergize with bas-1 suppression of tauopathy phenotypes. Loss of the DDC bas-1 reduced tau-induced toxicity in a C. elegans model of tauopathy, while loss of no other dopamine or serotonin synthesis genes tested had this effect. Because loss of activity upstream of DDC could reduce suppression of tau by DDC, this suggests the possibility that loss of DDC suppresses tau via the combined accumulation of dopamine precursor levodopa and serotonin precursor 5-hydroxytryptophan. Published by Elsevier Inc.

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

    DOEpatents

    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.

  10. Temperature and drug treatments in mevalonate kinase deficiency: an ex vivo study.

    PubMed

    Tricarico, Paola Maura; Kleiner, Giulio; Piscianz, Elisa; Zanin, Valentina; Monasta, Lorenzo; Crovella, Sergio; Marcuzzi, Annalisa

    2013-01-01

    Mevalonate Kinase Deficiency (MKD) is a rare autosomal recessive inborn disorder of cholesterol biosynthesis caused by mutations in the mevalonate kinase (MK) gene, leading to MK enzyme decreased activity. The consequent shortage of mevalonate-derived isoprenoid compounds results in an inflammatory phenotype, caused by the activation of the NALP3 inflammasome that determines an increased caspase-1 activation and IL-1 β release. In MKD, febrile temperature can further decrease the residual MK activity, leading to mevalonate pathway modulation and to possible disease worsening. We previously demonstrated that the administration of exogenous isoprenoids such as geraniol or the modulation of the enzymatic pathway with drugs, such as Tipifarnib, partially rescues the inflammatory phenotype associated with the defective mevalonic pathway. However, it has not been investigated yet how temperature can affect the success of these treatments. Thus, we investigated the effect of temperature on primary human monocytes from MKD patients. Furthermore the ability of geraniol and Tipifarnib to reduce the abnormal inflammatory response, already described at physiological temperature in MKD, was studied in a febrile condition. We evidenced the role of temperature in the modulation of the inflammatory events and suggested strongly considering this variable in future researches aimed at finding a treatment for MKD.

  11. Temperature and Drug Treatments in Mevalonate Kinase Deficiency: An Ex Vivo Study

    PubMed Central

    Tricarico, Paola Maura; Piscianz, Elisa; Crovella, Sergio

    2013-01-01

    Mevalonate Kinase Deficiency (MKD) is a rare autosomal recessive inborn disorder of cholesterol biosynthesis caused by mutations in the mevalonate kinase (MK) gene, leading to MK enzyme decreased activity. The consequent shortage of mevalonate-derived isoprenoid compounds results in an inflammatory phenotype, caused by the activation of the NALP3 inflammasome that determines an increased caspase-1 activation and IL-1β release. In MKD, febrile temperature can further decrease the residual MK activity, leading to mevalonate pathway modulation and to possible disease worsening. We previously demonstrated that the administration of exogenous isoprenoids such as geraniol or the modulation of the enzymatic pathway with drugs, such as Tipifarnib, partially rescues the inflammatory phenotype associated with the defective mevalonic pathway. However, it has not been investigated yet how temperature can affect the success of these treatments. Thus, we investigated the effect of temperature on primary human monocytes from MKD patients. Furthermore the ability of geraniol and Tipifarnib to reduce the abnormal inflammatory response, already described at physiological temperature in MKD, was studied in a febrile condition. We evidenced the role of temperature in the modulation of the inflammatory events and suggested strongly considering this variable in future researches aimed at finding a treatment for MKD. PMID:24073415

  12. NMR-based quantification of organic diphosphates

    PubMed Central

    Lenevich, Stepan

    2010-01-01

    Phosphorylated compounds are ubiquitous in life. Given their central role, many such substrates and analogues 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 31P 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. PMID:20833124

  13. A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes.

    PubMed

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

    2009-07-01

    Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate 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 Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC(50) values of 5.8 (meta isomer) and 3.0 microM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K(I) of 0.46 microM. Radiolabeled forms of both analogues selectively labeled the beta-subunit of ScPFTase. The para isomer was also shown to label Escherichia 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.

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

    PubMed Central

    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

  15. Preclinical evidence for nitrogen-containing bisphosphonate inhibition of farnesyl diphosphate (FPP) synthase in the kidney: implications for renal safety.

    PubMed

    Lühe, Anke; Künkele, Klaus-Peter; Haiker, Monika; Schad, Karen; Zihlmann, Christine; Bauss, Frieder; Suter, Laura; Pfister, Thomas

    2008-06-01

    Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and play an important role in the treatment of osteoporosis, metastatic bone disease, and Paget disease. However, nephrotoxicity has been reported with some bisphosphonates. Nitrogen-containing bisphosphonates directly inhibit farnesyl diphosphate (FPP) synthase activity (mevalonate pathway) and reduce protein prenylation leading to osteoclast cell death. The aim here was to elucidate if this inhibition also occurs in kidney cells and may directly account for nephrotoxicity. In an exploratory study in rats receiving zoledronate or ibandronate an approximate 2-fold increase in FPP synthase mRNA levels was observed in the kidney. The involvement of the mevalonate pathway was confirmed in subsequent in vitro studies with zoledronate, ibandronate, and pamidronate, using the non-nitrogen containing bisphosphonate clodronate as a comparator. In vitro changes in FPP synthase mRNA expression, enzyme activity, and levels of prenylated proteins were assessed. Using two cell lines (a rat normal kidney cell line, NRK-52E, and a human kidney proximal tubule cell line, HK-2), ibandronate and zoledronate were identified as most cytotoxic (EC50: 23/>1000 microM and 16/82 microM, respectively) and as the most potent inhibitors of FPP synthase (IC50; 1.6/7.4 microM and 0.5/0.7 microM, respectively). In both cell lines, inhibition of FPP synthase activity occurred prior to a decrease in levels of prenylated proteins followed by cytotoxicity. This further supports that the mechanism responsible for osteoclast inhibition (therapeutic effect) might also underlie the mechanism of nephrotoxicity.

  16. Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

    PubMed Central

    Serrano-Carreon, L.; Hathout, Y.; Bensoussan, M.; Belin, J.-M.

    1993-01-01

    The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-14C]linoleic acid or [5-14C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [14C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [14C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-14C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species. PMID:16349040

  17. Statins, Mevalonate Pathway and its Intermediate Products in Placental Development and Preeclampsia.

    PubMed

    Ermini, Leonardo; Post, Martin; Caniggia, Isabella

    2017-01-01

    The mevalonate pathway synthesizes intermediates and products such as cholesterol and nonsterol isoprenoids that are crucial for cell survival and function. In the human placenta, the prenylation of proteins, rather than cholesterol synthesis, represents the main "metabolic target" of mevalonate metabolism. Major cellular functions depend on isoprenylation including proliferation, migration, metabolism and protein glycosylation that are all crucial for proper development of the embryo and the placenta. Statins are inhibitors of HMG-CoA reductase, the enzyme that catalyzes the reduction of HMG-CoA to mevalonic acid by NADPH. In vitro experiments using human placental explants suggest that statins elicit a detrimental effect on placental growth. However, animal and epidemiologic studies show no increase of fetal malformations after exposure to statins during pregnancy. Moreover, emerging evidence from mouse studies suggest that statins may be useful in preventing serious pregnancy complications like preeclampsia. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Biosynthesis of the Juvenile Hormones of Manduca sexta: Labeling Pattern from Mevalonate, Propionate, and Acetate

    PubMed Central

    Schooley, David A.; Judy, Kenneth J.; Bergot, B. John; Hall, M. Sharon; Siddall, John B.

    1973-01-01

    Using organ culture, high-resolution liquid chromatography, and microchemical techniques, we demonstrated the efficient incorporation in vitro of several radiolabeled precursors into the two juvenile hormones of Manduca sexta. JH II, a homosesquiterpene hormone, reported from M. sexta as well as several other insects, incorporates radiolabel from acetate, mevalonate, and propionate. JH III, a sesquiterpene hormone recently reported as a natural product of M. sexta, incorporates label from acetate and mevalonate, but not from propionate. Based on the position of the labeled atoms in the precursors and upon the position of incorporation obtained from label-distribution data, a scheme for juvenile hormone biosynthesis is advanced. PMID:16592112

  19. Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects.

    PubMed

    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.

  20. Type II Isopentenyl Diphosphate Isomerase: Probing the Mechanism with Alkyne/Allene Diphosphate Substrate Analogues†

    PubMed Central

    Sharma, Nagendra K.; Pan, Jian-Jung; Poulter, C. Dale

    2010-01-01

    Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic five-carbon building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction. In contrast, the type II enzyme (IDI-2) requires reduced flavin, raising the possibility that the reaction catalyzed by IDI-2 involves the net addition/abstraction of a hydrogen atom. As part of our studies of the mechanism of isomerization for IDI-2, we synthesized allene and alkyne substrate analogues for the enzyme. These molecules are predicted to be substantially less reactive toward proton addition than IPP and DMAPP, but have similar reactivities toward hydrogen atom addition. This prediction was verified by calculations of gas phase heats of reaction for addition of a proton and of a hydrogen atom to 1-butyne (3) and 1,2-butadiene (4) to form the 1-buten-2-yl carbocation and radical, respectively, and related affinities for 2-methyl-1-butene (5) and 2-methyl-2-butene (6) using G3MP2B3 and CBS-QB3 protocols. Alkyne 1-OPP and allene 2-OPP were not substrates for Thermus thermophilus IDI-2 or Escherichia coli IDI-1, but instead were competitive inhibitors. The experimental and computational results are consistent with a protonation-deprotonation mechanism for the enzyme-catalyzed isomerization of IPP and DMAPP. PMID:20560533

  1. Defect in mevalonate pathway induces pyroptosis in Raw 264.7 murine monocytes.

    PubMed

    Marcuzzi, Annalisa; Piscianz, Elisa; Girardelli, Martina; Crovella, Sergio; Pontillo, Alessandra

    2011-09-01

    The inhibition of mevalonate pathway by the aminobisphosphonate alendronate (ALD) has been previously associated with an augmented lipopolysaccharide-induced interleukin-1beta (IL-1β) secretion in monocytes, as demonstrated in an auto-inflammatory disease known as mevalonate kinase deficiency (MKD). In this study we investigated the effect of ALD + LPS on monocyte cell line (Raw 264.7) death. ALD strongly augmented LPS-induced programmed cell death (PCD) as well as IL-1β secretion in Raw murine monocytes, whereas necrosis was rather unaffected. ALD + LPS induced caspase-3 activation. Inhibition of IL-1β stimulation partially restored cell viability. These findings suggest that the inhibition of mevalonate pathway, together with a bacterial stimulus, induce a PCD partly sustained by the caspase-3-related apoptosis and partly by caspase-1-associated pyroptosis. The involvement of pyroptosis is a novel hit in our cell model and opens discussions about its role in inflammatory cells with chemical or genetic inhibition of mevalonate pathway.

  2. Fruit carotenoid-deficient mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis.

    PubMed

    Pankratov, Ilya; McQuinn, Ryan; Schwartz, Jochanan; Bar, Einat; Fei, Zhangjun; Lewinsohn, Efraim; Zamir, Dani; Giovannoni, James J; Hirschberg, Joseph

    2016-10-01

    Isoprenoids consist of a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP). In plants, IDP is synthesized in the cytoplasm from mevalonic acid via the MVA pathway, and in plastids from 2-C-methyl-d-erythritol-4-phosphate through the MEP pathway. The enzyme IDP isomerase (IDI) catalyzes the interconversion between IDP and DMADP. Most plants contain two IDI enzymes, the functions of which are characteristically compartmentalized in the cells. Carotenoids are isoprenoids that play essential roles in photosynthesis and provide colors to flowers and fruits. They are synthesized in the plastids via the MEP pathway. Fruits of Solanum lycopersicum (tomato) accumulate high levels of the red carotene lycopene. We have identified mutations in tomato that reduce overall carotenoid accumulation in fruits. Four alleles of a locus named FRUIT CAROTENOID DEFICIENT 1 (fcd1) were characterized. Map-based cloning of fcd1 indicated that this gene encodes the plastidial enzyme IDI1. Lack of IDI1 reduced the concentration of carotenoids in fruits, flowers and cotyledons, but not in mature leaves. These results indicate that the plastidial IDI plays an important function in carotenoid biosynthesis, thus highlighting its role in optimizing the ratio between IDP and DMADP as precursors for different downstream isoprenoid pathways.

  3. Geranylfarnesyl diphosphate synthase from Methanosarcina mazei: Different role, different evolution

    SciTech Connect

    Ogawa, Takuya; Yoshimura, Tohru; Hemmi, Hisashi

    2010-02-26

    The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Goe1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.

  4. Biosynthetic arginine decarboxylase in phytopathogenic fungi.

    PubMed

    Khan, A J; Minocha, S C

    1989-01-01

    It has been reported that while bacteria and higher plants possess two different pathways for the biosynthesis of putrescine, via ornithine decarboxylase (ODC) and arginine decarboxylase (ADC); the fungi, like animals, only use the former pathway. We found that contrary to the earlier reports, two of the phytopathogenic fungi (Ceratocystis minor and Verticillium dahliae) contain significant levels of ADC activity with very little ODC. The ADC in these fungi has high pH optimum (8.4) and low Km (0.237 mM for C. minor, 0.103 mM for V. dahliae), and is strongly inhibited by alpha-difluoromethylarginine (DFMA), putrescine and spermidine, further showing that this enzyme is probably involved in the biosynthesis of polyamines and not in the catabolism of arginine as in Escherichia coli. The growth of these fungi is strongly inhibited by DFMA while alpha-difluoromethylornithine (DFMO) has little effect.

  5. ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae.

    PubMed

    Rodriguez, Sarah; Denby, Charles M; Van Vu, T; Baidoo, Edward E K; Wang, George; Keasling, Jay D

    2016-03-03

    With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsic physiological constraints-in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints. We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate. By combining the push/pull/block strategies, we significantly improved mevalonate

  6. ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae

    SciTech Connect

    Rodriguez, Sarah; Denby, Charles M.; Van Vu, T.; Baidoo, Edward E. K.; Wang, George; Keasling, Jay D.

    2016-03-03

    With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsic physiological constraints—in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints. We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate. In conclusion, by combining the push/pull/block strategies, we significantly improved

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

    PubMed Central

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

    2009-01-01

    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 β-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. PMID:19487664

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

    PubMed

    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.

  9. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    SciTech Connect

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong

    2011-12-31

    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  10. Complex evolution of orthologous and paralogous decarboxylase genes.

    PubMed

    Sáenz-de-Miera, L E; Ayala, F J

    2004-01-01

    The decarboxylases are involved in neurotransmitter synthesis in animals, and in pathways of secondary metabolism in plants. Different decarboxylase proteins are characterized for their different substrate specificities, but are encoded by homologous genes. We study, within a maximum-likelihood framework, the evolutionary relationships among dopa decarboxylase (Ddc), histidine decarboxylase (Hdc) and alpha-methyldopa hypersensitive (amd) in animals, and tryptophan decarboxylase (Wdc) and tyrosine decarboxylase (Ydc) in plants. The evolutionary rates are heterogeneous. There are differences between paralogous genes in the same lineages: 4.13 x 10(-10) nucleotide substitutions per site per year in mammalian Ddc vs. 1.95 in Hdc; between orthologous genes in different lineages, 7.62 in dipteran Ddc vs. 4.13 in mammalian Ddc; and very large temporal variations in some lineages, from 3.7 up to 54.9 in the Drosophila Ddc lineage. Our results are inconsistent with the molecular clock hypothesis.

  11. Properties of ribulose diphosphate carboxylase immobilized on porous glass

    NASA Technical Reports Server (NTRS)

    Shapira, J.; Hanson, C. L.; Lyding, J. M.; Reilly, P. J.

    1974-01-01

    Ribulose-1,5-diphosphate carboxylase from spinach has been bound to arylamine porous glass with a diazo linkage and to alklamine porous glass with glutaraldehyde. Stability at elevated temperatures and responses to changes of pH and ribulose-1,5-diphosphate, Mg(2+), and dithiothreitol concentrations were not significantly different from the soluble enzyme, though stability at 4 C was somewhat improved.

  12. Elucidation of the chemistry of enzyme-bound thiamin diphosphate prior to substrate binding: defining internal equilibria among tautomeric and ionization states.

    PubMed

    Nemeria, Natalia; Korotchkina, Lioubov; McLeish, Michael J; Kenyon, George L; Patel, Mulchand S; Jordan, Frank

    2007-09-18

    Both solution and crystallographic studies suggest that the 4'-aminopyrimidine ring of the thiamin diphosphate coenzyme participates in catalysis, likely as an intramolecular general acid-base catalyst via the unusual 1',4'-iminopyrimidine tautomer. It is indeed uncommon for a coenzyme to be identified in its rare tautomeric form on its reaction pathways, yet this has been possible with thiamin diphosphate, in some cases even in the absence of substrate [Nemeria, N., Chakraborty, S., Baykal, A., Korotchkina, L., Patel, M. S., and Jordan, F. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 78-82.]. The ability to detect both the aminopyrimidine and iminopyrimidine tautomeric forms of thiamin diphosphate on enzymes has enabled us to assign the predominant tautomeric form present in individual intermediates on the pathway. Herein, we report the pH dependence of these tautomeric forms providing the first data for the internal thermodynamic equilibria on thiamin diphosphate enzymes for the various ionization and tautomeric forms of this coenzyme on four enzymes: benzaldehyde lyase, benzoylformate decarboxylase, pyruvate oxidase, and the E1 component of the human pyruvate dehydrogenase multienzyme complex. Evidence is provided for an important function of the enzyme environment in altering both the ionization and tautomeric equilibria on the coenzyme even prior to addition of substrate. The pKa for the 4'-aminopyrimidinium moiety coincides with the pH for optimum activity thereby ensuring that all ionization states and tautomeric states are accessible during the catalytic cycle. The dramatic influence of the protein on the internal equilibria also points to conditions under which the long-elusive ylide intermediate could be stabilized.

  13. Induction of aromatic-L-amino acid decarboxylase by decarboxylase inhibitors in idiopathic parkinsonism.

    PubMed

    Boomsma, F; Meerwaldt, J D; Man in 't Veld, A J; Hovestadt, A; Schalekamp, M A

    1989-06-01

    We evaluated the effect of administration of L-dopa, alone or in combination with a peripheral decarboxylase inhibitor, on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD). After single-dose administration of L-dopa plus benserazide (Madopar) in healthy subjects and in chronically treated patients with parkinsonism, plasma ALAAD followed for 2 to 3 hours fell, but returned to predosing levels within 90 minutes. Four groups of patients with idiopathic parkinsonism were studied during chronic treatment: Group I, no L-dopa treatment (n = 31); Group II, L-dopa alone (n = 15); Group III, L-dopa plus benserazide (n = 28); and Group IV, L-dopa plus carbidopa (Sinemet, n = 30). Plasma ALAAD 2 hours after dosing was normal in Groups I and II. ALAAD was increased threefold in Groups III and IV, suggesting induction of ALAAD by the coadministration of a peripheral decarboxylase inhibitor. In a study of 3 patients in whom L-dopa/benserazide was started, plasma ALAAD rose gradually over 3 to 4 weeks. Further detailed pharmacokinetic studies of L-dopa, dopamine, and ALAAD in plasma and cerebrospinal fluid are required to determine if the apparent ALAAD induction by a peripheral decarboxylase inhibitor may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena.

  14. Compromized geranylgeranylation of RhoA and Rac1 in mevalonate kinase deficiency

    PubMed Central

    Henneman, L.; Schneiders, M. S.; Turkenburg, M.

    2010-01-01

    Mevalonate kinase deficiency (MKD) is an autoinflammatory disorder caused by mutations in the MVK gene resulting in decreased activity of the enzyme mevalonate kinase (MK). Although MK is required for biosynthesis of all isoprenoids, in MKD, in particular, the timely synthesis of geranylgeranyl pyrophosphate appears to be compromised. Because small guanosine triphosphatases (GTPases) depend on geranylgeranylation for their proper signaling function, we studied the effect of MK deficiency on geranylgeranylation and activation of the two small GTPases, RhoA and Rac1. We demonstrate that both geranylgeranylation and activation of the two GTPases are more easily disturbed in MKD cells than in control cells when the flux though the isoprenoid biosynthesis pathway is suppressed by low concentrations of simvastatin. The limited capacity of geranylgeranylation in MKD cells readily leads to markedly increased levels of nonisoprenylated and activated GTPases, which will affect proper signaling by these GTPases. PMID:20814828

  15. Two UDP-glucuronic acid decarboxylases involved in the biosynthesis of a bacterial exopolysaccharide in Paenibacillus elgii.

    PubMed

    Li, Ou; Qian, Chao-Dong; Zheng, Dao-Qiong; Wang, Pin-Mei; Liu, Yu; Jiang, Xin-Hang; Wu, Xue-Chang

    2015-04-01

    Xylose is described as a component of bacterial exopolysaccharides in only a limited number of bacterial strains. A bacterial strain, Paenibacillus elgii, B69 was shown to be efficient in producing a xylose-containing exopolysaccharide. Sequence analysis was performed to identify the genes encoding the uridine diphosphate (UDP)-glucuronic acid decarboxylase required for the synthesis of UDP-xylose, the precursor of the exopolysaccharide. Two sequences, designated as Peuxs1 and Peuxs2, were found as the candidate genes for such enzymes. The activities of the UDP-glucuronic acid decarboxylases were proven by heterologous expression and real-time nuclear magnetic resonance analysis. The intracellular activity and effect of these genes on the synthesis of exopolysaccharide were further investigated by developing a thymidylate synthase based knockout system. This system was used to substitute the conventional antibiotic resistance gene system in P. elgii, a natural multi-antibiotic resistant strain. Results of intracellular nucleotide sugar analysis showed that the intracellular UDP-xylose and UDP-glucuronic acid levels were affected in Peuxs1 or Peuxs2 knockout strains. The knockout of either Peuxs1 or Peuxs2 reduced the polysaccharide production and changed the monosaccharide ratio. No polysaccharide was found in the Peuxs1/Peuxs2 double knockout strain. Our results show that P. elgii can be efficient in forming UDP-xylose, which is then used for the synthesis of xylose-containing exopolysaccharide.

  16. Regulation of the Mevalonate Pathway for the Prevention of Breast Cancer

    DTIC Science & Technology

    2000-08-01

    polyunsaturated fatty acids ( PUFAs ) is associated with a decreased risk of breast cancer in women. These fatty acids also inhibit the development of... acids ( PUFAs ) can be accounted for by their inhibitory effect on the cholesterol biosynthesis (mevalonate) pathway. In Task 1, we have shown that the...this essential fatty acid for maximal mammary tumor growth. While the 7% safflower oil diet we used previously contains adequate linoleic acid , the 7

  17. Genetics Home Reference: malonyl-CoA decarboxylase deficiency

    MedlinePlus

    ... link) ACT Sheet: Elevated C3-DC acylcarnitine (PDF) Genetic Testing (1 link) Genetic Testing Registry: Deficiency of malonyl-CoA decarboxylase Other ... Topic: Lipid Metabolism Disorders Health Topic: Newborn Screening Genetic and Rare ... decarboxylase deficiency Educational Resources (3 links) ...

  18. DNAJA1 controls the fate of misfolded mutant p53 through the mevalonate pathway

    PubMed Central

    Parrales, Alejandro; Ranjan, Atul; Iyer, Swathi V.; Padhye, Subhash; Weir, Scott J.; Roy, Anuradha; Iwakuma, Tomoo

    2017-01-01

    Stabilization of mutant p53 (mutp53) in tumours greatly contributes to malignant progression. However, little is known about the underlying mechanisms and therapeutic approaches to destabilize mutp53. Here, through high-throughput screening we identify statins, cholesterol-lowering drugs, as degradation inducers for conformational or misfolded p53 mutants with minimal effects on wild-type p53 (wtp53) and DNA contact mutants. Statins preferentially suppress mutp53-expressing cancer cell growth. Specific reduction of mevalonate-5-phosphate by statins or mevalonate kinase knockdown induces CHIP ubiquitin ligase-mediated nuclear export, ubiquitylation, and degradation of mutp53 by impairing interaction of mutp53 with DNAJA1, a Hsp40 family member. Knockdown of DNAJA1 also induces CHIP-mediated mutp53 degradation, while its overexpression antagonizes statin-induced mutp53 degradation. Our study reveals that DNAJA1 controls the fate of misfolded mutp53, provides insights into potential strategies to deplete mutp53 through the mevalonate pathway–DNAJA1 axis, and highlights the significance of p53 status in impacting statins’ efficacy on cancer therapy. PMID:27775703

  19. Long-term outcome of a successful cord blood stem cell transplant in mevalonate kinase deficiency.

    PubMed

    Giardino, Stefano; Lanino, Edoardo; Morreale, Giuseppe; Madeo, Annalisa; Di Rocco, Maja; Gattorno, Marco; Faraci, Maura

    2015-01-01

    Mevalonate kinase deficiency (MKD) is a rare autosomal recessive inborn error of metabolism with an autoinflammatory phenotype that may be expressed as a spectrum of disease phenotypes, from those with prevailing autoinflammatory syndrome and variable response to anti-inflammatory therapies, to mevalonic aciduria, which is associated with dysmorphic features, severe neurologic involvement, and the worst prognosis. We describe a boy, aged 2 years, 10 months, with severe phenotype of mevalonate kinase deficiency who underwent allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-identical unrelated cord blood because his condition had failed to improve with antiinflammatory treatment as first-line therapy and an anticytokine drug as second-line therapy. The child had a sustained remission of febrile attacks and inflammation after transplant, and during a 5-year follow-up period, psychomotor and neurologic development were normal, without signs of underlying disease or late transplant-related effects. This case confirms that allogeneic HSCT is a safe and effective cure for patients affected by MKD in whom anticytokine drugs alone are insufficient for the management of autoinflammatory syndrome and for the unfavorable outcome of the disease.

  20. Diurnal variations in the plasma concentrations of mevalonic acid in patients with abetalipoproteinaemia.

    PubMed

    Pappu, A S; Illingworth, D R

    1994-10-01

    Previous studies have demonstrated that changes in the rates of cholesterol biosynthesis can be evaluated by the determination of plasma concentrations of sterol intermediates, including mevalonic acid and lathosterol and that, in normal human subjects, a diurnal rhythm exists in which the highest concentrations of sterol intermediates are observed at night. The factors responsible for this diurnal rhythm in cholesterol synthesis are, however, unknown. To test the hypothesis that the nocturnal increase in cholesterol biosynthesis is attributable to a reduced rate of hepatic uptake of chylomicron remnants at night as compared to higher rates of uptake during the daytime in response to alimentary lipaemia, we have examined the diurnal rhythm of mevalonic acid in six normal volunteers and three patients with phenotypic abetalipoproteinaemia. The latter patients do not absorb appreciable amounts of dietary cholesterol and are unable to synthesize chylomicron particles. Plasma concentrations of mevalonic acid exhibited a diurnal rhythm in the normal subjects, and the highest plasma concentrations were observed between 24.00 hours/04.00 hours. A similar rhythm was observed in the plasma of patients with abetalipoproteinaemia. These results suggest that the nocturnal increase in cholesterol biosynthesis which occurs in humans is not attributable to reduced hepatic uptake of chylomicron remnants at night; further studies are needed to better define those factors which influence the periodicity of cholesterol biosynthesis in humans.

  1. Inhibition of the mevalonate pathway affects epigenetic regulation in cancer cells

    PubMed Central

    Karlic, Heidrun; Thaler, Roman; Gerner, Christopher; Grunt, Thomas; Proestling, Katharina; Haider, Florian; Varga, Franz

    2015-01-01

    The mevalonate pathway provides metabolites for post-translational modifications such as farnesylation, which are critical for the activity of RAS downstream signaling. Subsequently occurring regulatory processes can induce an aberrant stimulation of DNA methyltransferase (DNMT1) as well as changes in histone deacetylases (HDACs) and microRNAs in many cancer cell lines. Inhibitors of the mevalonate pathway are increasingly recognized as anticancer drugs. Extensive evidence indicates an intense cross-talk between signaling pathways, which affect growth, differentiation, and apoptosis either directly or indirectly via epigenetic mechanisms. Herein, we show data obtained by novel transcriptomic and corresponding methylomic or proteomic analyses from cell lines treated with pharmacologic doses of respective inhibitors (i.e., simvastatin, ibandronate). Metabolic pathways and their epigenetic consequences appear to be affected by a changed concentration of NADPH. Moreover, since the mevalonate metabolism is part of a signaling network, including vitamin D metabolism or fatty acid synthesis, the epigenetic activity of associated pathways is also presented. This emphasizes the far-reaching epigenetic impact of metabolic therapies on cancer cells and provides some explanation for clinical observations, which indicate the anticancer activity of statins and bisphosphonates. PMID:25978957

  2. Incorporation of Mevalonic Acid into Ribosylzeatin in Tobacco Callus Ribonucleic Acid Preparations 1

    PubMed Central

    Murai, Norimoto; Armstrong, Donald J.; Skoog, Folke

    1975-01-01

    The incorporation of 14C-2-mevalonic acid into transfer RNA and ribosomal RNA (high molecular weight RNA) in rapidly growing, cytokinin-dependent tobacco (Nicotiana tabacum var. Wisconsin No. 38) callus cultures has been investigated. Approximately 40% of the label incorporated into transfer RNA was present in a ribonucleoside with chromatographic properties identical to those of cis-ribosylzeatin. The remainder of the label in the transfer RNA appears to be nonspecific incorporation resulting from degradation and metabolism of 14C-2-mevalonic acid by the tobacco callus tissue. Although the total radioactivity incorporated into ribosomal RNA was roughly the same as in transfer RNA, the specific radioactivity of the transfer RNA was about four times higher than that of the ribosomal RNA, and the ribosomal RNA labeling could be distinguished from the cytokinin labeling observed in transfer RNA. The distributions of the 14C-2-mevalonic acid label and cytokinin activity in tobacco callus transfer RNA fractionated by benzoylated diethylaminoethylcellulose chromatography indicate that at least two cytokinin-containing transfer RNA species are present in this tissue. PMID:16659180

  3. Alendronate, a double-edged sword acting in the mevalonate pathway

    PubMed Central

    TRICARICO, PAOLA MAURA; GIRARDELLI, MARTINA; KLEINER, GIULIO; KNOWLES, ALESSANDRA; VALENCIC, ERICA; CROVELLA, SERGIO; MARCUZZI, ANNALISA

    2015-01-01

    Aminobisphosphonate aledronate is a compound commonly used clinically for the treatment of osteoporosis and other bone diseases, as a result of it preventing bone resorption. However, in previous years it has also been used to obtain cellular and animal models of a rare genetic disorder termed Mevalonate Kinase Deficiency (MKD). MKD is caused by mutations affecting the mevalonate kinase enzyme, in the cholesterol pathway and alendronate can be used to biochemically mimic the genetic defect as it inhibits farnesyl pyrophosphate synthase in the same pathway. Despite evidence in favor of the inhibition exerted on the mevalonate pathway, there is at least one clinical case of MKD in which alendronate improved not only skeletal and bone fractures, as expected, but also MKD clinical features. Based on this finding, the present study assessed the anti-inflammatory properties of this aminobisphosphonate in vitro. No anti-inflammatory effects of alendronate were observed in the in vitro experiments. Since MKD lacks specific treatments, these results may assist scientists and physicians in making the decision as to the most suitable choice of therapeutic compounds for this neglected disease. PMID:26096667

  4. Alendronate, a double-edged sword acting in the mevalonate pathway.

    PubMed

    Tricarico, Paola Maura; Girardelli, Martina; Kleiner, Giulio; Knowles, Alessandra; Valencic, Erica; Crovella, Sergio; Marcuzzi, Annalisa

    2015-09-01

    Aminobisphosphonate aledronate is a compound commonly used clinically for the treatment of osteoporosis and other bone diseases, as a result of it preventing bone resorption. However, in previous years it has also been used to obtain cellular and animal models of a rare genetic disorder termed Mevalonate Kinase Deficiency (MKD). MKD is caused by mutations affecting the mevalonate kinase enzyme, in the cholesterol pathway and alendronate can be used to biochemically mimic the genetic defect as it inhibits farnesyl pyrophosphate synthase in the same pathway. Despite evidence in favor of the inhibition exerted on the mevalonate pathway, there is at least one clinical case of MKD in which alendronate improved not only skeletal and bone fractures, as expected, but also MKD clinical features. Based on this finding, the present study assessed the anti‑inflammatory properties of this aminobisphosphonate in vitro. No anti‑inflammatory effects of alendronate were observed in the in vitro experiments. Since MKD lacks specific treatments, these results may assist scientists and physicians in making the decision as to the most suitable choice of therapeutic compounds for this neglected disease.

  5. Block of the Mevalonate Pathway Triggers Oxidative and Inflammatory Molecular Mechanisms Modulated by Exogenous Isoprenoid Compounds

    PubMed Central

    Tricarico, Paola Maura; Kleiner, Giulio; Valencic, Erica; Campisciano, Giuseppina; Girardelli, Martina; Crovella, Sergio; Knowles, Alessandra; Marcuzzi, Annalisa

    2014-01-01

    Deregulation of the mevalonate pathway is known to be involved in a number of diseases that exhibit a systemic inflammatory phenotype and often neurological involvements, as seen in patients suffering from a rare disease called mevalonate kinase deficiency (MKD). One of the molecular mechanisms underlying this pathology could depend on the shortage of isoprenoid compounds and the subsequent mitochondrial damage, leading to oxidative stress and pro-inflammatory cytokines’ release. Moreover, it has been demonstrated that cellular death results from the balance between apoptosis and pyroptosis, both driven by mitochondrial damage and the molecular platform inflammasome. In order to rescue the deregulated pathway and decrease inflammatory markers, exogenous isoprenoid compounds were administered to a biochemical model of MKD obtained treating a murine monocytic cell line with a compound able to block the mevalonate pathway, plus an inflammatory stimulus. Our results show that isoprenoids acted in different ways, mainly increasing the expression of the evaluated markers [apoptosis, mitochondrial dysfunction, nucleotide-binding oligomerization-domain protein-like receptors 3 (NALP3), cytokines and nitric oxide (NO)]. Our findings confirm the hypothesis that inflammation is triggered, at least partially, by the shortage of isoprenoids. Moreover, although further studies are necessary, the achieved results suggest a possible role for exogenous isoprenoids in the treatment of MKD. PMID:24758928

  6. Block of the mevalonate pathway triggers oxidative and inflammatory molecular mechanisms modulated by exogenous isoprenoid compounds.

    PubMed

    Tricarico, Paola Maura; Kleiner, Giulio; Valencic, Erica; Campisciano, Giuseppina; Girardelli, Martina; Crovella, Sergio; Knowles, Alessandra; Marcuzzi, Annalisa

    2014-04-22

    Deregulation of the mevalonate pathway is known to be involved in a number of diseases that exhibit a systemic inflammatory phenotype and often neurological involvements, as seen in patients suffering from a rare disease called mevalonate kinase deficiency (MKD). One of the molecular mechanisms underlying this pathology could depend on the shortage of isoprenoid compounds and the subsequent mitochondrial damage, leading to oxidative stress and pro-inflammatory cytokines' release. Moreover, it has been demonstrated that cellular death results from the balance between apoptosis and pyroptosis, both driven by mitochondrial damage and the molecular platform inflammasome. In order to rescue the deregulated pathway and decrease inflammatory markers, exogenous isoprenoid compounds were administered to a biochemical model of MKD obtained treating a murine monocytic cell line with a compound able to block the mevalonate pathway, plus an inflammatory stimulus. Our results show that isoprenoids acted in different ways, mainly increasing the expression of the evaluated markers [apoptosis, mitochondrial dysfunction, nucleotide-binding oligomerization-domain protein-like receptors 3 (NALP3), cytokines and nitric oxide (NO)]. Our findings confirm the hypothesis that inflammation is triggered, at least partially, by the shortage of isoprenoids. Moreover, although further studies are necessary, the achieved results suggest a possible role for exogenous isoprenoids in the treatment of MKD.

  7. Distribution of the mevalonate and glyceraldehyde phosphate/pyruvate pathways for isoprenoid biosynthesis in unicellular algae and the cyanobacterium Synechocystis PCC 6714.

    PubMed Central

    Disch, A; Schwender, J; Müller, C; Lichtenthaler, H K; Rohmer, M

    1998-01-01

    Isopentenyl diphosphate, the universal isoprenoid precursor, can be produced by two different biosynthetic routes: either via the acetate/mevalonate (MVA) pathway, or via the more recently identified MVA-independent glyceraldehyde phosphate/pyruvate pathway. These two pathways are easily differentiated by incorporation of [1-13C]glucose and analysis of the resulting labelling patterns found in the isoprenoids. This method was successfully applied to several unicellular algae raised under heterotrophic growth conditions and allowed for the identification of the pathways that were utilized for isoprenoid biosynthesis. All isoprenoids examined (sterols, phytol, carotenoids) of the green algae Chlorella fusca and Chlamydomonas reinhardtii were synthesized via the GAP/pyruvate pathway, as in another previously investigated green alga, Scenedesmus obliquus, which was also shown in this study to synthesize ubiquinone by the same MVA-independent route. In the red alga Cyanidium caldarium and in the Chrysophyte Ochromonas danica a clear dichotomy was observed: as in higher plants, sterols were formed via the MVA route, whereas chloroplast isoprenoids (phytol in Cy. caldarium and O. danica and beta-carotene in O. danica) were synthesized via the GAP/pyruvate route. In contrast, the Euglenophyte Euglena gracilis synthesized ergosterol, as well as phytol, via the acetate/MVA route. Similar feeding experiments were performed with the cyanobacterium Synechocystis PCC 6714 using [1-13C]- and [6-13C]-glucose. The two isoprenoids examined, phytol and beta-carotene, were shown to have the typical labelling pattern derived from the GAP/pyruvate route. PMID:9657979

  8. Arginine Decarboxylase Is Localized in Chloroplasts.

    PubMed Central

    Borrell, A.; Culianez-Macia, F. A.; Altabella, T.; Besford, R. T.; Flores, D.; Tiburcio, A. F.

    1995-01-01

    Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC. PMID:12228631

  9. ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae

    DOE PAGES

    Rodriguez, Sarah; Denby, Charles M.; Van Vu, T.; ...

    2016-03-03

    With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsicmore » physiological constraints—in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints. We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate. In conclusion, by combining the push/pull/block strategies, we significantly

  10. Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae)

    NASA Astrophysics Data System (ADS)

    Keeling, Christopher I.; Blomquist, Gary J.; Tittiger, Claus

    In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.

  11. [Spectroscopic study of the structure and intramolecular mobility of yeast pyruvate decarboxylase].

    PubMed

    Maskevich, S A; Maskevich, A A; Kivach, L N; Chernikevich, I P; Zabrodskaia, S V; Oparin, D A

    1993-12-01

    Steady-state and time-resolved fluorimetry were used to study the properties of holo- and apopyruvate decarboxylase (EC 4.1.1.1, PDC) from Brewer's yeast after interaction with substrate (pyruvate), cofactor (thiamine diphosphate, ThDP) and Mg2+ ions. The analysis of the enzyme's intrinsic fluorescence as well as of its complex with the probe 2-(p-toluidinylnaphthalene)-6-sulphonate (TNS) revealed that ThDP was found at the polar region of the PDC active sites, inducing a decrease in the mobility of the protein's nearest surroundings. The fluorescent probe had three different sites of binding to the protein apoform, two of which being located at the catalytic site and having different rotation freedom. The study of the PDC complex with thiochrome pyrophosphate, a ThDP structural analogue, pointed to the occurrence of a non-polar region of the enzyme active site for pyruvate absorption besides the polar region. The binding of pyruvate to the protein does not depend upon the cofactor's binding. On the basis of the fluorescent studies a model of the ThDP and pyruvate arrangement at the PDC active site is suggested.

  12. Arginine kinase shows nucleoside diphosphate kinase-like activity toward deoxythymidine diphosphate.

    PubMed

    Lopez-Zavala, Alonso A; Sotelo-Mundo, Rogerio R; Hernandez-Flores, Jose M; Lugo-Sanchez, Maria E; Sugich-Miranda, Rocio; Garcia-Orozco, Karina D

    2016-06-01

    Arginine kinase (AK) (ATP: L-arginine phosphotransferase, E.C. 2.7.3.3) catalyzes the reversible transfer of ATP γ-phosphate group to L-arginine to synthetize phospho-arginine as a high-energy storage. Previous studies suggest additional roles for AK in cellular processes. Since AK is found only in invertebrates and it is homologous to creatine kinase from vertebrates, the objective of this work was to demonstrate nucleoside diphosphate kinase-like activity for shrimp AK. For this, AK from marine shrimp Litopenaeus vannamei (LvAK) was purified and its activity was assayed for phosphorylation of TDP using ATP as phosphate donor. Moreover, by using high-pressure liquid chromatography (HPLC) the phosphate transfer reaction was followed. Also, LvAK tryptophan fluorescence emission changes were detected by dTDP titration, suggesting that the hydrophobic environment of Trp 221, which is located in the top of the active site, is perturbed upon dTDP binding. The kinetic constants for both substrates Arg and dTDP were calculated by isothermal titration calorimetry (ITC). Besides, docking calculations suggested that dTDP could bind LvAK in the same cavity where ATP bind, and LvAK basic residues (Arg124, 126 and 309) stabilize the dTDP phosphate groups and the pyrimidine base interact with His284 and Ser122. These results suggest that LvAK bind and phosphorylate dTDP being ATP the phosphate donor, thus describing a novel alternate nucleoside diphosphate kinase-like activity for this enzyme.

  13. Regulation of Ribulose Diphosphate Formation in Vivo by Light

    PubMed Central

    Klob, W.; Kandler, O.; Tanner, W.

    1972-01-01

    Light-dependent formation of ribulose-1,5 diphosphate is completely inhibited by low concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea which do not severely affect cyclic photophosphorylation. Also in Scenedesmus mutant number 11, capable of cyclic photophosphorylation, cellular ribulose-1,5 diphosphate-levels do not increase upon illumination. When mutant cells are H2 adapted, however, a light-dependent formation of ribulose-1,5 diphosphate is observed in the presence of H2. From these results it has been concluded that at least part of the Calvin cycle does not operate in the dark, since a reductant is lacking which is generated in the light. PMID:16658080

  14. Isopentenyl diphosphate isomerase: A checkpoint to isoprenoid biosynthesis.

    PubMed

    Berthelot, Karine; Estevez, Yannick; Deffieux, Alain; Peruch, Frédéric

    2012-08-01

    Even if the isopentenyl diphosphate (IPP) isomerases have been discovered in the 50s, it is only in the last decade that the genetical, enzymatical, structural richness and cellular importance of this large family of crucial enzymes has been uncovered. Present in all living kingdoms, they can be classified in two subfamilies: type 1 and type 2 IPP isomerases, which show clearly distinct characteristics. They all perform the regulatory isomerization of isopentenyl diphosphate into dimethylallyl diphosphate, a key rate-limiting step of the terpenoid biosynthesis, via a protonation/deprotonation mechanism. Due to their importance in the isoprenoid metabolism and the increasing interest of industry devoted to terpenoid production, it is foreseen that the biotechnological development of such enzymes should be under intense scrutiny in the near future.

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

    PubMed

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Determination of isopentenyl diphosphate and farnesyl diphosphate in tissue samples with a comment on secondary regulation of polyisoprenoid biosynthesis

    SciTech Connect

    Bruenger, E.; Rilling, H.C.

    1988-09-01

    A double-isotope dilution procedure is described for the determination of two isoprenoid precursors, isopentenyl and farnesyl diphosphate. Recovery of each is determined by the addition of the appropriate radioactive diphosphate to the tissue sample. After partial purification, each is coupled by a prenyltransferase with a cosubstrate of known specific activity. The products, doubly labeled farnesyl and geranylgeranyl diphosphates, are cleaved to the parent alcohols by alkaline phosphatase. The resulting polyprenols are isolated by reversed-phase thin-layer chromatography and their radioisotopic content is determined. The levels of these precursors have been measured in livers of rats and mice that have been maintained on several different diets. The concentration of each was about 0.5 mumol/g wet tissue and varied as much as 10-fold under the different test conditions. The levels of isopentenyl diphosphate isomerase, farnesyl diphosphate synthetase, and squalene synthetase were also measured in these animals. The changes in levels of these enzymes, in conjunction with the variation in substrate concentrations, are such that they could substantially influence the rate of cholesterol synthesis in liver.

  17. Ribulose diphosphate carboxylase/oxygenase. III. Isolation and properties.

    PubMed

    Ryan, F J; Tolbert, N E

    1975-06-10

    Similarities in properties of ribulose diphosphate carboxylase and oxygenase activities further substantiate the hypothesis that the same protein catalyzes both reactions. The Km (ribulose diphosphate) is 0.33 mM for the ribulose diphosphate oxygenase, when assayed in air with an oxygen electrode. Maximum activity is obtained with 10 to 35 mM MgCl2. Higher MgCl2 concentrations are inhibitory, but they shift the pH optimum from 9.3 or 9.4 to 8.7 or 9.0. MnCl2 is an effective cofactor of the oxygenase and some activity is obtained with CoCl2. Both the ribulose diphosphate carboxylase and oxygenase activity of the purified protein from spinach leaves are slowly inactivated by storage at 0 degrees and reactivated in 10 min at 50 degrees, provided both 25 mM MgCl2 and 1 mM dithiothreitol are present. The sulfhydryl groups of the enzyme which react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) are approximately 4 at pH 7.8 and 11 at pH 9.4. At both pH values ribulose diphosphate prevents two of these sulfhydryl groups from reacting with this reagent. About 50% inhibition of the oxygenase activity at pH 9.0 occurs with 50 mM bicarbonate in the presence of 3 mM ribulose diphosphate, and from variations in these parameters the inhibition is attributed to the CO2 species. The purified enzyme of acrylamide gels prevented the reduction of nitroblue tetrazolium in the presence of the superoxide radical, but the enzyme in solution did not react as a superoxide dismutase.

  18. A review on the chemical synthesis of pyrophosphate bonds in bioactive nucleoside diphosphate analogs.

    PubMed

    Xu, Zhihong

    2015-09-15

    Currently, there is an ongoing interest in the synthesis of nucleoside diphosphate analogs as important regulators in catabolism/anabolism, and their potential applications as mechanistic probes and chemical tools for bioassays. However, the pyrophosphate bond formation step remains as the bottleneck. In this Digest, the chemical synthesis of the pyrophosphate bonds of representative bioactive nucleoside diphosphate analogs, i.e. phosphorus-modified analogs, nucleoside cyclic diphosphates, and nucleoside diphosphate conjugates, will be described.

  19. Characterization of ribulose diphosphate carboxylase and phosphoribulokinase from Thiobacillus thioparus and Thiobacillus neapolitanus.

    NASA Technical Reports Server (NTRS)

    Johnson, E. J.; Johnson, M. K.; Macelroy, R. D.

    1968-01-01

    Ribulose diphosphate carboxylase and phosphoribulokinase activity in chemosynthetic autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus, noting sedimentation and gel filtration characteristics

  20. Characterization of ribulose diphosphate carboxylase and phosphoribulokinase from Thiobacillus thioparus and Thiobacillus neapolitanus.

    NASA Technical Reports Server (NTRS)

    Johnson, E. J.; Johnson, M. K.; Macelroy, R. D.

    1968-01-01

    Ribulose diphosphate carboxylase and phosphoribulokinase activity in chemosynthetic autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus, noting sedimentation and gel filtration characteristics

  1. Three Distinct Glutamate Decarboxylase Genes in Vertebrates

    PubMed Central

    Grone, Brian P.; Maruska, Karen P.

    2016-01-01

    Gamma-aminobutyric acid (GABA) is a widely conserved signaling molecule that in animals has been adapted as a neurotransmitter. GABA is synthesized from the amino acid glutamate by the action of glutamate decarboxylases (GADs). Two vertebrate genes, GAD1 and GAD2, encode distinct GAD proteins: GAD67 and GAD65, respectively. We have identified a third vertebrate GAD gene, GAD3. This gene is conserved in fishes as well as tetrapods. We analyzed protein sequence, gene structure, synteny, and phylogenetics to identify GAD3 as a homolog of GAD1 and GAD2. Interestingly, we found that GAD3 was lost in the hominid lineage. Because of the importance of GABA as a neurotransmitter, GAD3 may play important roles in vertebrate nervous systems. PMID:27461130

  2. Dopa decarboxylase activity of the living human brain

    SciTech Connect

    Gjedde, A.; Reith, J.; Dyve, S.; Leger, G.; Guttman, M.; Diksic, M.; Evans, A.; Kuwabara, H. )

    1991-04-01

    Monoaminergic neurons use dopa decarboxylase to form dopamine from L-3,4-dihydroxyphenylalanine (L-dopa). We measured regional dopa decarboxylase activity in brains of six healthy volunteers with 6-({sup 18}F)fluoro-L-dopa and positron emission tomography. We calculated the enzyme activity, relative to its Km, with a kinetic model that yielded the relative rate of conversion of 6-({sup 18}F)fluoro-L-dopa to ({sup 18}F)fluorodopamine. Regional values of relative dopa decarboxylase activity ranged from nil in occipital cortex to 1.9 h-1 in caudate nucleus and putamen, in agreement with values obtained in vitro.

  3. Using site-saturation mutagenesis to explore mechanism and substrate specificity in thiamin diphosphate-dependent enzymes.

    PubMed

    Andrews, Forest H; McLeish, Michael J

    2013-12-01

    For almost 20 years, site-saturation mutagenesis (SSM) has been used to evolve stereoselective enzymes as catalysts for synthetic organic chemistry. Much of this work has focused on enzymes such as lipases and esterases, although the range is rapidly expanding. By contrast, using SSM to study enzyme mechanisms is much less common. Instead, site-directed mutagenesis is more generally employed, with a particular emphasis on alanine variants. In the present review, we provide examples of the growing use of SSM to study not only substrate and reaction selectivity, but also the reaction mechanism of thiamin diphosphate (ThDP)-dependent enzymes. We report that the use of SSM to examine the roles of the catalytic residues of benzoylformate decarboxylase gave rise to results that were at odds with earlier kinetic and structural studies using alanine substitutions and also questioned their conclusions. SSM was also employed to examine the long held tenet that a bulky hydrophobic residue provides a fulcrum by which the V-conformation of the ThDP cofactor is maintained. X-ray structures showed that ThDP stayed in the V-conformation even when the replacement residues were charged or did not contact the cofactor. We also summarize the results obtained when SSM was used to evolve new substrate specificity and/or enantioselectivity in ThDP-dependent enzymes such as benzoylformate decarboxylase, transketolase, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase and the E1 component of the 2-oxoglutarate dehydrogenase complex.

  4. Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates

    NASA Technical Reports Server (NTRS)

    Mar, A.; Dworkin, J.; Oro, J.

    1987-01-01

    Using urea and cyanamide, the two condensing agents considered to have been present on the primitive earth, uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P), and glucose-6-phosphate (G6P) were synthesized under simulated prebiotic conditions. The reaction products were separated and identified using paper chromatography, thin layer chromatography, enzymatic analyses, and ion-pair reverse-phase high performance liquid chromatography. The possibility of nonenzymatic synthesis of metabolic intermediates on the primitive earth from simple precursors was thus demonstrated.

  5. Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates

    NASA Technical Reports Server (NTRS)

    Mar, A.; Dworkin, J.; Oro, J.

    1987-01-01

    Using urea and cyanamide, the two condensing agents considered to have been present on the primitive earth, uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P), and glucose-6-phosphate (G6P) were synthesized under simulated prebiotic conditions. The reaction products were separated and identified using paper chromatography, thin layer chromatography, enzymatic analyses, and ion-pair reverse-phase high performance liquid chromatography. The possibility of nonenzymatic synthesis of metabolic intermediates on the primitive earth from simple precursors was thus demonstrated.

  6. Biosynthesis of isoprenoids in Escherichia coli: stereochemistry of the reaction catalyzed by farnesyl diphosphate synthase.

    PubMed

    Leyes, A E; Baker, J A; Poulter, C D

    1999-10-07

    [formula: see text] Farnesyl diphosphate (FPP) synthase from Escherichia coli catalyzes the condensation of isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP) with selective removal of the pro-R hydrogen at C2 of IPP, the same stereochemistry observed for the pig liver, yeast, and avian enzymes.

  7. Enzymatic process optimization for the in vitro production of isoprene from mevalonate.

    PubMed

    Cheng, Tao; Liu, Hui; Zou, Huibin; Chen, Ningning; Shi, Mengxun; Xie, Congxia; Zhao, Guang; Xian, Mo

    2017-01-09

    As an important bulk chemical for synthetic rubber, isoprene can be biosynthesized by robust microbes. But rational engineering and optimization are often demanded to make the in vivo process feasible due to the complexities of cellular metabolism. Alternative synthetic biochemistry strategies are in fast development to produce isoprene or isoprenoids in vitro. This study set up an in vitro enzyme synthetic chemistry process using 5 enzymes in the lower mevalonate pathway to produce isoprene from mevalonate. We found the level and ratio of individual enzymes would significantly affect the efficiency of the whole system. The optimized process using 10 balanced enzyme unites (5.0 µM of MVK, PMK, MVD; 10.0 µM of IDI, 80.0 µM of ISPS) could produce 6323.5 µmol/L/h (430 mg/L/h) isoprene in a 2 ml in vitro system. In a scale up process (50 ml) only using 1 balanced enzyme unit (0.5 µM of MVK, PMK, MVD; 1.0 µM of IDI, 8.0 µM of ISPS), the system could produce 302 mg/L isoprene in 40 h, which showed higher production rate and longer reaction phase with comparison of the in vivo control. By optimizing the enzyme levels of lower MVA pathway, synthetic biochemistry methods could be set up for the enzymatic production of isoprene or isoprenoids from mevalonate.

  8. Improved Purification and Spectroscopic Properties of Squash Glutamate Decarboxylase.

    PubMed

    Matsumoto, T; Yamaura, I; Funatsu, M

    1996-01-01

    Squash glutamate decarboxylase was purified by DEAE-Cellulose batchwise followed by Blue-Sepharose, Cellulofine GCL-2000, and Toyopearl HW-55F column chromatography. The purified glutamate decarboxylase had a high specific activity (95.0 u/mg). The absorption spectrum of glutamate decarboxylase had an absorption maximum at 420 nm in the range 300-500 nm. A pH change from 5.3 to 7.8 was accompanied by a decrease in absorbancy at 420 nm. One mole of glutamate decarboxylase contained 3.8 and 1.3 mol of pyridoxal 5'-phosphate at pH 5.8 and pH 7.8, respectively.

  9. Peripheral neuropathy associated with antiglutamic acid decarboxylase antibodies.

    PubMed

    Saltık, Sema; Türkeş, Muzaffer; Tüzün, Erdem; Cakır, Arif; Ulusoy, Canan

    2013-05-01

    Autoantibodies to glutamic acid decarboxylase are found in some rare neurological diseases. However, acute peripheral neuropathy associated with antiglutamic acid decarboxylase autoimmunity has not been reported previously. Here we report a case of a patient who presented with acute cranial and peripheral neuropathy in association with the presence of serum antiglutamic acid decarboxylase antibodies. A 13-year-old boy was admitted to our pediatric neurology clinic with diplopia due to sixth cranial nerve palsy and ascending motor weakness in all extremities. The nerve conduction studies showed bilateral motor and sensory demyelinating neuropathy. Full recovery was achieved following intravenous immunoglobulin treatment. Glutamic acid decarboxylase autoimmunity-associated neurological diseases spectrum may also include acute demyelinating peripheral neuropathy. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Keto-isovalerate decarboxylase enzymes and methods of use thereof

    DOEpatents

    McElvain, Jessica; O'Keefe, Daniel P.; Paul, Brian James; Payne, Mark S.; Rothman, Steven Cary; He, Hongxian

    2016-01-19

    Provided herein are polypeptides and polynucleotides encoding such polypeptides which have ketoisovalerate decarboxylase activity. Also provided are recombinant host cells comprising such polypeptides and polynucleotides and methods of use thereof.

  11. Identification of Novel Benzoylformate Decarboxylases by Growth Selection▿†

    PubMed Central

    Henning, Helge; Leggewie, Christian; Pohl, Martina; Müller, Michael; Eggert, Thorsten; Jaeger, Karl-Erich

    2006-01-01

    A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the β-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde. PMID:17012586

  12. Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates

    PubMed Central

    López-Zavala, Alonso A.; Quintero-Reyes, Idania E.; Carrasco-Miranda, Jesús S.; Stojanoff, Vivian; Weichsel, Andrzej; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.

    2014-01-01

    Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012 ▶), J. Bioenerg. Biomembr. 44, 325–331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2′-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism. PMID:25195883

  13. Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates.

    PubMed

    López-Zavala, Alonso A; Quintero-Reyes, Idania E; Carrasco-Miranda, Jesús S; Stojanoff, Vivian; Weichsel, Andrzej; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R

    2014-09-01

    Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012), J. Bioenerg. Biomembr. 44, 325-331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2'-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism.

  14. Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate☆

    PubMed Central

    Karp, Frank; Zhao, Yuxin; Santhamma, Bindu; Assink, Bryce; Coates, Robert M.; Croteau, Rodney B.

    2007-01-01

    The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (±)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (−)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase. PMID:17949678

  15. GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway.

    PubMed

    Cárdenas, Pablo D; Sonawane, Prashant D; Pollier, Jacob; Vanden Bossche, Robin; Dewangan, Veena; Weithorn, Efrat; Tal, Lior; Meir, Sagit; Rogachev, Ilana; Malitsky, Sergey; Giri, Ashok P; Goossens, Alain; Burdman, Saul; Aharoni, Asaph

    2016-02-15

    Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops.

  16. GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway

    PubMed Central

    Cárdenas, Pablo D.; Sonawane, Prashant D.; Pollier, Jacob; Vanden Bossche, Robin; Dewangan, Veena; Weithorn, Efrat; Tal, Lior; Meir, Sagit; Rogachev, Ilana; Malitsky, Sergey; Giri, Ashok P.; Goossens, Alain; Burdman, Saul; Aharoni, Asaph

    2016-01-01

    Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops. PMID:26876023

  17. Mevalonate kinase genotype in children with recurrent fevers and high serum IgD level.

    PubMed

    Stabile, Achille; Compagnone, Adele; Napodano, Salvatore; Raffaele, Carmela Gerarda Luana; Patti, Maria; Rigante, Donato

    2013-12-01

    In selected cases, childhood's recurrent fevers of unknown origin can be referred to systemic autoinflammatory diseases as mevalonate kinase deficiency (MKD), caused by mutations in the mevalonate kinase gene (MVK), previously named "hyper-IgD syndrome" due to its characteristic increase in serum IgD level. There is no clear evidence for studying MVK genotype in these patients. From a cohort of 305 children evaluated for recurrent fevers in our outpatient clinic during the decade 2001-2011, we have retrospectively selected 10 unrelated Italian children displaying febrile episodes, associated with recurrent inflammatory signs (variably involving gastrointestinal tube, joints, lymph nodes, and skin) and persistently increased serum IgD levels. All these patients were examined for MVK genotype: only 2 presented bonafide MVK mutations, 5 showed the same S52N MVK polymorphism, while the remaining 3 had a wild-type MVK sequence. Clinical details of these patients have been reviewed through the critical analysis of their medical charts. Our report underscores the pitfalls of MKD diagnosis based on clinical grounds and IgD levels, emphasizing the uncertain contribution of MVK polymorphisms in the diagnostic assessment of the syndrome.

  18. Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway.

    PubMed

    Freed-Pastor, William A; Mizuno, Hideaki; Zhao, Xi; Langerød, Anita; Moon, Sung-Hwan; Rodriguez-Barrueco, Ruth; Barsotti, Anthony; Chicas, Agustin; Li, Wencheng; Polotskaia, Alla; Bissell, Mina J; Osborne, Timothy F; Tian, Bin; Lowe, Scott W; Silva, Jose M; Børresen-Dale, Anne-Lise; Levine, Arnold J; Bargonetti, Jill; Prives, Carol

    2012-01-20

    p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast epithelial cells form spheroids reminiscent of acinar structures found in vivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed sterol biosynthesis genes in human breast tumors. These findings implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.

  19. Effect of mevalonic acid on cholesterol synthesis in bovine intramuscular and subcutaneous adipocytes.

    PubMed

    Liu, Xiaomu; You, Wei; Cheng, Haijian; Zhang, Qingfeng; Song, Enliang; Wan, Fachun; Han, Hong; Liu, Guifen

    2016-02-01

    Mevalonic acid (MVA) is a key material in the synthesis of cholesterol; indeed, intracellular cholesterol synthesis is also called the mevalonic acid pathway. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is an essential enzyme in cholesterol biosynthesis. This study suggests that MVA may play an important role in the differentiation of bovine adipose tissue in vivo. We investigated differential mRNA expression in bovine intramuscular preadipocytes (BIPs) and bovine subcutaneous preadipocytes (BSPs) by culturing cells from the longissimus dorsi muscle and subcutaneous fat tissues of Luxi yellow cattle. The morphology of lipid accumulation of bovine preadipocytes was detected by Oil Red O staining, and total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), and high-density lipoprotein cholesterol (HDLC) levels were measured. Temporospatial expression of HMGR was investigated by real-time quantitative polymerase chain reaction (PCR). The TC, LDLC, and HDLC content did not significantly differ over time but increased slowly with increasing MVA concentration. HMGR expression increased over time and with increasing concentrations of MVA. MVA increased adipose cell proliferation in a dose-dependent and time-dependent manner. MVA stimulated HMGR expression in two cell types and its influence on adipocyte differentiation.

  20. A Porphodimethene Chemical Inhibitor of Uroporphyrinogen Decarboxylase

    PubMed Central

    Yip, Kenneth W.; Zhang, Zhan; Sakemura-Nakatsugawa, Noriko; Huang, Jui-Wen; Vu, Nhu Mai; Chiang, Yi-Kun; Lin, Chih-Lung; Kwan, Jennifer Y. Y.; Yue, Shijun; Jitkova, Yulia; To, Terence; Zahedi, Payam; Pai, Emil F.; Schimmer, Aaron D.; Lovell, Jonathan F.; Sessler, Jonathan L.; Liu, Fei-Fei

    2014-01-01

    Uroporphyrinogen decarboxylase (UROD) catalyzes the conversion of uroporphyrinogen to coproporphyrinogen during heme biosynthesis. This enzyme was recently identified as a potential anticancer target; its inhibition leads to an increase in reactive oxygen species, likely mediated by the Fenton reaction, thereby decreasing cancer cell viability and working in cooperation with radiation and/or cisplatin. Because there is no known chemical UROD inhibitor suitable for use in translational studies, we aimed to design, synthesize, and characterize such a compound. Initial in silico-based design and docking analyses identified a potential porphyrin analogue that was subsequently synthesized. This species, a porphodimethene (named PI-16), was found to inhibit UROD in an enzymatic assay (IC50 = 9.9 µM), but did not affect porphobilinogen deaminase (at 62.5 µM), thereby exhibiting specificity. In cellular assays, PI-16 reduced the viability of FaDu and ME-180 cancer cells with half maximal effective concentrations of 22.7 µM and 26.9 µM, respectively, and only minimally affected normal oral epithelial (NOE) cells. PI-16 also combined effectively with radiation and cisplatin, with potent synergy being observed in the case of cisplatin in FaDu cells (Chou-Talalay combination index <1). This work presents the first known synthetic UROD inhibitor, and sets the foundation for the design, synthesis, and characterization of higher affinity and more effective UROD inhibitors. PMID:24587102

  1. Properties of oxaloacetate decarboxylase from Veillonella parvula.

    PubMed Central

    Ng, S K; Wong, M; Hamilton, I R

    1982-01-01

    Oxaloacetate decarboxylase was purified to 136-fold from the oral anaerobe Veillonella parvula. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity of the enzyme was exhibited at pH 7.0 for both carboxylation and decarboxylation. At this pH, the Km values for oxaloacetate and Mg2+ were at 0.06 and 0.17 mM, respectively, whereas the Km values for pyruvate, CO2, and Mg2+ were 3.3, 1.74, and 1.85 mM, respectively. Hyperbolic kinetics were observed with all of the aforementioned compounds. The Keq' was 2.13 X 10(-3) mM-1 favoring the decarboxylation of oxaloacetate. In the carboxylation step, avidin, acetyl coenzyme A, biotin, and coenzyme A were not required. ADP and NADH had no effect on either the carboxylation or decarboxylation step, but ATP inhibited the carboxylation step competitively and the decarboxylation step noncompetitively. These types of inhibition fitted well with the overall lactate metabolism of the non-carbohydrate-fermenting anaerobe. PMID:7076619

  2. Localization of arginine decarboxylase in tobacco plants.

    PubMed

    Bortolotti, Cristina; Cordeiro, Alexandra; Alcázar, Rubén; Borrell, Antoni; Culiañez-Macià, Francisco A.; Tiburcio, Antonio F.; Altabella, Teresa

    2004-01-01

    The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.

  3. Mapping of glutamic acid decarboxylase (GAD) genes

    SciTech Connect

    Edelhoff, S.; Adler, D.A.; Disteche, C.M.; Grubin, C.E.; Karlsen, A.E.; Lernmark, A.; Foster, D. )

    1993-07-01

    Glutamic acid decarboxylase (GAD) catalyzes the synthesis of [gamma]-aminobutyric acid (GABA), which is known as a major inhibitory neurotransmitter in the central nervous system (CNS), but is also present outside the CNS. Recent studies showed that GAD is the major target of autoantibodies associated with the development of insulin-dependent diabetes mellitus and of the rare stiff man syndrome. Studies of GAD expression have demonstrated multiple transcripts, suggesting several isoforms of GAD. In this study, three different genes were mapped by in situ hybridization to both human and mouse chromosomes. The GAD1 gene was mapped to human chromosome 2q31 and to mouse chromosome 2D in a known region of conservation between human and mouse. GAD2, previously mapped to human chromosome 10p11.2-p12, was mapped to mouse chromosome 2A2-B, which identifies a new region of conservation between human and mouse chromosomes. A potential GAD3 transcript was mapped to human chromosome 22q13 and to mouse chromosome 15E in a known region of conservation between human and mouse. It is concluded that the GAD genes may form a family with as many as three related members. 30 refs., 5 figs.

  4. Phosphatidylserine decarboxylases, key enzymes of lipid metabolism.

    PubMed

    Schuiki, Irmgard; Daum, Günther

    2009-02-01

    Phosphatidylserine decarboxylases (PSDs) (E.C. 4.1.1.65) are enzymes which catalyze the formation of phosphatidylethanolamine (PtdEtn) by decarboxylation of phosphatidylserine (PtdSer). This enzymatic activity has been identified in both prokaryotic and eukaryotic organisms. PSDs occur as two types of proteins depending on their localization and the sequence of a conserved motif. Type I PSDs include enzymes of eukaryotic mitochondria and bacterial origin which contain the amino acid sequence LGST as a characteristic motif. Type II PSDs are found in the endomembrane system of eukaryotes and contain a typical GGST motif. These characteristic motifs are considered as autocatalytic cleavage sites where proenzymes are split into alpha- and beta-subunits. The S-residue set free by this cleavage serves as an attachment site of a pyruvoyl group which is required for the activity of the enzymes. Moreover, PSDs harbor characteristic binding sites for the substrate PtdSer. Substrate supply to eukaryotic PSDs requires lipid transport because PtdSer synthesis and decarboxylation are spatially separated. Targeting of PSDs to their proper locations requires additional intramolecular domains. Mitochondrially localized type I PSDs are directed to the inner mitochondrial membrane by N-terminal targeting sequences. Type II PSDs also contain sequences in their N-terminal extensions which might be required for subcellular targeting. Lack of PSDs causes various defects in different cell types. The physiological relevance of these findings and the central role of PSDs in lipid metabolism will be discussed in this review.

  5. Post-transcriptional regulation of ornithine decarboxylase

    PubMed Central

    Nowotarski, Shannon L.; Origanti, Sofia; Shantz, Lisa M.

    2013-01-01

    Activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC), and intracellular levels of ODC protein are controlled very tightly. Numerous studies have described ODC regulation at the levels of transcription, translation and protein degradation in normal cells, and dysregulation of these processes in response to oncogenic stimuli. Although post-transcriptional regulation of ODC has been well-documented, the RNA binding proteins (RBPs) that interact with ODC mRNA and control synthesis of the ODC protein have not been defined. Using Ras-transformed rat intestinal epithelial cells (Ras12V cells) as a model, we have begun identifying the RBPs that associate with the ODC transcript. Binding of RBPs could potentially regulate ODC synthesis by either changing mRNA stability or rate of mRNA translation. Techniques for measuring RBP binding and translation initiation are described here. Targeting control of ODC translation or mRNA decay could be a valuable method of limiting polyamine accumulation and subsequent tumor development in a variety of cancers. PMID:21318880

  6. Cysteinesulfinate decarboxylase: Characterization, inhibition, and metabolic role in taurine formation

    SciTech Connect

    Weinstein, C.L.

    1988-01-01

    Cysteinesulfinate decarboxylase, an enzyme that plays a major role in the formation of taurine from cysteine, has been purified from rat liver to homogeneity and characterized. The physical properties of the enzyme were studied, along with its substrate specificity. Multiple forms of the enzyme were found in rat liver, kidney, and brain with isoelectric points ranging from pH 5.6 to 4.9. These multiple forms did not differ in their substrate specificity. It was found by using gel electrofocusing and polyclonal antibodies raised to the liver enzyme that the different forms of cysteinesulfinate decarboxylase are identical in the various rat tissues studied. Various inhibitors of the enzyme were tested both in vitro and in vivo in order to evaluate the role of cysteinesulfinate decarboxylase in taurine formation in mammalian tissues. In in vitro studies, cysteinesulfinate decarboxylase was irreversibly inhibited by {beta}-ethylidene-DL-aspartate (Ki = 10 mM), and competitive inhibition was found using mercaptomethylsuccinate (Ki = 0.1 mM) and D-cysteinesulfinate (Ki = 0.32 mM) when L-cysteinesulfinate was used as a substrate. In order to be able to test these inhibitors in vivo, L-(1-{sup 14}C)cysteinesulfonate was evaluated as a probe for the in vivo measurement of cysteinesulfinate decarboxylase activity. The metabolism of cysteinesulfonate and the product of its transamination, {beta}-sulfopyruvate, was studied, and it was found that L-(1-{sup 14}C)cysteinesulfonate is an accurate and convenient probe for cysteinesulfinate decarboxylase activity. Using L-(1-{sup 14}C)cysteinesulfonate, it was found that D-cysteinesulfinate inhibits cysteinesulfinate decarboxylase activity by greater than 90% in the intact mouse and that inhibition lasts for up to fifteen hours.

  7. The role of residues glutamate-50 and phenylalanine-496 in Zymomonas mobilis pyruvate decarboxylase.

    PubMed Central

    Candy, J M; Koga, J; Nixon, P F; Duggleby, R G

    1996-01-01

    Several enzymes require thiamine diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification, homotetrameric subunit structure and simple kinetic properties. Crystallographic analyses of three ThDP-dependent enzymes [Müller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103] have suggested that an invariant glutamate participates in catalysis. In order to evaluate the role of this residue, identified in PDC from Zymomonas mobilis as Glu-50, it has been altered to glutamine and aspartate by site-directed mutagenesis of the cloned gene. The mutant proteins were expressed in Escherichia coli. Here we demonstrate that substitution with aspartate yields an enzyme with 3% of the activity of the wild-type, but with normal kinetics for pyruvate. Replacement of Glu-50 with glutamine yields an enzyme with only 0.5% of the catalytic activity of the wild-type enzyme. Each of these mutant enzymes has a decreased affinity for both ThDP and Mg2+. It has been reported that the binding of cofactors to apoPDC quenches the intrinsic tryptophan fluorescence [Diefenbach and Duggleby (1991) Biochem. J. 276, 439-445] and we have identified the residue responsible as Trp-487 [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98]. Although this residue is some distance from the cofactor binding site, it lies in the dimer interface, and the proposal has been put forward [Dyda, Furey, Swaminathan, Sax, Farrenkopf and Jordan (1993) Biochemistry 32, 6165-6170] that alteration of ring stacking with Phe-496 of the adjacent subunit is the mechanism of fluorescence quenching when cofactors bind. The closely related enzyme indolepyruvate decarboxylase (from Enterobacter cloacae) has a leucine residue at the position corresponding to Phe-496 but shows

  8. Coupling between catalysis and oligomeric structure in nucleoside diphosphate kinase.

    PubMed

    Mesnildrey, S; Agou, F; Karlsson, A; Bonne, D D; Véron, M

    1998-02-20

    A dimeric Dictyostelium nucleoside diphosphate kinase has been stabilized by the double mutation P100S-N150stop which targets residues involved in the trimer interface (Karlsson, A., Mesnildrey, S., Xu, Y., Moréra, S., Janin, J., and Veron, M. (1996) J. Biol. Chem. 271, 19928-19934). The reassociation of this dimeric form into a hexamer similar to the wild-type enzyme is induced by the presence of a nucleotide substrate. Equilibrium sedimentation and gel filtration experiments, as well as enzymatic activity measurements, show that reactivation of the enzyme closely parallels its reassociation. A phosphorylatable intermediate with low activity participates in the association pathway while the dimeric form is shown totally devoid of enzymatic activity. Our results support the hypothesis that different oligomeric species of nucleoside diphosphate kinase are involved in different cellular processes where the enzymatic activity is not required.

  9. Expression of the cytoplasmic mevalonate pathway in chloroplasts to reduce substrate limitations for cytoplasmically-produced terpenoid secondary products

    USDA-ARS?s Scientific Manuscript database

    All products of isoprenoid metabolism originate with the C5 non-allylic substrate, isopentenyl pyrophosphate (IPP). IPP is produced in plants by two distinct pathways, the mevalonate pathway (MEV) in the cytosol and the 2 C methyl-D-erythritol 4 phosphate (MEP) pathway in plastids. A multi-gene a...

  10. Mevalonate deprivation mediates the impact of lovastatin on the differentiation of murine 3T3-F442A preadipocytes.

    PubMed

    Elfakhani, Manal; Torabi, Sheida; Hussein, Deema; Mills, Nathaniel; Verbeck, Guido F; Mo, Huanbiao

    2014-03-01

    The statins competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity and consequently the synthesis of mevalonate. The use of statins is associated with insulin resistance, presumably due to the impaired differentiation and diminished glucose utilization of adipocytes. We hypothesize that mevalonate is essential to adipocyte differentiation and adipogenic gene expression. Adipo-Red assay and Oil Red O staining showed that an eight-day incubation with 0-2.5 µmol/L lovastatin dose-dependently reduced the intracellular triglyceride content of murine 3T3-F442A adipocytes. Concomitantly, lovastatin downregulated the expression of peroxisome proliferator-activated receptor γ (Pparγ), leptin (Lep), fatty acid binding protein 4 (Fabp4), and adiponectin (AdipoQ) as measured by quantitative real-time polymerase chain reaction (real-time qPCR). The expression of sterol regulatory element binding protein 1 (Srebp-1), a transcriptional regulator of Pparγ and Lep genes, was also suppressed by lovastatin. Western-blot showed that lovastatin reduced the level of CCAAT/enhancer binding protein α (C/EBPα) while inducing a compensatory over-expression of HMG CoA reductase. The impact of lovastatin on intracellular triglyceride content and expression of the adipogenic genes was reversed by supplemental mevalonate. Mevalonate-derived metabolites have essential roles in promoting adipogenic gene expression and adipocyte differentiation.

  11. The farnesyltransferase inhibitors tipifarnib and lonafarnib inhibit cytokines secretion in a cellular model of mevalonate kinase deficiency.

    PubMed

    Marcuzzi, Annalisa; De Leo, Luigina; Decorti, Giuliana; Crovella, Sergio; Tommasini, Alberto; Pontillo, Alessandra

    2011-07-01

    The shortage of geranylgeranyl-pyrophosphate (GGPP) was associated to an increased IL-1β release in the autoinflammatory syndrome mevalonate kinase deficiency (MKD), a rare inherited disease that has no specific therapy. Farnesyltransferase inhibitors (FTIs) act at the end of mevalonate pathway. Two FTIs, tipifarnib (Tip) and lonafarnib (Lon), were therefore evaluated as possible therapeutical choices for the treatment of MKD. FTIs could lead to a redirection of the limited available number of mevalonate intermediates preferentially to GGPP synthesis, eventually preventing the uncontrolled inflammatory response. The effect of Tip and Lon on intracellular cholesterol level (ICL) and on proinflammatory cytokines secretion was evaluated in a cellular model of MKD, chemically obtained treating RAW 264.7 cells with lovastatin (Lova) and alendronate (Ald). The combination of FTIs with the isoprenoid geraniol (GOH) was also tested both in this model and in monocytes isolated from MKD patients. Tip and Lon proved to revert the ICL lowering and to significantly reduce the lipopolysaccharide-induced cytokines secretion in Ald-Lova -RAW 264.7 cells. This anti-inflammatory effect was amplified combining the use of GOH with FTIs. The effect of GOH and Tip was successfully replicated in MKD patients' monocytes. Tip and Lon showed a dramatic anti-inflammatory effect in monocytes where mevalonate pathway was chemically or genetically impaired.

  12. Characterization of a second lysine decarboxylase isolated from Escherichia coli.

    PubMed Central

    Kikuchi, Y; Kojima, H; Tanaka, T; Takatsuka, Y; Kamio, Y

    1997-01-01

    We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli. PMID:9226257

  13. Ornithine Decarboxylase, Polyamines, and Pyrrolizidine Alkaloids in Senecio and Crotalaria

    PubMed Central

    Birecka, Helena; Birecki, Mieczyslaw; Cohen, Eric J.; Bitonti, Alan J.; McCann, Peter P.

    1988-01-01

    When tested for ornithine and arginine decarboxylases, pyrrolizidine alkaloid-bearing Senecio riddellii, S. longilobus (Compositae), and Crotalaria retusa (Leguminosae) plants exhibited only ornithine decarboxylase activity. This contrasts with previous studies of four species of pyrrolizidine alkaloid-bearing Heliotropium (Boraginaceae) in which arginine decarboxylase activity was very high relative to that of ornithine decarboxylase. Unlike Heliotropium angiospermum and Heliotropium indicum, in which endogenous arginine was the only detectable precursor of putrescine channeled into pyrrolizidines, in the species studied here—using difluoromethylornithine and difluoromethylarginine as the enzyme inhibitors—endogenous ornithine was the main if not the only precursor of putrescine converted into the alkaloid aminoalcohol moiety. In S. riddellii and C. retusa at flowering, ornithine decarboxylase activity was present mainly in leaves, especially the young ones. However, other very young organs such as inflorescence and growing roots exhibited much lower or very low activities; the enzyme activity in stems was negligible. There was no correlation between the enzyme activity and polyamine or alkaloid content in either species. In both species only free polyamines were detected except for C. retusa roots and inflorescence—with relatively very high levels of these compounds—in which conjugated putrescine, spermidine, and spermine were also found; agmatine was not identified by HPLC in any plant organ except for C. retusa roots with rhizobial nodules. Organ- or age-dependent differences in the polyamine levels were small or insignificant. The highest alkaloid contents were found in young leaves and inflorescence. PMID:16665870

  14. Effects of aliphatic diamines on rat liver ornithine decarboxylase activity.

    PubMed Central

    Pegg, A E; Conover, C; Wrona, A

    1978-01-01

    Rat liver ornithine decarboxylase activity was decreased by administration of putrescine (1,4-diaminobutane) or other diamines, including 1,3-diaminopropane, 1,5-diaminopentane and 1,6-diaminohexane. This effect was seen in control rats and in rats in which hepatic ornithine decarboxylase activity had been increased by administration of growth hormone (somatotropin) or thioacetamide. Loss of activity was not dependent on the conversion of putrescine into polyamines and was short-lived. Within 6h after intraperitoneal administration of 0.8 mmol/kg body wt., ornithine decarboxylase activity had returned to normal values. This return correlated with the rapid loss of the diamines from the liver, and the decrease in activity could be slightly prolonged by treatment with aminoguanidine, a diamine oxidase inhibitor. A decrease in ornithine decarboxylase activity by these diamines was accompanied by the accumulation in the liver of a nondiffusible inhibitor that decreased the activity of a purified ornithine decarboxylase preparation. The possibility that administration of non-physiological diamines that are not converted into polyamines might be useful for the inhibition of polyamine synthesis is discussed. PMID:646807

  15. Formation of a Novel Macrocyclic Alkaloid from the Unnatural Farnesyl Diphosphate Analogue Anilinogeranyl Diphosphate by 5-Epi-Aristolochene Synthase

    PubMed Central

    Rising, Kathleen A.; Crenshaw, Charisse M.; Koo, Hyun Jo; Subramanian, Thangaiah; Chehade, Kareem A. H.; Starks, Courtney; Allen, Keith D.; Andres, Douglas A.; Spielmann, H. Peter; Noel, Joseph P.; Chappell, Joe

    2015-01-01

    As part of an effort to identify substrate analogs suitable for helping to resolve structural features important for terpene synthases, the inhibition of 5-epi-aristolochene biosynthesis from farnesyl diphosphate (FPP) by the tobacco 5-epi-aristolochene synthase incubated with anilinogeranyl diphosphate (AGPP) was examined. The apparent noncompetitive nature of the inhibition supported further assessment of how AGPP might be bound to crystallographic forms of the enzyme. Surprisingly, the bound form of the inhibitor appeared to have undergone a cyclization event consistent with the native mechanism associated with FPP catalysis. Biocatalytic formation of a novel 13-membered macrocyclic paracyclophane alkaloid was confirmed by high-resolution GC-MS and NMR analysis. This work provides insights into new biosynthetic means for generating novel, functionally diversified, medium-sized terpene alkaloids. PMID:25897591

  16. Interplay of mevalonate and Hippo pathways regulates RHAMM transcription via YAP to modulate breast cancer cell motility

    PubMed Central

    Wang, Zhongyuan; Wu, Yanping; Wang, Haifeng; Zhang, Yangqing; Mei, Lin; Fang, Xuexun; Zhang, Xudong; Zhang, Fang; Chen, Hongbo; Liu, Ying; Jiang, Yuyang; Sun, Shengnan; Zheng, Yi; Li, Na; Huang, Laiqiang

    2014-01-01

    Expression of receptor for hyaluronan-mediated motility (RHAMM), a breast cancer susceptibility gene, is tightly controlled in normal tissues but elevated in many tumors, contributing to tumorigenesis and metastases. However, how the expression of RHAMM is regulated remains elusive. Statins, inhibitors of mevalonate metabolic pathway widely used for hypercholesterolemia, have been found to also have antitumor effects, but little is known of the specific targets and mechanisms. Moreover, Hippo signaling pathway plays crucial roles in organ size control and cancer development, yet its downstream transcriptional targets remain obscure. Here we show that RHAMM expression is regulated by mevalonate and Hippo pathways converging onto Yes-associated protein (YAP)/TEAD, which binds RHAMM promoter at specific sites and controls its transcription and consequently breast cancer cell migration and invasion (BCCMI); and that simvastatin inhibits BCCMI via targeting YAP-mediated RHAMM transcription. Required for ERK phosphorylation and BCCMI, YAP-activated RHAMM transcription is dependent on mevalonate and sensitive to simvastatin, which modulate RHAMM transcription by modulating YAP phosphorylation and nuclear-cytoplasmic localization. Further, modulation by mevalonate/simvastatin of YAP-activated RHAMM transcription requires geranylgeranylation, Rho GTPase activation, and actin cytoskeleton rearrangement, but is largely independent of MST and LATS kinase activity. These findings from in vitro and in vivo investigations link mevalonate and Hippo pathways with RHAMM as a downstream effector, a YAP-transcription and simvastatin-inhibition target, and a cancer metastasis mediator; uncover a mechanism regulating RHAMM expression and cancer metastases; and reveal a mode whereby simvastatin exerts anticancer effects; providing potential targets for cancer therapeutic agents. PMID:24367099

  17. Inhibition of mevalonate pathway prevents ischemia-induced cardiac dysfunction in rats via RhoA-independent signaling pathway.

    PubMed

    Yang, Ying; Rong, Xiqing; Lv, Xue; Jiang, Wenbing; Yang, Yuan; Lai, Dongwu; Xu, Shiming; Fu, Guosheng

    2017-10-01

    We previously demonstrated that anoxia-mediated Ca(2+) handling dysfunction could be ameliorated through inhibition of mevalonate pathway via RhoA- and Ras-related mechanisms in H9c2 cells. In this study, we further explored whether inhibition of mevalonate pathway is associated with cardiac remodeling and dysfunction in ischemic cardiomyopathy, and discussed the possible role of Ras, Rac and RhoA in cardiac dysfunction. We investigated the role of mevalonate pathway in cardiac remodeling and cardiomyocyte Ca(2+) handling proteins expression in a rat model of cardiac dysfunction due to myocardial infarction (MI). After MI, adult male Sprague-Dawley rats were treated with drugs that antagonize key components in mevalonate pathway, including 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, and Rho-kinase for 10 weeks. The protein expression of ryanodine receptor 2 (RyR2), sarcoplasmic reticulum Ca(2+) ATPase (SERCA) 2a, phospholamban (PLB), phospho-PLB at serine-16 (PSer16-PLB), FKBP12.6, and RhoA as well as RyR2 and FKBP12.6 mRNA levels was evaluated. Rosuvastatin and alendronate treatment prevented myocardial remodeling, improved cardiac function and reduced infarct size. Furthermore, rosuvastatin and alendronate promoted an increase in the protein expression of SERCA2a and PSer16-PLB/PLB ratio as well as partially restored the RyR2 and FKBP12.6 gene and protein expression. Fasudil failed to exert these beneficial effects. These findings indicate that mevalonate pathway inhibition by rosuvastatin and alendronate prevents cardiac remodeling and dysfunction possibly through RhoA-independent mechanisms. © 2017 John Wiley & Sons Ltd.

  18. Snapshot of a Reaction Intermediate: Analysis of Benzoylformate Decarboxylase in Complex with a Benzoylphosphonate Inhibitor

    SciTech Connect

    Brandt, Gabriel S.; Kneen, Malea M.; Chakraborty, Sumit; Baykal, Ahmet T.; Nemeria, Natalia; Yep, Alejandra; Ruby, David I.; Petsko, Gregory A.; Kenyon, George L.; McLeish, Michael J.; Jordan, Frank; Ringe, Dagmar

    2009-04-22

    Benzoylformate decarboxylase (BFDC) is a thiamin diphosphate- (ThDP-) dependent enzyme acting on aromatic substrates. In addition to its metabolic role in the mandelate pathway, BFDC shows broad substrate specificity coupled with tight stereo control in the carbon-carbon bond-forming reverse reaction, making it a useful biocatalyst for the production of chiral-hydroxy ketones. The reaction of methyl benzoylphosphonate (MBP), an analogue of the natural substrate benzoylformate, with BFDC results in the formation of a stable analogue (C2{alpha}-phosphonomandelyl-ThDP) of the covalent ThDP-substrate adduct C2{alpha}-mandelyl-ThDP. Formation of the stable adduct is confirmed both by formation of a circular dichroism band characteristic of the 1',4'-iminopyrimidine tautomeric form of ThDP (commonly observed when ThDP forms tetrahedral complexes with its substrates) and by high-resolution mass spectrometry of the reaction mixture. In addition, the structure of BFDC with the MBP inhibitor was solved by X-ray crystallography to a spatial resolution of 1.37 {angstrom} (PDB ID 3FSJ). The electron density clearly shows formation of a tetrahedral adduct between the C2 atom of ThDP and the carbonyl carbon atom of the MBP. This adduct resembles the intermediate from the penultimate step of the carboligation reaction between benzaldehyde and acetaldehyde. The combination of real-time kinetic information via stopped-flow circular dichroism with steady-state data from equilibrium circular dichroism measurements and X-ray crystallography reveals details of the first step of the reaction catalyzed by BFDC. The MBP-ThDP adduct on BFDC is compared to the recently solved structure of the same adduct on benzaldehyde lyase, another ThDP-dependent enzyme capable of catalyzing aldehyde condensation with high stereospecificity.

  19. Saturation mutagenesis of putative catalytic residues of benzoylformate decarboxylase provides a challenge to the accepted mechanism

    PubMed Central

    Yep, Alejandra; Kenyon, George L.; McLeish, Michael J.

    2008-01-01

    Benzoylformate decarboxylase from Pseudomonas putida (PpBFDC) is a thiamin diphosphate-dependent enzyme that carries out the nonoxidative decarboxylation of aromatic 2-keto acids. The x-ray structure of PpBFDC suggested that Ser-26, His-70, and His-281 would play important roles in its catalytic mechanism, and the S26A, H70A, and H281A variants all exhibited greatly impaired catalytic activity. Based on stopped-flow studies with the alanine mutants, it was proposed that the histidine residues acted as acid-base catalysts, whereas Ser-26 was involved in substrate binding and played a significant, albeit less well defined, role in catalysis. While developing a saturation mutagenesis protocol to examine residues involved in PpBFDC substrate specificity, we tested the procedure on His-281. To our surprise, we found that His-281, which is thought to be necessary for protonation of the carbanion/enamine intermediate, could be replaced by phenyl alanine with only a 5-fold decrease in kcat. Even more surprising were our subsequent observations (i) that His-70 could be replaced by threonine or leucine with approximately a 30-fold decrease in kcat/Km compared with a 4,000-fold decrease for the H70A variant and (ii) that Ser-26, which forms hydrogen bonds with the substrate carboxylate, could be replaced by threonine, leucine, or methionine without significant loss of activity. These results call into question the assigned roles for Ser-26, His-70, and His-281. Further, they demonstrate the danger in assigning catalytic function based solely on results with alanine mutants and show that saturation mutagenesis is a valuable tool in assessing the role and relative importance of putative catalytic residues. PMID:18398009

  20. Roles of the Mevalonate Pathway and Cholesterol Trafficking in Pulmonary Host Defense.

    PubMed

    Gabor, Kristin A; Fessler, Michael B

    2017-01-01

    The mevalonic acid synthesis pathway, cholesterol, and lipoproteins play fundamental roles in lung physiology and the innate immune response. Recent literature investigating roles for cholesterol synthesis and trafficking in host defense against respiratory infection was critically reviewed. The innate immune response and the cholesterol biosynthesis/trafficking network regulate one another, with important implications for pathogen invasion and host defense in the lung. The activation of pathogen recognition receptors and downstream cellular host defense functions are critically sensitive to cellular cholesterol. Conversely, microorganisms can co-opt the sterol/lipoprotein network in order to facilitate replication and evade immunity. Emerging literature suggests the potential for harnessing these insights towards therapeutic development. Given that >50% of adults in the U.S. have serum cholesterol abnormalities and pneumonia remains a leading cause of death, the potential impact of cholesterol on pulmonary host defense is of tremendous public health significance and warrants further mechanistic and translational investigation.

  1. Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L. (Dunal).

    PubMed

    Chaurasiya, Narayan D; Sangwan, Neelam S; Sabir, Farzana; Misra, Laxminarain; Sangwan, Rajender S

    2012-10-01

    Withanolides are pharmaceutically important C(28)-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of (13)C label from (13)C(1)-D-glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of (13)C abundance at each carbon of withaferin A from (13)C(1)-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific (13)C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids. Key message The study elucidates significant contribution of DOXP pathway to withanolide biosynthesis. A new connotation of biosynthetic load-based role of DOXP/MVA recruitment in isoprenoid biosynthesis has been proposed.

  2. The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway

    PubMed Central

    Rauthan, Manish; Ranji, Parmida; Aguilera Pradenas, Nataly; Pitot, Christophe; Pilon, Marc

    2013-01-01

    Statins are cholesterol-lowering drugs that inhibit 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the synthesis of cholesterol via the mevalonate pathway. This pathway also produces coenzyme Q (a component of the respiratory chain), dolichols (important for protein glycosylation), and isoprenoids (lipid moieties responsible for the membrane association of small GTPases). We previously showed that the nematode Caenorhabditis elegans is useful to study the noncholesterol effects of statins because its mevalonate pathway lacks the sterol synthesis branch but retains all other branches. Here, from a screen of 150,000 mutagenized genomes, we isolated four C. elegans mutants resistant to statins by virtue of gain-of-function mutations within the first six amino acids of the protein ATFS-1, the key regulator of the mitochondrial unfolded protein response that includes activation of the chaperones HSP-6 and HSP-60. The atfs-1 gain-of-function mutants are also resistant to ibandronate, an inhibitor of an enzyme downstream of HMG-CoA reductase, and to gliotoxin, an inhibitor acting on a subbranch of the pathway important for protein prenylation, and showed improved mitochondrial function and protein prenylation in the presence of statins. Additionally, preinduction of the mitochondrial unfolded protein response in wild-type worms using ethidium bromide or paraquat triggered statin resistance, and similar observations were made in Schizosaccharomyces pombe and in a mammalian cell line. We conclude that statin resistance through maintenance of mitochondrial homeostasis is conserved across species, and that the cell-lethal effects of statins are caused primarily through impaired protein prenylation that results in mitochondria dysfunction. PMID:23530189

  3. Pyruvate decarboxylases from the petite-negative yeast Saccharomyces kluyveri.

    PubMed

    Møller, K; Langkjaer, R B; Nielsen, J; Piskur, J; Olsson, L

    2004-01-01

    Saccharomyces kluyveri is a petite-negative yeast, which is less prone to form ethanol under aerobic conditions than is S. cerevisiae. The first reaction on the route from pyruvate to ethanol is catalysed by pyruvate decarboxylase, and the differences observed between S. kluyveri and S. cerevisiae with respect to ethanol formation under aerobic conditions could be caused by differences in the regulation of this enzyme activity. We have identified and cloned three genes encoding functional pyruvate decarboxylase enzymes (PDCgenes) from the type strain of S. kluyveri (Sk- PDC11, Sk- PDC12 and Sk- PDC13). The regulation of pyruvate decarboxylase in S. kluyveri was studied by measuring the total level of Sk- PDC mRNA and the overall enzyme activity under various growth conditions. It was found that the level of Sk- PDC mRNA was enhanced by glucose and oxygen limitation, and that the level of enzyme activity was controlled by variations in the amount of mRNA. The mRNA level and the pyruvate decarboxylase activity responded to anaerobiosis and growth on different carbon sources in essentially the same fashion as in S. cerevisiae. This indicates that the difference in ethanol formation between these two yeasts is not due to differences in the regulation of pyruvate decarboxylase(s), but rather to differences in the regulation of the TCA cycle and the respiratory machinery. However, the PDC genes of Saccharomyces/ Kluyveromyces yeasts differ in their genetic organization and phylogenetic origin. While S. cerevisiae and S. kluyveri each have three PDC genes, these have apparently arisen by independent duplications and specializations in each of the two yeast lineages.

  4. Unexpected reactivity of 2-fluorolinalyl diphosphate in the active site of crystalline 2-methylisoborneol synthase

    PubMed Central

    Köksal, Mustafa; Chou, Wayne K. W.; Cane, David E.; Christianson, David W.

    2013-01-01

    The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in its unliganded state and in complex with 2 Mg2+ ions and cis-2-fluorogeranyl diphosphate at 1.85 Å and 2.00 Å resolution, respectively. Under normal circumstances, MIBS catalyzes the cyclization of the naturally-occurring, non-canonical 11-carbon isoprenoid substrate, 2-methylgeranyl diphosphate, which first undergoes an ionization-isomerization-ionization sequence through the tertiary diphosphate intermediate 2-methyllinalyl diphosphate to enable subsequent cyclization chemistry. MIBS does not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and we recently reported the crystal structure of MIBS complexed with this unreactive substrate analogue [Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D. W. (2012) Biochemistry 51, 3011–3020]. However, cocrystallization of MIBS with the fluorinated analogue of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl diphosphate, reveals unexpected reactivity for the intermediate analogue and yields the crystal structure of the complex with the primary allylic diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure of the unliganded enzyme reveals that the crystalline enzyme active site remains partially open, presumably due to the binding of only 2 Mg2+ ions. Assays in solution indicate that MIBS catalyzes the generation of (1R)-(+)-camphor from the substrate 2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization mechanism leading to 2-substituted isoborneol products; however, the initially generated 2-fluoroisoborneol cyclization product is unstable and undergoes elimination of hydrogen fluoride to yield (1R)-(+)-camphor. PMID:23844678

  5. Comparison between activation of ornithine decarboxylase and histidine decarboxylase in rat stomach.

    PubMed

    Ding, X Q; Chen, D; Rosengren, E; Persson, L; Hakanson, R

    1996-03-01

    We compared the responses of rat stomach ornithine decarboxylase (ODC) and histidine decarboxylase (HDC) to food intake, oral treatment with antisecretagogues, NaHCO3, and hypertonic NaCl, antrectomy, intravenous infusion of gastrin-17, the selective cholecystokinin (CCK)-B/gastrin receptor antagonist L-365,260, and the somatostatin analogue RC-160. The serum gastrin concentration and oxyntic mucosal ODC and HDC activities were higher in freely fed rats than in fasted rats. Food intake in fasted rats raised the serum gastrin concentration and the ODC and HDC activities. Ranitidine, omeprazole, and NaHCO3 raised the serum gastrin concentration and activated ODC and HDC. Hypertonic NaCl raised the ODC activity 200-fold, whereas circulating gastrin and HDC activity were increased only moderately. Infusion of gastrin-17 activated HDC but not ODC. L-365,260 prevented the activation of HDC but not of ODC in response to food intake and treatment with omeprazole, NaHCO3, or hypertonic NaCl. Antrectomy prevented the food- and omeprazole-evoked rise in oxyntic mucosal HDC activity but not the rise in ODC activity. RC-160 suppressed HDC activity after food intake and treatment with omeprazole, NaHCO3, or NaCl. In contrast, RC-160 suppressed omeprazole- and NaHCO3-evoked ODC activation but not that evoked by food intake or NaCl. The results support the view that HDC in the oxyntic mucosa is activated by gastrin and suppressed by somatostatin. The induction of ODC is not mediated by gastrin; ODC activation appears to be related to acid inhibition per se or to mucosal maintenance and repair; somatostatin, or rather the lack of it, might contribute to the induction of ODC after acid blockade. The mechanism behind the activation of rat stomach ODC seems to differ depending on the type of stimulus.

  6. Nucleotide sequence of the pyruvate decarboxylase gene from Zymomonas mobilis.

    PubMed

    Neale, A D; Scopes, R K; Wettenhall, R E; Hoogenraad, N J

    1987-02-25

    Pyruvate decarboxylase (EC 4.1.1.1), the penultimate enzyme in the alcoholic fermentation pathway of Zymomonas mobilis, converts pyruvate to acetaldehyde and carbon dioxide. The complete nucleotide sequence of the structural gene encoding pyruvate decarboxylase from Zymomonas mobilis has been determined. The coding region is 1704 nucleotides long and encodes a polypeptide of 567 amino acids with a calculated subunit mass of 60,790 daltons. The amino acid sequence was confirmed by comparison with the amino acid sequence of a selection of tryptic fragments of the enzyme. The amino acid composition obtained from the nucleotide sequence is in good agreement with that obtained experimentally.

  7. Catecholamine toxicity in aromatic L-amino acid decarboxylase deficiency.

    PubMed

    Anselm, Irina A; Darras, Basil T

    2006-08-01

    This report presents the case of an adult male with aromatic L-amino acid decarboxylase deficiency who developed serious cardiac rhythm disturbances during treatment with intravenous dopamine and norepinephrine for severe hypotension. Three weeks later, he spontaneously developed atrial fibrillation while not receiving exogenous catecholamines. He died suddenly after several months. We presume cardiac arrhythmia was the most likely cause of his death. Patients with aromatic L-amino acid decarboxylase deficiency may be prone to cardiac arrhythmias at rest and also may be exceptionally sensitive to exogenous catecholamines. Therefore, close cardiac monitoring is indicated at baseline and during treatment with pressors.

  8. Domain relationships in thiamine diphosphate-dependent enzymes.

    PubMed

    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.

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

    PubMed

    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.

  10. Kinetic studies of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase reaction using 3-desmethyl allylic substrate analogs.

    PubMed

    Fujikura, Keitaro; Maki, Yuji; Ohya, Norimasa; Satoh, Mikiya; Koyama, Tanetoshi

    2008-03-01

    In order to investigate the substrate binding feature of undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26 with respect to farnesyl diphosphate and a reaction intermediate, (Z,E,E)-geranylgeranyl diphosphate, we examined the reactivity of artificial substrate analogs, 3-desmethyl farnesyl diphosphate and 3-desmethyl Z-geranylgeranyl diphosphate, which lack the methyl group at the 3-position of farnesyl diphosphate and Z-geranylgeranyl diphosphate, respectively. Undecaprenyl diphosphate synthase did not accept either of the 3-desmethyl analogs as the allylic substrate, indicating that the methyl group at the 3-position of the allylic substrate is important in the undecaprenyl diphosphate synthase reaction. These analogs showed different inhibition patterns in the cis-prenyl chain elongation reaction with respect to the reactions of farnesyl diphosphate and Z-geranylgeranyl diphosphate as allylic substrate. These results suggest that the binding site for the natural substrate farnesyl diphosphate and those for the intermediate allylic diphosphate, which contains the cis-prenyl unit, are different during the cis-prenyl chain elongation reaction.

  11. Mevalonic acid is partially synthesized from amino acids in Halobacterium cutirubrum: a /sup 13/C nuclear magnetic resonance study

    SciTech Connect

    Ekiel, I.; Sprott, G.D.; Smith, I.C.P.

    1986-05-01

    /sup 13/C nuclear magnetic resonance revealed an unusual pathway for the biosynthesis of lipids in Halobacterium cutirubrum and H. halobium. Mevalonic acid was not synthesized from three acetyl-coenzyme A molecules, as has been suggested previously, and the branch-methyl and methine carbons in phytanyl chains were derived from neither acetate nor glycerol. Instead, they were supplied by the degradation of amino acids, in particular of lysine. Presumably, two different types of two-carbon fragments were used simultaneously by halobacteria for the biosynthesis of mevalonate. The labeling pattern of squalene supported the above conclusions. Based on these data, a general scheme is proposed to account for the contribution of lysine-to-lipid biosynthesis.

  12. Ovarian tumour growth is characterized by mevalonate pathway gene signature in an orthotopic, syngeneic model of epithelial ovarian cancer

    PubMed Central

    Greenaway, James B.; Virtanen, Carl; Osz, Kata; Revay, Tamas; Hardy, Daniel; Shepherd, Trevor; DiMattia, Gabriel; Petrik, Jim

    2016-01-01

    Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer and often is not detected until late stages when cancer cells transcoelomically metastasize to the abdomen and typically become resistant to therapy resulting in very low survival rates. We utilize an orthotopic, syngeneic mouse model to study late stage disease and have discovered that the tumor cells within the abdominal ascites are irreversibly re-programmed, with an increased tumorigenicity and resistance to apoptosis. The goal of this study was to characterize the reprogramming that occurred in the aggressive ascites-derived cells (28-2 cells) compared to the original cell line used for tumor induction (ID8 cells). Microarray experiments showed that the majority of genes upregulated in the 28-2 cells belonged to the mevalonate pathway, which is involved in cholesterol biosynthesis, protein prenylation, and activation of small GTPases. Upregulation of mevalonate appeared to be associated with the acquisition of a p53 mutation in the ascites-derived cells. Treatment with simvastatin to inhibit HMG CoA reductase, the rate limiting enzyme of this pathway, induced apoptosis in the 28-2 cell line. Rescue experiments revealed that mevalonate, but not cholesterol, could inhibit the simvastatin-mediated effects. In vivo, daily intraperitoneal simvastatin treatment significantly regressed advanced stage disease and induced death of metastatic tumor cells. These data suggest that ovarian cancer cells become reprogrammed, with genetic mutations, and upregulation of the mevalonate pathway, which facilitates the development of advanced stage disease. The use of statins to inhibit HMGCR may provide novel therapeutic opportunities for the treatment of advanced stage EOC. PMID:27329838

  13. Improving nucleoside diphosphate kinase for antiviral nucleotide analogs activation.

    PubMed

    Gallois-Montbrun, Sarah; Schneider, Benoit; Chen, Yuxing; Giacomoni-Fernandes, Veronique; Mulard, Laurence; Morera, Solange; Janin, Joel; Deville-Bonne, Dominique; Veron, Michel

    2002-10-18

    Antiviral nucleoside analog therapies rely on their incorporation by viral DNA polymerases/reverse transcriptase leading to chain termination. The analogs (3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T), and other dideoxynucleosides) are sequentially converted into triphosphate by cellular kinases of the nucleoside salvage pathway and are often poor substrates of these enzymes. Nucleoside diphosphate (NDP) kinase phosphorylates the diphosphate derivatives of the analogs with an efficiency some 10(4) lower than for its natural substrates. Kinetic and structural studies of Dictyostelium and human NDP kinases show that the sugar 3'-OH, absent from all antiviral analogs, is required for catalysis. To improve the catalytic efficiency of NDP kinase on the analogs, we engineered several mutants with a protein OH group replacing the sugar 3'-OH. The substitution of Asn-115 in Ser and Leu-55 in His results in an NDP kinase mutant with an enhanced ability to phosphorylate antiviral derivatives. Transfection of the mutant enzyme in Escherichia coli results in an increased sensitivity to AZT. An x-ray structure at 2.15-A resolution of the Dictyostelium enzyme bearing the serine substitution in complex with the R(p)-alpha-borano-triphosphate derivative of AZT shows that the enhanced activity reflects an improved geometry of binding and a favorable interaction of the 3'-azido group with the engineered serine.

  14. Cloning, Expression, and Characterization of cis-Polyprenyl Diphosphate Synthase from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius

    PubMed Central

    Hemmi, Hisashi; Yamashita, Satoshi; Shimoyama, Takefumi; Nakayama, Toru; Nishino, Tokuzo

    2001-01-01

    cis-polyprenyl diphosphate synthases are involved in the biosynthesis of the glycosyl carrier lipid in most organisms. However, only little is known about this enzyme of archaea. In this report, we isolated the gene of cis-polyprenyl diphosphate synthase from a thermoacidophilic archaeon, Sulfolobus acidocaldarius, and characterized the recombinant enzyme. PMID:11114943

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

    PubMed

    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.

  16. [Thiamine diphosphate pool and its biosynthesis in the liver in galactosamine hepatitis].

    PubMed

    Trebukhina, R V; Moiseenok, A G; Petushok, V G; Tumanov, V N; Gorenshteĭn, B I; Sheĭbak, V M; Omel'ianchik, S N

    1989-01-01

    The hepatitis-like changes were induced in the liver of albino female rats weighing 120-150 g and fed on the appropriate vivarium diet by single parenteral administration of hydrochloride galactosamine in a dose of 0.9 or 1.8 mmol per 1 kg of body weight. The thiamine diphosphate level in the cytosol fraction of the liver decreased 24 h after the preparation administration, the same in blood but with the higher dose used. The activity of pyruvate dehydrogenase, a thiamine diphosphate dependent enzyme, decreased similarly. The cytosol transketolase activity lowered by 38-39%. The coenzyme biosynthesis disturbance due to a fall by 49-58% in the thiamine pyrophosphatase activity is considered to be responsible for hydrochloride galactosamine-induced decrease in the thiamine diphosphate pool. Specificity of the thiamine diphosphate pool disturbance and discoordination of thiamine diphosphate dependent enzymes in the liver are observed under administration of hydrochloride galactosamine.

  17. Fluorescent Farnesyl Diphosphate Analogue: A Probe To Validate trans-Prenyltransferase Inhibitors.

    PubMed

    Teng, Kuo-Hsun; Hsu, Erh-Ting; Chang, Ying-Hsuan; Lin, Sheng-Wei; Liang, Po-Huang

    2016-08-09

    Some trans-prenyltransferases, such as long-chain C40 octaprenyl diphosphate synthase (OPPS), short-chain C15 farnesyl diphosphate synthase (FPPS), and C20 geranylgeranyl diphosphate synthase (GGPPS), are important drug targets. These enzymes catalyze chain elongation of FPP or geranyl diphosphate (GPP) through condensation reactions with isopentenyl diphosphate (IPP), forming designated numbers of trans-double bonds in the final products. To facilitate drug discovery, we report here a sensitive and reliable fluorescence-based assay for monitoring their activities in real time. MANT-O-GPP, a fluorescent analogue of FPP, was used as an alternative substrate and converted by the wild-type OPPS and the engineered FPPS and GGPPS into sufficiently long products with enhanced fluorescence intensities. This fluorescence probe was used to reveal the inhibitory mechanism of zoledronate, a bisphosphonate drug that targets human FPPS and possibly GGPPS.

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

    SciTech Connect

    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.

  19. Synthesis of the coenzymes adenosine diphosphate glucose, guanosine diphosphate glucose, and cytidine diphosphoethanolamine under primitive Earth conditions

    NASA Technical Reports Server (NTRS)

    Mar, A.; Oro, J.

    1991-01-01

    The nonenzymatic synthesis of the coenzymes adenosine diphosphate glucose (ADPG), guanosine diphosphate glucose (GDPG), and cytidine diphosphoethanolamine (CDP-ethanolamine) has been carried out under conditions considered to have been prevalent on the early Earth. The production of these compounds was performed by allowing simple precursor molecules to react under aqueous solutions, at moderate temperatures and short periods of time, with mediation by cyanamide or urea. These two condensing agents are considered to have been present in significant amounts on the primitive Earth and have been previously used in the nonenzymatic synthesis of several other important biochemical compounds. In our experiments, ADPG was obtained by heating glucose-1-phosphate (G1P) and ATP in the presence of cyanamide for 24 h at 70 degrees C. The reaction of G1P and GTP under the same conditions yielded GDPG. The cyanamide-mediated production of CDP-ethanolamine was carried out by reacting a mixture of ethanolamine phosphate and CTP for 24 h at 70 degrees C. The separation and identification of the reaction products was carried out by paper chromatography, thin-layer chromatography, high performance thin-layer chromatography, high performance liquid chromatography, both normal and reverse-phase, UV spectroscopy, enzymatic assays, and acid hydrolysis. Due to the mild conditions employed, and to the relative ease of these reactions, these studies offer a simple attractive system for the nonenzymatic synthesis of phosphorylated high-energy metabolic intermediates under conditions considered to have been prevalent on the ancient Earth.

  20. Synthesis of the coenzymes adenosine diphosphate glucose, guanosine diphosphate glucose, and cytidine diphosphoethanolamine under primitive Earth conditions

    NASA Technical Reports Server (NTRS)

    Mar, A.; Oro, J.

    1991-01-01

    The nonenzymatic synthesis of the coenzymes adenosine diphosphate glucose (ADPG), guanosine diphosphate glucose (GDPG), and cytidine diphosphoethanolamine (CDP-ethanolamine) has been carried out under conditions considered to have been prevalent on the early Earth. The production of these compounds was performed by allowing simple precursor molecules to react under aqueous solutions, at moderate temperatures and short periods of time, with mediation by cyanamide or urea. These two condensing agents are considered to have been present in significant amounts on the primitive Earth and have been previously used in the nonenzymatic synthesis of several other important biochemical compounds. In our experiments, ADPG was obtained by heating glucose-1-phosphate (G1P) and ATP in the presence of cyanamide for 24 h at 70 degrees C. The reaction of G1P and GTP under the same conditions yielded GDPG. The cyanamide-mediated production of CDP-ethanolamine was carried out by reacting a mixture of ethanolamine phosphate and CTP for 24 h at 70 degrees C. The separation and identification of the reaction products was carried out by paper chromatography, thin-layer chromatography, high performance thin-layer chromatography, high performance liquid chromatography, both normal and reverse-phase, UV spectroscopy, enzymatic assays, and acid hydrolysis. Due to the mild conditions employed, and to the relative ease of these reactions, these studies offer a simple attractive system for the nonenzymatic synthesis of phosphorylated high-energy metabolic intermediates under conditions considered to have been prevalent on the ancient Earth.

  1. Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures.

    PubMed Central

    Clastre, M.; Bantignies, B.; Feron, G.; Soler, E.; Ambid, C.

    1993-01-01

    A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate, was isolated from Vitis vinifera L. cv Muscat de Frontignan cell cultures. Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels. The enzyme formed only geranyl diphosphate as a product. In all cases, neither neryl diphosphate, the cis isomer, nor farnesyl diphosphate was detected. The enzyme showed a native molecular mass of 68 [plus or minus] 5 kD as determined by gel permeation. On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 [plus or minus] 2 kD. Michaelis constants for isopentenyl diphosphate and dimethylallyl diphosphate were 8.5 and 56.8 [mu]M, respectively. The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100. Inorganic pyrophosphate, aminophenylethyl diphosphate, and geranyl diphosphate had inhibitory effects on the enzyme. PMID:12231811

  2. [In vitro study over statins effects on cellular growth curves and its reversibility with mevalonate].

    PubMed

    Millan Núñez-Cortés, Jesús; Alvarez Rodriguez, Ysmael; Alvarez Novés, Granada; Recarte Garcia-Andrade, Carlos; Alvarez-Sala Walther, Luis

    2014-01-01

    HMG-CoA-Reductase inhibitors, also known as statins, are currently the most powerful cholesterol-lowering drugs available on the market. Clinical trials and experimental evidence suggest that statins have heavy anti-atherosclerotic effects. These are in part consequence of lipid lowering but also result from pleiotropic actions of the drugs. These so-called pleiotropic properties affect various aspects of cell function, inflammation, coagulation, and vasomotor activity. These effects are mediated either indirectly through LDL-c reduction or via a direct effect on cellular functions. Although many of the pleiotropic properties of statins may be a class effect, some may be unique to certain agents and account for differences in their pharmacological activity. So, although statins typically have similar effects on LDL-c levels, differences in chemical structure and pharmacokinetic profile can lead to variations in pleiotropic effects. In this paper we analize the in vitro effects of different statins over different cell lines from cells implicated in atherosclerotic process: endothelial cells, fibroblasts, and vascular muscular cells. In relation with our results we can proof that the effects of different dosis of different statins provides singular effects over growth curves of different cellular lines, a despite of a class-dependent effects. So, pleiotropic effects and its reversibility with mevalonate are different according with the molecule and the dosis. Copyright © 2013 Elsevier España, S.L. y SEA. All rights reserved.

  3. (13)C-metabolic flux analysis for mevalonate-producing strain of Escherichia coli.

    PubMed

    Wada, Keisuke; Toya, Yoshihiro; Banno, Satomi; Yoshikawa, Katsunori; Matsuda, Fumio; Shimizu, Hiroshi

    2017-02-01

    Mevalonate (MVA) is used to produce various useful products such as drugs, cosmetics and food additives. An MVA-producing strain of Escherichia coli (engineered) was constructed by introducing mvaES genes from Enterococcus faecalis. The engineered strain produced 1.84 mmol/gDCW/h yielding 22% (C-mol/C-mol) of MVA from glucose in the aerobic exponential growth phase. The mass balance analysis revealed that the MVA yield of the engineered strain was close to the upper limit at the biomass yield. Since MVA is synthesized from acetyl-CoA using NADPH as a cofactor, the production of MVA affects central metabolism in terms of carbon utilization and NADPH requirements. The reason for this highly efficient MVA production was investigated based on (13)C-metabolic flux analysis. The estimated flux distributions revealed that the fluxes of acetate formation and the TCA cycle in the engineered strain were lower than those in the control strain. Although the oxidative pentose phosphate pathway is considered as the NADPH generating pathway in E. coli, no difference of the flux was observed between the control and engineered strains. The production/consumption balance of NADPH suggested that additional requirement of NADPH for MVA synthesis was obtained from the transhydrogenase reaction in the engineered strain. Comparison between the measured flux distribution and the ideal values for MVA production proposes a strategy for further engineering to improve the MVA production in E. coli.

  4. A role for the mevalonate pathway in early plant symbiotic signaling

    PubMed Central

    Venkateshwaran, Muthusubramanian; Jayaraman, Dhileepkumar; Chabaud, Mireille; Genre, Andrea; Balloon, Allison J.; Maeda, Junko; Forshey, Kari; den Os, Désirée; Kwiecien, Nicholas W.; Coon, Joshua J.; Barker, David G.; Ané, Jean-Michel

    2015-01-01

    Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca2+ concentration (Ca2+ spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca2+ spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume–rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca2+ spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca2+ spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca2+ spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca2+ spiking in this heterologous system. PMID:26199419

  5. Arginine decarboxylase as the source of putrescine for tobacco alkaloids

    NASA Technical Reports Server (NTRS)

    Tiburcio, A. F.; Galston, A. W.

    1986-01-01

    The putrescine which forms a part of nicotine and other pyrrolidine alkaloids is generally assumed to arise through the action of ornithine decarboxylase (ODC). However, we have previously noted that changes in the activity of arginine decarboxylase (ADC), an alternate source of putrescine, parallel changes in tissue alkaloids, while changes in ODC activity do not. This led us to undertake experiments to permit discrimination between ADC and ODC as enzymatic sources of putrescine destined for alkaloids. Two kinds of evidence presented here support a major role for ADC in the generation of putrescine going into alkaloids: (a) A specific 'suicide inhibitor' of ADC effectively inhibits the biosynthesis of nicotine and nornicotine in tobacco callus, while the analogous inhibitor of ODC is less effective, and (b) the flow of 14C from uniformly labelled arginine into nicotine is much more efficient than that from ornithine.

  6. Lotus hairy roots expressing inducible arginine decarboxylase activity.

    PubMed

    Chiesa, María A; Ruiz, Oscar A; Sánchez, Diego H

    2004-05-01

    Biotechnological uses of plant cell-tissue culture usually rely on constitutive transgene expression. However, such expression of transgenes may not always be desirable. In those cases, the use of an inducible promoter could be an alternative approach. To test this hypothesis, we developed two binary vectors harboring a stress-inducible promoter from Arabidopsis thaliana, driving the beta-glucuronidase reporter gene and the oat arginine decarboxylase. Transgenic hairy roots of Lotus corniculatus were obtained with osmotic- and cold-inducible beta-glucuronidase and arginine decarboxylase activities. The increase in the activity of the latter was accompanied by a significant rise in total free polyamines level. Through an organogenesis process, we obtained L. corniculatus transgenic plants avoiding deleterious phenotypes frequently associated with the constitutive over-expression of arginine decarboxylation and putrescine accumulation.

  7. Arginine decarboxylase as the source of putrescine for tobacco alkaloids

    NASA Technical Reports Server (NTRS)

    Tiburcio, A. F.; Galston, A. W.

    1986-01-01

    The putrescine which forms a part of nicotine and other pyrrolidine alkaloids is generally assumed to arise through the action of ornithine decarboxylase (ODC). However, we have previously noted that changes in the activity of arginine decarboxylase (ADC), an alternate source of putrescine, parallel changes in tissue alkaloids, while changes in ODC activity do not. This led us to undertake experiments to permit discrimination between ADC and ODC as enzymatic sources of putrescine destined for alkaloids. Two kinds of evidence presented here support a major role for ADC in the generation of putrescine going into alkaloids: (a) A specific 'suicide inhibitor' of ADC effectively inhibits the biosynthesis of nicotine and nornicotine in tobacco callus, while the analogous inhibitor of ODC is less effective, and (b) the flow of 14C from uniformly labelled arginine into nicotine is much more efficient than that from ornithine.

  8. Effect of L-glutamate on 2-oxoglutarate decarboxylase in Euglena gracilis.

    PubMed Central

    Shigeoka, S; Hanaoka, T; Kishi, N; Nakano, Y

    1992-01-01

    The effect of tricarboxylic acid-cycle intermediates and related compounds on 2-oxoglutarate decarboxylase activity was investigated. The addition of L-glutamate to Euglena cells grown on glucose/(NH4)2SO4 medium resulted in an increase in 2-oxoglutarate decarboxylase activity, which was abolished by the simultaneous addition of cycloheximide. Immunochemical titration, immunoblot analysis and labelling in vivo with antibody raised against 2-oxoglutarate decarboxylase showed that the increase in 2-oxoglutarate decarboxylase activity was due to synthesis of new protein and not to activation of pre-existing protein. The experimental results reported here demonstrate that L-glutamate is assimilated by the pathway, via 2-oxoglutarate, that consists of L-glutamate-oxaloacetate aminotransferase, 2-oxoglutarate decarboxylase and succinate semialdehyde dehydrogenase, rather than by the gamma-aminobutyrate shunt, consisting of L-glutamate decarboxylase and gamma-aminobutyrate aminotransferase. Images Fig. 4. PMID:1347680

  9. Ornithine decarboxylase activity and: [125I]iododeoxyuridine incorporation in rat prostate.

    PubMed Central

    Fuller, D J; Donaldson, L J; Thomas, G H

    1975-01-01

    The relationship between ornithine decarboxylase activity and [125I]iododexyuridine incorporation was studied in prostates from castrated rats (aged 5, 26 and 80 weeks) injected daily with testosterone for up to 10 days. The results suggest that ornithine decarboxylase activity is a parameter of secretory activity, rather than growth, in the ventral prostate. In the dorsolateral prostate, ornithine decarboxylase activity tends to parallel [125I]iododeoxyuridine incorporation. PMID:1212206

  10. Overexpression of erg20 gene encoding farnesyl pyrophosphate synthase has contrasting effects on activity of enzymes of the dolichyl and sterol branches of mevalonate pathway in Trichoderma reesei.

    PubMed

    Piłsyk, Sebastian; Perlińska-Lenart, Urszula; Górka-Nieć, Wioletta; Graczyk, Sebastian; Antosiewicz, Beata; Zembek, Patrycja; Palamarczyk, Grażyna; Kruszewska, Joanna S

    2014-07-10

    The mevalonate pathway is the most diverse metabolic route resulting in the biosynthesis of at least 30,000 isoprenoid compounds, many of which, such as sterols or dolichols, are indispensable for living cells. In the filamentous fungus Trichoderma of major biotechnological interest isoprenoid metabolites are also involved in the biocontrol processes giving the mevalonate pathway an additional significance. On the other hand, little is known about genes coding for enzymes of the mevalonate pathway in Trichoderma. Here, we present cloning and functional analysis of the erg20 gene from Trichoderma reesei coding for farnesyl pyrophosphate (FPP) synthase (EC 2.5.1.10), an enzyme located at the branching point of the mevalonate pathway. Expression of the gene in a thermosensitive erg20-2 mutant of Saccharomyces cerevisiae impaired in the FPP synthase activity suppressed the thermosensitive phenotype. The same gene overexpressed in T. reesei significantly enhanced the FPP synthase activity and also stimulated the activity of cis-prenyltransferase, an enzyme of the dolichyl branch of the mevalonate pathway. Unexpectedly, the activity of squalene synthase from the other, sterol branch, was significantly decreased without, however, affecting ergosterol level.

  11. Crystal structure of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase, an enzyme in the non-mevalonate pathway of isoprenoid synthesis.

    PubMed

    Wada, Takashi; Kuzuyama, Tomohisa; Satoh, Shinya; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Unzai, Satoru; Tame, Jeremy R H; Park, Sam-Yong

    2003-08-08

    The crystal structure of the enzyme 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME) kinase from the thermophilic bacterium Thermus thermophilus HB8 has been determined at 1.7-A resolution. This enzyme catalyzes phosphorylation of the 2-hydroxyl group of CDP-ME, the fourth step of the non-mevalonate pathway, which is essential for isoprenoid biosynthesis in several pathogenic microorganisms. Since this pathway is absent in humans, it is an important target for the development of novel antimicrobial compounds. The structure of the enzyme is similar to the structures of mevalonate kinase and homoserine kinase, members of the GHMP superfamily. Lys8 and Asp125 are active site residues in mevalonate kinase that also appear to play a catalytic role in CDP-ME kinase. Both the mevalonate and the non-mevalonate pathways therefore involve closely related kinases with similar mechanisms. Assaying the enzyme showed that CDP-ME kinase will phosphorylate CDP-ME but not 4-(uridine 5'-diphospho)-2-C-methyl-D-erythritol, indicating the substrate pyrimidine moiety is involved in important interactions with the enzyme.

  12. Mevalonosomes: specific vacuoles containing the mevalonate pathway in Plocamium brasiliense cortical cells (Rhodophyta).

    PubMed

    Paradas, Wladimir Costa; Crespo, Thalita Mendes; Salgado, Leonardo Tavares; de Andrade, Leonardo Rodrigues; Soares, Angélica Ribeiro; Hellio, Claire; Paranhos, Ricardo Rogers; Hill, Lilian Jorge; de Souza, Geysa Marinho; Kelecom, Alphonse Germaine Albert Charles; Da Gama, Bernardo Antônio Perez; Pereira, Renato Crespo; Amado-Filho, Gilberto Menezes

    2015-04-01

    This paper has identified, for the first time in a member of the Rhodophyta, a vacuolar organelle containing enzymes that are involved in the mevalonate pathway-an important step in red algal isoprenoid biosynthesis. These organelles were named mevalonosomes (Mev) and were found in the cortical cells (CC) of Plocamium brasiliense, a marine macroalgae that synthesizes several halogenated monoterpenes. P. brasiliense specimens were submitted to a cytochemical analysis of the activity of the 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS). Using transmission electron microscopy (TEM), we confirmed the presence of HMGS activity within the Mev. Because HMGS is necessary for the biosynthesis of halogenated monoterpenes, we isolated a hexanic fraction (HF) rich in halogenated monoterpenes from P. brasiliense that contained a pentachlorinated monoterpene as a major metabolite. Because terpenes are often related to chemical defense, the antifouling (AF) activity of pentachlorinated monoterpene was tested. We found that the settlement of the mussel Perna perna was reduced by HF treatment (2.25 times less than control; 40% and 90% of fouled surface, respectively; P = 0.001; F9,9 = 1.13). The HF (at 10 μg · mL(-1) ) also inhibited three species of fouling microalgae (Chlorarachnion reptans, Cylindrotheca cloisterium, and Exanthemachrysis gayraliae), while at a higher concentration (50 μg · mL(-1) ), it inhibited the bacteria Halomonas marina, Polaribacter irgensii, Pseudoalteromonas elyakovii, Shewanella putrefaciens, and Vibrio aestuarianus. The AF activity of P. brasiliense halogenated monoterpenes and the localization of HMGS activity inside Mev suggest that this cellular structure found in CC may play a role in thallus protection against biofouling. © 2015 Phycological Society of America.

  13. Targeting the Mevalonate Cascade as a New Therapeutic Approach in Heart Disease, Cancer and Pulmonary Disease

    PubMed Central

    Yeganeh, Behzad; Wiechec, Emmilia; Ande, Sudharsana R; Sharma, Pawan; Moghadam, Adel Rezaei; Post, Martin; Freed, Darren H.; Hashemi, Mohammad; Shojaei, Shahla; Zeki, Amir A.; Ghavami, Saeid

    2014-01-01

    The cholesterol biosynthesis pathway, also known as the mevalonate (MVA) pathway, is an essential cellular pathway that is involved in diverse cell functions. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) is the rate-limiting step in cholesterol biosynthesis and catalyzes the conversion of HMG-CoA to MVA. Given its role in cholesterol and isoprenoid biosynthesis, the regulation of HMGCR has been intensely investigated. Because all cells require a steady supply of MVA, both the sterol (i.e. cholesterol) and non-sterol (i.e. isoprenoid) products of MVA metabolism exert coordinated feedback regulation on HMGCR through different mechanisms. The proper functioning of HMGCR as the proximal enzyme in the MVA pathway is essential under both normal physiologic conditions and in many diseases given its role in cell cycle pathways and cell proliferation, cholesterol biosynthesis and metabolism, cell cytoskeletal dynamics and stability, cell membrane structure and fluidity, mitochondrial function, proliferation, and cell fate. The blockbuster statin drugs (‘statins’) directly bind to and inhibit HMGCR, and their use for the past thirty years has revolutionized the treatment of hypercholesterolemia and cardiovascular diseases, in particular coronary heart disease. Initially thought to exert their effects through cholesterol reduction, recent evidence indicates that statins also have pleiotropic immunomodulatory properties independent of cholesterol lowering. In this review we will focus on the therapeutic applications and mechanisms involved in the MVA cascade including Rho GTPase and Rho kinase (ROCK) signaling, statin inhibition of HMGCR, geranylgeranyltransferase (GGTase) inhibition, and farnesyltransferase (FTase) inhibition in cardiovascular disease, pulmonary diseases (e.g. asthma and chronic obstructive pulmonary disease (COPD), and cancer. PMID:24582968

  14. Mevalonate metabolism regulates Basal breast cancer stem cells and is a potential therapeutic target.

    PubMed

    Ginestier, Christophe; Monville, Florence; Wicinski, Julien; Cabaud, Olivier; Cervera, Nathalie; Josselin, Emmanuelle; Finetti, Pascal; Guille, Arnaud; Larderet, Gaelle; Viens, Patrice; Sebti, Said; Bertucci, François; Birnbaum, Daniel; Charafe-Jauffret, Emmanuelle

    2012-07-01

    There is increasing evidence that breast tumors are organized in a hierarchy, with a subpopulation of tumorigenic cancer cells, the cancer stem cells (CSCs), which sustain tumor growth. The characterization of protein networks that govern CSC behavior is paramount to design new therapeutic strategies targeting this subpopulation of cells. We have sought to identify specific molecular pathways of CSCs isolated from 13 different breast cancer cell lines of luminal or basal/mesenchymal subtypes. We compared the gene expression profiling of cancer cells grown in adherent conditions to those of matched tumorsphere cultures. No specific pathway was identified to be commonly regulated in luminal tumorspheres, resulting from a minor CSC enrichment in tumorsphere passages from luminal cell lines. However, in basal/mesenchymal tumorspheres, the enzymes of the mevalonate metabolic pathway were overexpressed compared to those in cognate adherent cells. Inhibition of this pathway with hydroxy-3-methylglutaryl CoA reductase blockers resulted in a reduction of breast CSC independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Further modulation of this metabolic pathway demonstrated that protein geranylgeranylation (GG) is critical to breast CSC maintenance. A small molecule inhibitor of the geranylgeranyl transferase I (GGTI) enzyme reduced the breast CSC subpopulation both in vitro and in primary breast cancer xenografts. We found that the GGTI effect on the CSC subpopulation is mediated by inactivation of Ras homolog family member A (RHOA) and increased accumulation of P27(kip1) in the nucleus. The identification of protein GG as a major contributor to CSC maintenance opens promising perspectives for CSC targeted therapy in basal breast cancer.

  15. An activity, stability and selectivity comparison of propioin synthesis by thiamine diphosphate-dependent enzymes in a solid/gas bioreactor.

    PubMed

    Mikolajek, Renaud; Spiess, Antje C; Pohl, Martina; Lamare, Sylvain; Büchs, Jochen

    2007-06-18

    Enzymatic carboligation in a solid/gas bioreactor represents a new challenge in biotechnology. In this paper, the continuous gas-phase production of propioin from two propanal molecules by using thiamine diphosphate-dependent enzymes was studied. Two enzymes were used, namely benzaldehyde lyase (BAL) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD) from Pseudomonas putida. The enzymes are homologous and catalyze carboligase and carbolyase reactions in which no external cofactor regeneration is needed. The influence of water and substrate activity on the initial reaction rate and biocatalyst stability was investigated. An increase in water activity raised the initial reaction rates to the maximal values of 250 and 80 U g(-1) for BAL and BFD, respectively. The half-life showed the same trend with maximal values of 50 and 78 min for BAL and BFD, respectively. The increase in the half-life by increasing water activity was unexpected. It was also observed that BFD is more stable than BAL in the presence of the substrate propanal. Both enzymes showed substrate inhibition in the kinetic studies, and BAL was also deactivated during the reaction. Unexpectedly, the stereoselectivity of both enzymes (ee of 19 % for BAL and racemic mixture for BFD) was significantly impaired in the gas phase compared to the liquid phase.

  16. Potentiated suppression of Dickkopf-1 in breast cancer by combined administration of the mevalonate pathway inhibitors zoledronic acid and statins.

    PubMed

    Göbel, Andy; Browne, Andrew J; Thiele, Stefanie; Rauner, Martina; Hofbauer, Lorenz C; Rachner, Tilman D

    2015-12-01

    The Wnt-inhibitor dickkopf-1 (DKK-1) promotes cancer-induced osteolytic bone lesions by direct inhibition of osteoblast differentiation and indirect activation of osteoclasts. DKK-1 is highly expressed in human breast cancer cells and can be suppressed by inhibitors of the mevalonate pathway such as statins and amino-bisphosphonates. However, supraphysiological concentrations are required to suppress DKK-1. We show that a sequential mevalonate pathway blockade using statins and amino-bisphosphonates suppresses DKK-1 more significantly than the individual agents alone. Thus, the reduction of the DKK-1 expression and secretion in the human osteotropic tumor cell lines MDA-MB-231, MDA-MET, and MDA-BONE by zoledronic acid was potentiated by the combination with low concentrations of statins (atorvastatin, simvastatin, and rosuvastatin) by up to 75% (p < 0.05). The specific rescue of prenylation using farnesyl pyrophosphate or geranylgeranyl pyrophosphate revealed that these effects were mediated by suppressed geranylgeranylation rather than by suppressed farnesylation. Moreover, combining low concentrations of statins (1 µM atorvastatin or 0.25 µM simvastatin) and zoledronic acid at low concentrations resulted in an at least 50% reversal of breast cancer-derived DKK-1-mediated inhibition of osteogenic markers in C2C12 cells (p < 0.05). Finally, the intratumoral injection of atorvastatin and zoledronic acid in as subcutaneous MDA-MB-231 mouse model reduced the serum level of human DKK-1 by 25% compared to untreated mice. Hence our study reveals that a sequential mevalonate pathway blockade allows for the combined use of low concentration of statins and amino-bisphosphonates. This combination still significantly suppresses breast cancer-derived DKK-1 to levels where it can no longer inhibit Wnt-mediated osteoblast differentiation.

  17. Priming by Hexanoic Acid Induce Activation of Mevalonic and Linolenic Pathways and Promotes the Emission of Plant Volatiles.

    PubMed

    Llorens, Eugenio; Camañes, Gemma; Lapeña, Leonor; García-Agustín, Pilar

    2016-01-01

    Hexanoic acid (Hx) is a short natural monocarboxylic acid present in some fruits and plants. Previous studies reported that soil drench application of this acid induces effective resistance in tomato plants against Botrytis cinerea and Pseudomonas syringae and in citrus against Alternaria alternata and Xanthomonas citri. In this work, we performed an in deep study of the metabolic changes produced in citrus by the application of Hx in response to the challenge pathogen A. alternata, focusing on the response of the plant. Moreover, we used (13)C labeled hexanoic to analyze its behavior inside the plants. Finally, we studied the volatile emission of the treated plants after the challenge inoculation. Drench application of (13)C labeled hexanoic demonstrated that this molecule stays in the roots and is not mobilized to the leaves, suggesting long distance induction of resistance. Moreover, the study of the metabolic profile showed an alteration of more than 200 molecules differentially induced by the application of the compound and the inoculation with the fungus. Bioinformatics analysis of data showed that most of these altered molecules could be related with the mevalonic and linolenic pathways suggesting the implication of these pathways in the induced resistance mediated by Hx. Finally, the application of this compound showed an enhancement of the emission of 17 volatile metabolites. Taken together, this study indicates that after the application of Hx this compound remains in the roots, provoking molecular changes that may trigger the defensive response in the rest of the plant mediated by changes in the mevalonic and linolenic pathways and enhancing the emission of volatile compounds, suggesting for the first time the implication of mevalonic pathway in response to hexanoic application.

  18. Priming by Hexanoic Acid Induce Activation of Mevalonic and Linolenic Pathways and Promotes the Emission of Plant Volatiles

    PubMed Central

    Llorens, Eugenio; Camañes, Gemma; Lapeña, Leonor; García-Agustín, Pilar

    2016-01-01

    Hexanoic acid (Hx) is a short natural monocarboxylic acid present in some fruits and plants. Previous studies reported that soil drench application of this acid induces effective resistance in tomato plants against Botrytis cinerea and Pseudomonas syringae and in citrus against Alternaria alternata and Xanthomonas citri. In this work, we performed an in deep study of the metabolic changes produced in citrus by the application of Hx in response to the challenge pathogen A. alternata, focusing on the response of the plant. Moreover, we used 13C labeled hexanoic to analyze its behavior inside the plants. Finally, we studied the volatile emission of the treated plants after the challenge inoculation. Drench application of 13C labeled hexanoic demonstrated that this molecule stays in the roots and is not mobilized to the leaves, suggesting long distance induction of resistance. Moreover, the study of the metabolic profile showed an alteration of more than 200 molecules differentially induced by the application of the compound and the inoculation with the fungus. Bioinformatics analysis of data showed that most of these altered molecules could be related with the mevalonic and linolenic pathways suggesting the implication of these pathways in the induced resistance mediated by Hx. Finally, the application of this compound showed an enhancement of the emission of 17 volatile metabolites. Taken together, this study indicates that after the application of Hx this compound remains in the roots, provoking molecular changes that may trigger the defensive response in the rest of the plant mediated by changes in the mevalonic and linolenic pathways and enhancing the emission of volatile compounds, suggesting for the first time the implication of mevalonic pathway in response to hexanoic application. PMID:27148319

  19. Targeting farnesyl-transferase as a novel therapeutic strategy for mevalonate kinase deficiency: in vitro and in vivo approaches.

    PubMed

    De Leo, Luigina; Marcuzzi, Annalisa; Decorti, Giuliana; Tommasini, Alberto; Crovella, Sergio; Pontillo, Alessandra

    2010-06-01

    Mevalonate kinase deficiency (MKD) is a rare inborn auto-inflammatory disease due to the impairment of the pathway for the biosynthesis of cholesterol and non-sterol isoprenoids. The shortage of isoprenoids compounds and in particular of geranylgeranylpyrophosphate (GGPP) was recently associated to the MKD characteristic inflammatory attacks. The aim of this study is to demonstrate that the normalization of the mevalonate pathway intermediates levels and in particular of GGPP, through the specific inhibition of farnesyl-transferase (FT) with Manumycin A could ameliorate the inflammatory phenotype of MKD patients. The effect of Manumycin A was first evaluated in MKD mouse and cellular models, chemically obtained using the aminobisphosphonate alendronate (ALD), and then in monocytes isolated from 2 MKD patients. Our findings were compared to those obtained by using natural exogenous isoprenoids (NEIs). Manumycin A was able to significantly reduce the inflammatory marker serum amyloid A in ALD-treated Balb/c mice, as well as IL-1 beta secretion in ALD-monocytes and in MKD patients. These results clearly showed that, through the inhibition of FT, an increased number of mevalonate pathway intermediates could be redirected towards the synthesis of GGPP diminishing the inflammatory response. The importance in limiting the shortage of GGPP was emphasized by the anti-inflammatory effect of NEIs that, due to their biochemical structure, can enter the MKD pathway. In conclusion, manumycin A, as well as NEIs, showed anti-inflammatory effect in MKD models and especially in MKD-monocytes, suggesting novel approaches in the treatment of MKD, an orphan disease without any efficacious treatment currently available.

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

    PubMed

    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.

  1. The cloning, characterization, and functional analysis of a gene encoding an isopentenyl diphosphate isomerase involved in triterpene biosynthesis in the Lingzhi or reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes).

    PubMed

    Wu, Feng-Li; Shi, Liang; Yao, Jian; Ren, Ang; Zhou, Chao; Mu, Da-Shuai; Zhao, Ming-Wen

    2013-01-01

    An isopentenyl diphosphate isomerase (IDI) gene, GlIDI, was isolated from Ganoderma lucidum, which produces triterpenes through the mevalonate pathway. The open reading frame of GlIDI encodes a 252 amino acid polypeptide with a theoretical molecular mass of 28.71 kDa and a theoretical isoelectric point of 5.36. GlIDI is highly homologous to other fungal IDIs and contains conserved active residues and nudix motifs shared by the IDI protein family. The color complementation assay indicated that GlIDI can accelerate the accumulation of β-carotene and confirmed that the cloned complementary DNA encoded a functional GlIDI protein. Gene expression analysis showed that the GlIDI transcription level was relatively low in the mycelia and reached a relatively high level in the mushroom primordia. In addition, its expression level could be up-regulated by 254 µM methyl jasmonate. Our results suggest that this enzyme may play an important role in triterpene biosynthesis.

  2. Product Rearrangement from Altering a Single Residue in the Rice syn-Copalyl Diphosphate Synthase

    PubMed Central

    2016-01-01

    Through site-directed mutagenesis targeted at identification of the catalytic base in the rice (Oryza sativa) syn-copalyl diphosphate synthase OsCPS4, changes to a single residue (H501) were found to induce rearrangement rather than immediate deprotonation of the initially formed bicycle, leading to production of the novel compound syn-halimadienyl diphosphate. These mutational results are combined with quantum chemical calculations to provide insight into the underlying reaction mechanism. PMID:26878189

  3. Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba.

    PubMed

    Chen, Qiangwen; Yan, Jiaping; Meng, Xiangxiang; Xu, Feng; Zhang, Weiwei; Liao, Yongling; Qu, Jinwang

    2017-01-02

    Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT (GbAACT, GenBank Accession No. KX904942) and MVK (GbMVK, GenBank Accession No. KX904944) were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

  4. The Primary Effect on the Proteome of ARID1A-mutated Ovarian Clear Cell Carcinoma is Downregulation of the Mevalonate Pathway at the Post-transcriptional Level*

    PubMed Central

    Goldman, Aaron R.; Bitler, Benjamin G.; Schug, Zachary; Conejo-Garcia, Jose R.; Zhang, Rugang; Speicher, David W.

    2016-01-01

    Inactivating mutations in ARID1A, which encodes a subunit of the SWI/SNF chromatin-remodeling complex, are found in over half of ovarian clear cell carcinoma cases and more broadly across most types of cancers. To identify ARID1A-dependent changes in intracellular signaling pathways, we performed proteome analyses of isogenic ovarian clear cell carcinoma cell lines with or without ARID1A expression. Knockout of ARID1A in an ovarian clear cell carcinoma cell line with wild-type ARID1A, OVCA429, primarily resulted in downregulation of the mevalonate pathway, an important metabolic pathway involved in isoprenoid synthesis, cholesterol synthesis, and other downstream pathways. In a complementary experiment, expression of wild-type ARID1A in an ovarian clear cell carcinoma cell line containing mutated ARID1A, OVISE, affected the mevalonate pathway in a reciprocal manner. A striking aspect of these analyses was that, although only 5% of the detected proteome showed significant abundance changes, most proteins in the mevalonate pathway were coordinately affected by ARID1A status. There were generally corresponding changes when comparing the proteomics data to our previously published microarray data for ectopic expression of ARID1A in the OVISE cell line. However, ARID1A-dependent changes were not detected for genes within the mevalonate pathway. This discrepancy suggests that the mevalonate pathway is not regulated directly by ARID1A-mediated transcription and may be regulated post-transcriptionally. We conclude that ARID1A status indirectly influences the mevalonate pathway and probably influences other processes including glycogen metabolism and 14-3-3-mediated signaling. Further, our findings demonstrate that changes in mRNA levels are sometimes poor indicators of signaling pathways affected by gene manipulations in cancer cells. PMID:27654507

  5. Heterodimeric geranyl(geranyl)diphosphate synthase from hop (Humulus lupulus) and the evolution of monoterpene biosynthesis.

    PubMed

    Wang, Guodong; Dixon, Richard A

    2009-06-16

    Myrcene, which accounts for 30-50% of the essential oil in hop (Humulus lupulus L.) trichomes, derives from geranyl diphosphate (GPP), the common precursor of monoterpenes. Full-length sequences of heterodimeric GPP synthase small subunit (GPPS.SSU, belonging to the SSU I subfamily) and large subunit (LSU) cDNAs were mined from a hop trichome cDNA library. The SSU was inactive, whereas the LSU produced GPP, farnesyl diphosphate, and geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate and isopentenyl diphosphate in vitro. Coexpression of both subunits in Escherichia coli yielded a heterodimeric enzyme exhibiting altered ratios of GPP and GGPP synthase activities and greatly enhanced catalytic efficiency. Transcript analysis suggested that the heterodimeric geranyl(geranyl)diphosphate synthase [G(G)PPS] is involved in myrcene biosynthesis in hop trichomes. The critical role of the conserved CxxxC motif (where "x" can be any hydrophobic amino acid residue) in physical interactions between the 2 subunits was demonstrated by using site-directed mutagenesis, and this motif was used in informatic searches to reveal a previously undescribed SSU subfamily (SSU II) present in both angiosperms and gymnosperms. The evolution and physiological roles of SSUs are discussed.

  6. Mutagenesis at asp27 of pyruvate decarboxylase from Zymomonas mobilis. Effect on its ability to form acetoin and acetolactate.

    PubMed

    Wu, Y G; Chang, A K; Nixon, P F; Li, W; Duggleby, R G

    2000-11-01

    Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a bivalent cation as essential cofactors. The three-dimensional structure of PDC from Zymomonas mobilis (ZMPDC) shows that Asp27 (D27) is close to ThDP in the active site, and mutagenesis of this residue has suggested that it participates in catalysis. The normal product of the PDC reaction is acetaldehyde but it is known that the enzyme can also form acetoin as a by-product from the hydroxyethyl-ThDP reaction intermediate. This study focuses on the role of D27 in the production of acetoin and a second by-product, acetolactate. D27 in ZMPDC was altered to alanine (D27A) and this mutated protein, the wild-type, and two other previously constructed PDC mutants (D27E and D27N) were expressed and purified. Determination of the kinetic properties of D27A showed that the affinity of D27A for ThDP is decreased 30-fold, while the affinity for Mg2+ and the Michaelis constant for pyruvate were similar to those of the wild-type. The time-courses of their reactions were investigated. Each mutant has greatly reduced ability to produce acetaldehyde and acetoin compared with the wild-type PDC. However, the effect of these mutations on acetaldehyde production is greater than that on acetoin formation. The D27A mutant can also form acetolactate, whereas neither of the other mutants, nor the wild-type PDC, can do so. In addition, acetaldehyde formation and/or release are reversible in wild-type ZMPDC but irreversible for the mutants. The results are explained by a mechanism involving thermodynamic and geometric characteristics of the intermediates in the reaction.

  7. Resolution of brewers' yeast pyruvate decarboxylase into two isozymes.

    PubMed

    Kuo, D J; Dikdan, G; Jordan, F

    1986-03-05

    A novel purification method was developed for brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) that for the first time resolved the enzyme into two isozymes on DEAE-Sephadex chromatography. The isozymes were found to be distinct according to sodium dodecyl sulfate polyacrylamide gel electrophoresis: the first one to be eluted gave rise to one band, the second to two bands. The isozymes were virtually the same so far as specific activity, KM, inhibition kinetics and irreversible binding properties by the mechanism-based inhibitor (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid are concerned. This finding resolves a longstanding controversy concerning the quaternary structure of this enzyme.

  8. Cell biology, physiology and enzymology of phosphatidylserine decarboxylase.

    PubMed

    Di Bartolomeo, Francesca; Wagner, Ariane; Daum, Günther

    2017-01-01

    Phosphatidylethanolamine is one of the most abundant phospholipids whose major amounts are formed by phosphatidylserine decarboxylases (PSD). Here we provide a comprehensive description of different types of PSDs in the different kingdoms of life. In eukaryotes, type I PSDs are mitochondrial enzymes, whereas other PSDs are localized to other cellular compartments. We describe the role of mitochondrial Psd1 proteins, their function, enzymology, biogenesis, assembly into mitochondria and their contribution to phospholipid homeostasis in much detail. We also discuss briefly the cellular physiology and the enzymology of Psd2. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

  9. Acyloin formation by benzoylformate decarboxylase from Pseudomonas putida.

    PubMed Central

    Wilcocks, R; Ward, O P; Collins, S; Dewdney, N J; Hong, Y; Prosen, E

    1992-01-01

    Whole cells and cell extracts of Pseudomonas putida grown in a medium containing ammonium mandelate have the capacity to produce the acyloin compound 2-hydroxypropiophenone when incubated with benzoylformate and acetaldehyde. Benzaldehyde and benzyl alcohol were formed as reaction by-products. The enantiomeric excess of the 2-hydroxypropiophenone product was found to be 91 to 92%. The absolute configuration of the enzymatically prepared product at the carbinol carbon was found to be S. The thiamine PPi-linked enzyme benzoylformate decarboxylase, purified to give a single protein band on polyacrylamide gel electrophoresis, was shown to be responsible for the catalysis of this novel condensation reaction. Images PMID:1622241

  10. Ribulose diphosphate carboxylase of the cyanobacterium Spirulina platensis

    SciTech Connect

    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.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    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

  14. Inhibition of the mevalonate pathway enhances carvacrol biosynthesis and DXR gene expression in shoot cultures of Satureja khuzistanica Jamzad.

    PubMed

    Ramak, Parvin; Kazempour Osaloo, Shahrokh; Ebrahimzadeh, Hassan; Sharifi, Mozafar; Behmanesh, Mehrdad

    2013-09-01

    Carvacrol is a major component of Satureja khuzistanica Jamzad (≤90%) that has significant antimicrobial and antioxidant properties. Considering the specific capabilities of S. khuzistanica to produce highly pure carvacrol, this plant is an important potential source of carvacrol that could address the abundant consumption and increasing demand for this monoterpene in current world markets. This research was performed to better understand the process of biosynthesis and accumulation of carvacrol in S. khuzistanica. Tests were performed on shoot cultures of S. khuzistanica in Linsmaier-Skoog (LS) medium treated with different concentrations of fosmidomycin (an inhibitor of the non-mevalonate pathway) and mevinolin (an inhibitor of the mevalonate pathway) for 21 days at the following concentrations: 0, 10, 25, 50, 75 and 100 μM. The present study demonstrated that the MEP pathway is the major pathway that provides IPP for the biosynthesis of carvacrol, and the expression and activity levels of the DXR enzyme have a critical effect on carvacrol biosynthesis. Surprisingly, Mevinolin at concentrations of 75 and 100 μM increased the carvacrol content and the DXR activity and gene expression in S. khuzistanica plantlets. Copyright © 2013 Elsevier GmbH. All rights reserved.

  15. Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis.

    PubMed

    Dudley, Quentin M; Anderson, Kim C; Jewett, Michael C

    2016-12-16

    Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for accelerating the design and synthesis of biosynthetic pathways. However, as most cell-free biomolecule synthesis systems to date use purified enzymes, energy and cofactor balance can be limiting. To address this challenge, we report a new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or extracts. In our modular approach, cell-free lysates, each selectively enriched with an overexpressed enzyme, are generated in parallel and then combinatorically mixed to construct a full biosynthetic pathway. Endogenous enzymes in the cell-free extract fuel high-level energy and cofactor regeneration. As a model, we apply our framework to synthesize mevalonate, an intermediate in isoprenoid synthesis. We use our approach to rapidly screen enzyme variants, optimize enzyme ratios, and explore cofactor landscapes for improving pathway performance. Further, we show that genomic deletions in the source strain redirect metabolic flux in resultant lysates. In an optimized system, mevalonate was synthesized at 17.6 g·L(-1) (119 mM) over 20 h, resulting in a volumetric productivity of 0.88 g·L(-1)·hr(-1). We also demonstrate that this system can be lyophilized and retain biosynthesis capability. Our system catalyzes ∼1250 turnover events for the cofactor NAD(+) and demonstrates the ability to rapidly prototype and debug enzymatic pathways in vitro for compelling metabolic engineering and synthetic biology applications.

  16. Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production

    PubMed Central

    Song, Adelene Ai-Lian; Abdullah, Janna Ong; Abdullah, Mohd. Puad; Shafee, Norazizah; Othman, Roohaida; Tan, Ee-Fun; Noor, Normah Mohd.; Raha, Abdul Rahim

    2012-01-01

    Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction. PMID:23300671

  17. Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum

    USDA-ARS?s Scientific Manuscript database

    Aspartate 1-decarboxylase (ADC) and dopa decarboxylase (DDC) provide b–alanine and dopamine used in insect cuticle tanning. Beta-alanine is conjugated with dopamine to yield N-b-alanyldopamine (NBAD), a substrate for the phenoloxidase laccase that catalyzes the synthesis of cuticle protein cross-li...

  18. Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus

    USDA-ARS?s Scientific Manuscript database

    The integrative vector pINTRS was used to transfer glutamate decarboxylase (GAD) activity to Streptococcus thermophilus ST128, thus allowing for the production of '-aminobutyric acid (GABA). In pINTRS, the gene encoding glutamate decarboxylase, gadB, was flanked by DNA fragments homologous to a S. ...

  19. The biosynthetic origin of irregular monoterpenes in Lavandula: isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase.

    PubMed

    Demissie, Zerihun A; Erland, Lauren A E; Rheault, Mark R; Mahmoud, Soheil S

    2013-03-01

    Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 μm and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.

  20. D-tagatose 1,6-diphosphate aldolase from lactic streptococci: purification, properties, and use in measuring intracellular tagatose 1,6-diphosphate.

    PubMed Central

    Crow, V L; Thomas, T D

    1982-01-01

    Two D-ketohexose 1,6-diphosphate aldolases are present in Streptococcus cremoris E8 and S. lactis C10. One aldolase, which was induced by growth on either lactose or galactose, was active with both tagatose 1,6-diphosphate (TDP) and fructose 1,6-diphosphate (FDP), having a lower Km and a higher Vmax with TDP as the substrate. This enzyme, named TDP aldolase, had properties typical of a class I aldolase, being insensitive to EDTA and showing substrate-dependent inactivation by sodium borohydride. Sodium dodecyl sulfate-gel electrophoresis indicated a subunit molecular weight of 34,500. The amino acid composition of TDP aldolase is reported. When the enzyme was incubated with either triose phosphates or FDP, the equilibrium mixture contained an FDP/TDP ratio of 6.9:1. The other aldolase, which had properties typical of a class II aldolase, showed activity with FDP but not with TDP. The intracellular TDP concentration, measured with the purified TDP aldolase, was 0.4 to 4.0 mM in cells growing on lactose or galactose and was lower (0 to 1.0 mM) in cells growing on glucose. The intracellular concentration of FDP was always higher than that of TDP. The role of ketohexose diphosphates in the regulation of end product fermentation by lactic streptococci is discussed. PMID:6807956

  1. Crystal structure of pyruvate decarboxylase from Zymobacter palmae

    PubMed Central

    Buddrus, Lisa; Andrews, Emma S. V.; Leak, David J.; Danson, Michael J.; Arcus, Vickery L.; Crennell, Susan J.

    2016-01-01

    Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg2+ ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and R r.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were R work = 0.186 (0.271 in the highest resolution bin) and R free = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions. PMID:27599861

  2. Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity.

    PubMed

    Lietzan, Adam D; St Maurice, Martin

    2014-02-15

    Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

  3. Crystal Structure of Uroporphyrinogen Decarboxylase from Bacillus subtilis▿

    PubMed Central

    Fan, Jun; Liu, Qun; Hao, Quan; Teng, Maikun; Niu, Liwen

    2007-01-01

    Uroporphyrinogen decarboxylase (UROD) is a branch point enzyme in the biosynthesis of the tetrapyrroles. It catalyzes the decarboxylation of four acetate groups of uroporphyrinogen III to yield coproporphyrinogen III, leading to heme and chlorophyll biosynthesis. UROD is a special type of nonoxidative decarboxylase, since no cofactor is essential for catalysis. In this work, the first crystal structure of a bacterial UROD, Bacillus subtilis UROD (URODBs), has been determined at a 2.3 Å resolution. The biological unit of URODBs was determined by dynamic light scattering measurements to be a homodimer in solution. There are four molecules in the crystallographic asymmetric unit, corresponding to two homodimers. Structural comparison of URODBs with eukaryotic URODs reveals a variation of two loops, which possibly affect the binding of substrates and release of products. Structural comparison with the human UROD-coproporphyrinogen III complex discloses a similar active cleft, with five invariant polar residues (Arg29, Arg33, Asp78, Tyr154, and His322) and three invariant hydrophobic residues (Ile79, Phe144, and Phe207), in URODBs. Among them, Asp78 may interact with the pyrrole NH groups of the substrate, and Arg29 is a candidate for positioning the acetate groups of the substrate. Both residues may also play catalytic roles. PMID:17122346

  4. Tyrosine decarboxylase from Lactobacillus brevis: soluble expression and characterization.

    PubMed

    Zhang, Kai; Ni, Ye

    2014-02-01

    Tyrosine decarboxylase (TDC, EC 4.1.1.25) is an enzyme that catalyzes the decarboxylation of l-tyrosine to produce tyramine and CO2. In this study, a 1881-bp tdc gene from Lactobacillus brevis was cloned and heterologously expressed in Escherichia coli BL21 (DE3). Glucose was discovered to play an important role in the soluble expression of rLbTDC. After optimization, recombinant TDC (rLbTDC) was achieved in excellent solubility and a yield of 224mg rLbTDC/L broth. The C-terminal His-Tagged rLbTDC was one-step purified with 90% recovery. Based on SDS-PAGE and gel filtration analysis, rLbTDC is a dimer composed of two identical subunits of approximately 70kDa. Using l-tyrosine as substrate, the specific activity of rLbTDC was determined to be 133.5U/mg in the presence of 0.2mM pyridoxal-5'-phosphate at 40°C and pH 5.0. The Km and Vmax values of rLbTDC were 0.59mM and 147.1μmolmin(-1)mg(-1), respectively. In addition to l-tyrosine, rLbTDC also exhibited decarboxylase activity towards l-DOPA. This study has demonstrated, for the first time, the soluble expression of tdc gene from L. brevis in heterologous host.

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

    PubMed Central

    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

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

    PubMed

    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.

  7. A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate.

    PubMed

    Günnewich, Nils; Higashi, Yasuhiro; Feng, Xiaohong; Choi, Kum-Boo; Schmidt, Jürgen; Kutchan, Toni M

    2013-07-01

    The bicyclic diterpene (-)-sclareol is accumulated in glandular trichomes in Salvia sclarea (Schmiderer et al., 2008), and is a major terpenoid component of this plant species. It is used as the starting material for Ambrox synthesis, a synthetic ambergris analog used in the flavor and fragrance industry. In order to investigate the formation of sclareol, cDNA prepared from secretory cells of glandular trichomes from S. sclarea inflorescence were randomly sequenced. A putative copalyl diphosphate synthase encoding EST, SscTPS1, was functionally expressed in Escherichia coli. Whereas reaction of geranylgeranyl diphosphate with the putative copalyl diphosphate synthase followed by hydrolysis with alkaline phosphatase yielded a diastereomeric mixture of (13R)- and (13S)-manoyl oxide, HCl hydrolysis yielded (-)-sclareol (1) and 13-epi-sclareol as products. The product of the reaction of SscTPS1 with geranylgeranyl diphosphate was subjected to analysis by LC-negative ion ESI-MS/MS without prior hydrolysis. EPI scans were consistent with copalyl diphosphate to which 18 mass units had added (m/z 467 [M+H](-)). The enzymatic reaction was also carried out in the presence of 60% H2(18)O. LC-negative ion ESI-MS/MS analysis established an additional reaction product consistent with the incorporation of (18)O. Incubation in the presence of 60% (2)H2O resulted in the incorporation of one deuterium atom. These results suggest water capture of the carbocation intermediate, which is known to occur in reactions catalyzed by monoterpene synthases, but has been described only several times for diterpene synthases. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Intestinal alkaline phosphatase inhibits the proinflammatory nucleotide uridine diphosphate.

    PubMed

    Moss, Angela K; Hamarneh, Sulaiman R; Mohamed, Mussa M Rafat; Ramasamy, Sundaram; Yammine, Halim; Patel, Palak; Kaliannan, Kanakaraju; Alam, Sayeda N; Muhammad, Nur; Moaven, Omeed; Teshager, Abeba; Malo, Nondita S; Narisawa, Sonoko; Millán, José Luis; Warren, H Shaw; Hohmann, Elizabeth; Malo, Madhu S; Hodin, Richard A

    2013-03-15

    Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP.

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

    PubMed

    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.

  10. Lithium-cation conductivity and crystal structure of lithium diphosphate

    SciTech Connect

    Voronin, V.I.; Sherstobitova, E.A.; Blatov, V.A.; Shekhtman, G.Sh.

    2014-03-15

    The electrical conductivity of lithium diphosphate Li{sub 4}P{sub 2}O{sub 7} has been measured and jump-like increasing of ionic conductivity at 913 K has been found. The crystal structure of Li{sub 4}P{sub 2}O{sub 7} has been refined using high temperature neutron diffraction at 300–1050 K. At 913 K low temperature triclinic form of Li{sub 4}P{sub 2}O{sub 7} transforms into high temperature monoclinic one, space group P2{sub 1}/n, a=8.8261(4) Å, b=5.2028(4) Å, c=13.3119(2) Å, β=104.372(6)°. The migration maps of Li{sup +} cations based on experimental data implemented into program package TOPOS have been explored. It was found that lithium cations in both low- and high temperature forms of Li{sub 4}P{sub 2}O{sub 7} migrate in three dimensions. Cross sections of the migrations channels extend as the temperature rises, but at the phase transition point have a sharp growth showing a strong “crystal structure – ion conductivity” correlation. -- Graphical abstract: Crystal structure of Li{sub 4}P{sub 2}O{sub 7} at 950 K. Red balls represent oxygen atoms; black lines show Li{sup +} ion migration channels in the layers perpendicular to [001] direction. Highlights: • Structure of Li{sub 4}P{sub 2}O{sub 7} has been refined using high temperature neutron diffraction. • At 913 K triclinic form of Li{sub 4}P{sub 2}O{sub 7} transforms into high temperature monoclinic one. • The migration maps of Li{sup +} implemented into program package TOPOS have been explored. • Cross sections of the migrations channels at the phase transition have a sharp growth.

  11. Trans, trans-farnesol as a mevalonate-derived inducer of murine 3T3-F442A pre-adipocyte differentiation

    PubMed Central

    Torabi, Sheida

    2015-01-01

    Based on our finding that depletion of mevalonate-derived metabolites inhibits adipocyte differentiation, we hypothesize that trans, trans-farnesol (farnesol), a mevalonate-derived sesquiterpene, induces adipocyte differentiation. Farnesol dose-dependently (25–75 μmol/L) increased intracellular triglyceride content of murine 3T3-F442A pre-adipocytes measured by AdipoRed™ Assay and Oil Red-O staining. Concomitantly, farnesol dose-dependently increased glucose uptake and glucose transport protein 4 (GLUT4) expression without affecting cell viability. Furthermore, quantitative real-time polymerase chain reaction and Western blot showed that farnesol increased the mRNA and protein levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, and the mRNA levels of PPARγ-regulated fatty acid-binding protein 4 and adiponectin; in contrast, farnesol downregulated Pref-1 gene, a marker of pre-adipocytes. GW9662 (10 µmol/L), an antagonist of PPARγ, reversed the effects of farnesol on cellular lipid content, suggesting that PPARγ signaling pathway may mediate the farnesol effect. Farnesol (25–75 μmol/L) did not affect the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in the mevalonate pathway. Farnesol may be the mevalonate-derived inducer of adipocyte differentiation and potentially an insulin sensitizer via activation of PPARγ and upregulation of glucose uptake. PMID:26660152

  12. The pathway of leucine to mevalonate in halophilic archaea: efficient incorporation of leucine into isoprenoidal lipid with the involvement of isovaleryl-CoA dehydrogenase in Halobacterium salinarum.

    PubMed

    Yamauchi, Noriaki

    2010-01-01

    The pathway of leucine to mevalonate, which has attracted attention in the study of the biosynthesis of isoprenoid in parasitic protozoa and myxobacterium, was observed in the biosynthesis of the lipid core in halophilic archaea. The involvement of isovaleryl-CoA dehydrogenase was strongly suggested, with stereospecific conversion of the diastereotopic methyl group of leucine to isoprenoidal lipid.

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

    PubMed

    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.

  14. A corpora allata farnesyl diphosphate synthase in mosquitoes displaying a metal ion dependent substrate specificity

    PubMed Central

    Rivera-Perez, Crisalejandra; Nyati, Pratik; Noriega, Fernando G.

    2015-01-01

    Farnesyl diphosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis, it catalyzes the head-to-tail condensation of dimethylallyl diphosphate (DMAPP) with two molecules of isopentenyl diphosphate (IPP) to generate farnesyl diphosphate (FPP), a precursor of juvenile hormone (JH). In this study, we functionally characterized an Aedes aegypti FPPS (AaFPPS) expressed in the corpora allata. AaFPPS is the only FPPS gene present in the genome of the yellow fever mosquito, it encodes a 49.6 kDa protein exhibiting all the characteristic conserved sequence domains on prenyltransferases. AaFPPS displays its activity in the presence of metal cofactors; and the product condensation is dependent of the divalent cation. Mg2+ ions lead to the production of FPP, while the presence of Co2+ ions lead to geranyl diphosphate (GPP) production. In the presence of Mg2+ the AaFPPS affinity for allylic substrates is GPP>DMAPP>IPP. These results suggest that AaFPPS displays “catalytic promiscuity”, changing the type and ratio of products released (GPP or FPP) depending on allylic substrate concentrations and the presence of different metal cofactors. This metal ion-dependent regulatory mechanism allows a single enzyme to selectively control the metabolites it produces, thus potentially altering the flow of carbon into separate metabolic pathways. PMID:26188328

  15. A corpora allata farnesyl diphosphate synthase in mosquitoes displaying a metal ion dependent substrate specificity.

    PubMed

    Rivera-Perez, Crisalejandra; Nyati, Pratik; Noriega, Fernando G

    2015-09-01

    Farnesyl diphosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis, it catalyzes the head-to-tail condensation of dimethylallyl diphosphate (DMAPP) with two molecules of isopentenyl diphosphate (IPP) to generate farnesyl diphosphate (FPP), a precursor of juvenile hormone (JH). In this study, we functionally characterized an Aedes aegypti FPPS (AaFPPS) expressed in the corpora allata. AaFPPS is the only FPPS gene present in the genome of the yellow fever mosquito, it encodes a 49.6 kDa protein exhibiting all the characteristic conserved sequence domains on prenyltransferases. AaFPPS displays its activity in the presence of metal cofactors; and the product condensation is dependent of the divalent cation. Mg(2+) ions lead to the production of FPP, while the presence of Co(2+) ions lead to geranyl diphosphate (GPP) production. In the presence of Mg(2+) the AaFPPS affinity for allylic substrates is GPP > DMAPP > IPP. These results suggest that AaFPPS displays "catalytic promiscuity", changing the type and ratio of products released (GPP or FPP) depending on allylic substrate concentrations and the presence of different metal cofactors. This metal ion-dependent regulatory mechanism allows a single enzyme to selectively control the metabolites it produces, thus potentially altering the flow of carbon into separate metabolic pathways.

  16. Bis(benzoyloxybenzyl)-DiPPro nucleoside diphosphates of anti-HIV active nucleoside analogues.

    PubMed

    Weinschenk, Lina; Gollnest, Tristan; Schols, Dominique; Balzarini, Jan; Meier, Chris

    2015-05-01

    Nucleoside analogues are extensively used as antiviral and anticancer agents. Their efficiency is dependent on their metabolism into the ultimately active nucleoside triphosphates. Often one step or even more in the metabolism of the nucleoside to the triphosphate is inefficient. To overcome this hurdle, prodrugs of the nucleotides are needed. Bis(acyloxybenzyl)nucleoside diphosphates have been reported by us as a first example of an efficient nucleoside diphosphate prodrug (DiPPro nucleotides). Here, the synthesis and the properties of bis(benzoyloxybenzyl)nucleoside diphosphates of the nucleoside analogues d4T and AZT are disclosed. The synthesis was achieved by using a phosphoramidite/oxidation route. In chemical hydrolysis studies, most of the compounds formed a nucleoside diphosphate. This was confirmed in CEM cell extracts, although the prodrug stability in extracts was lower than in phosphate buffer. Furthermore, the stability and the amount of nucleoside diphosphate formed were dependent on the substituent in the benzoyl moiety. Some of the compounds were more active against HIV in thymidine kinase-deficient CEM/TK(-) cells than were d4T or AZT. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

    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.

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

    PubMed Central

    Dozier, Jonathan K; Distefano, Mark D

    2012-01-01

    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 use either 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 IC50 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. PMID:22085443

  19. Uncovering the Lactobacillus plantarum WCFS1 Gallate Decarboxylase Involved in Tannin Degradation

    PubMed Central

    Jiménez, Natalia; Curiel, José Antonio; Reverón, Inés; de las Rivas, Blanca

    2013-01-01

    Lactobacillus plantarum is a lactic acid bacterium able to degrade tannins by the subsequent action of tannase and gallate decarboxylase enzymes. The gene encoding tannase had previously been identified, whereas the gene encoding gallate decarboxylase is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of gallic-acid induced L. plantarum extracts showed a 54-kDa protein which was absent in the uninduced cells. This protein was identified as Lp_2945, putatively annotated UbiD. Homology searches identified ubiD-like genes located within three-gene operons which encoded the three subunits of nonoxidative aromatic acid decarboxylases. L. plantarum is the only bacterium in which the lpdC (lp_2945) gene and the lpdB and lpdD (lp_0271 and lp_0272) genes are separated in the chromosome. Combination of extracts from recombinant Escherichia coli cells expressing the lpdB, lpdC, and lpdC genes demonstrated that LpdC is the only protein required to yield gallate decarboxylase activity. However, the disruption of these genes in L. plantarum revealed that the lpdB and lpdC gene products are essential for gallate decarboxylase activity. Similar to L. plantarum tannase, which exhibited activity only in esters derived from gallic and protocatechuic acids, purified His6-LpdC protein from E. coli showed decarboxylase activity against gallic and protocatechuic acids. In contrast to the tannase activity, gallate decarboxylase activity is widely present among lactic acid bacteria. This study constitutes the first genetic characterization of a gallate decarboxylase enzyme and provides new insights into the role of the different subunits of bacterial nonoxidative aromatic acid decarboxylases. PMID:23645198

  20. An endosymbiont positively modulates ornithine decarboxylase in host trypanosomatids

    SciTech Connect

    Frossard, Mariana Lins; Seabra, Sergio Henrique; Matta, Renato Augusto da; Souza, Wanderley de; Garcia de Mello, Fernando; Motta, Maria Cristina Machado . E-mail: motta@biof.ufrj.br

    2006-05-05

    Summary: Some trypanosomatids, such as Crithidia deanei, are endosymbiont-containing species. Aposymbiotic strains are obtained after antibiotic treatment, revealing interesting aspects of this symbiotic association. Ornithine decarboxylase (ODC) promotes polyamine biosynthesis and contributes to cell proliferation. Here, we show that ODC activity is higher in endosymbiont-bearing trypanosomatids than in aposymbiotic cells, but isolated endosymbionts did not display this enzyme activity. Intriguingly, expressed levels of ODC were similar in both strains, suggesting that ODC is positively modulated in endosymbiont-bearing cells. When the aposymbiotic strain was grown in conditioned medium, obtained after cultivation of the endosymbiont-bearing strain, cellular proliferation as well as ODC activity and localization were similar to that observed in the endosymbiont-containing trypanosomatids. Furthermore, dialyzed-heated medium and trypsin treatment reduced ODC activity of the aposymbiont strain. Taken together, these data indicate that the endosymbiont can enhance the protozoan ODC activity by providing factors of protein nature, which increase the host polyamine metabolism.

  1. Altered subcellular localization of ornithine decarboxylase in Alzheimer's disease brain

    SciTech Connect

    Nilsson, Tatjana . E-mail: Tatjana.Nilsson@ki.se; Bogdanovic, Nenad; Volkman, Inga; Winblad, Bengt; Folkesson, Ronnie; Benedikz, Eirikur

    2006-06-02

    The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD) brains. In frontal cortex and hippocampus of control cases, the most pronounced ODC immunoreactivity was found in the nucleus. In possible and definite AD the immunoreactivity had shifted to the cytoplasm. In cerebellum of control cases, ODC staining was found in a small portion of Purkinje cells, mostly in the nucleus. In AD, both possible and definite, the number of stained Purkinje cells increased significantly and immunoreactivity was shifted to the cytoplasm, even though it was still prominent in the nucleus. In conclusion, our study reveals an early shift of the ODC immunoreactivity in AD from the nuclear compartment towards the cytoplasm.

  2. Kinetic challenges facing oxalate, malonate, acetoacetate, and oxaloacetate decarboxylases.

    PubMed

    Wolfenden, Richard; Lewis, Charles A; Yuan, Yang

    2011-04-20

    To compare the powers of the corresponding enzymes as catalysts, the rates of uncatalyzed decarboxylation of several aliphatic acids (oxalate, malonate, acetoacetate, and oxaloacetate) were determined at elevated temperatures and extrapolated to 25 °C. In the extreme case of oxalate, the rate of the uncatalyzed reaction at pH 4.2 was 1.1 × 10(-12) s(-1), implying a 2.5 × 10(13)-fold rate enhancement by oxalate decarboxylase. Whereas the enzymatic decarboxylation of oxalate requires O(2) and Mn(II), the uncatalyzed reaction is unaffected by the presence of these cofactors and appears to proceed by heterolytic elimination of CO(2).

  3. Levodopa combined with peripheral decarboxylase inhibition in Parkinson's disease

    PubMed Central

    Barbeau, André; Mars, Harold; Botez, Mihai I.; Joubert, Marie

    1972-01-01

    The authors report their experience, over a 26-month period, in the management of 60 parkinsonian patients with the combination of levodopa and an inhibitor of peripheral dopa-decarboxylase, Ro 4-4602. This approach to Parkinson's disease is useful, safe, and at least as effective as levodopa alone. To date there have been no recognizable toxic effects attributable to Ro 4-4602. This agent appears to prolong the duration of action of levodopa, smoothing out its therapeutic effects. The percentage of patients obtaining a very good and excellent response is slightly increased. There is a possible diminution in the late-occurring bradykinetic and hypotonic freezing episodes. Nausea and cardiac arrhythmias are lessened, as are the incidence and severity of hypotension. Abnormal involuntary movements remain the limiting adverse side effect. PMID:5034697

  4. Glutamic acid decarboxylase autoimmunity in Batten disease and other disorders.

    PubMed

    Pearce, David A; Atkinson, Mark; Tagle, Danilo A

    2004-12-14

    Degenerative diseases of the CNS, such as stiff-person syndrome (SPS), progressive cerebellar ataxia, and Rasmussen encephalitis, have been characterized by the presence of autoantibodies. Recent findings in individuals with Batten disease and in animal models for the disorder indicate that this condition may be associated with autoantibodies against glutamic acid decarboxylase (GAD), an enzyme that converts the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Anti-GAD autoantibodies could result in excess excitatory neurotransmitters, leading to the seizures and other symptoms observed in patients with Batten disease. The pathogenic potential of GAD autoantibodies is examined in light of what is known for other autoimmune disorders, such as multiple sclerosis, SPS, Rasmussen encephalitis, and type 1 diabetes, and may have radical implications for diagnosis and management of Batten disease.

  5. [Inhibitory effect of essential oils, food additives, peracetic acid and detergents on bacterial histidine decarboxylase].

    PubMed

    Kamii, Eri; Terada, Gaku; Akiyama, Jyunki; Isshiki, Kenji

    2011-01-01

    The aim of this study is to examine whether various essential oils, food additives, peracetic acid and detergents inhibit bacterial histidine decarboxylase. Crude extract of Morganella morganii NBRC3848 was prepared and incubated with various agents. Histidine decarboxylase activity was significantly inhibited (p<0.05) by 26 of 45 compounds tested. Among the 26 agents, sodium hypochlorite completely decomposed both histidine and histamine, while peracetic acid caused slight decomposition. Histidine and histamine were stable in the presence of the other 24 agents. These results indicated that 25 of the agents examined were inhibitors of histidine decarboxylase.

  6. The effects of statins on the mevalonic acid pathway in recombinant yeast strains expressing human HMG-CoA reductase.

    PubMed

    Maciejak, Agata; Leszczynska, Agata; Warchol, Ilona; Gora, Monika; Kaminska, Joanna; Plochocka, Danuta; Wysocka-Kapcinska, Monika; Tulacz, Dorota; Siedlecka, Joanna; Swiezewska, Ewa; Sojka, Maciej; Danikiewicz, Witold; Odolczyk, Norbert; Szkopinska, Anna; Sygitowicz, Grazyna; Burzynska, Beata

    2013-08-30

    The yeast Saccharomyces cerevisiae can be a useful model for studying cellular mechanisms related to sterol synthesis in humans due to the high similarity of the mevalonate pathway between these organisms. This metabolic pathway plays a key role in multiple cellular processes by synthesizing sterol and nonsterol isoprenoids. Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the cholesterol synthesis pathway. However, the effects of statins extend beyond their cholesterol-lowering action, since inhibition of HMGR decreases the synthesis of all products downstream in the mevalonate pathway. Using transgenic yeast expressing human HMGR or either yeast HMGR isoenzyme we studied the effects of simvastatin, atorvastatin, fluvastatin and rosuvastatin on the cell metabolism. Statins decreased sterol pools, prominently reducing sterol precursors content while only moderately lowering ergosterol level. Expression of genes encoding enzymes involved in sterol biosynthesis was induced, while genes from nonsterol isoprenoid pathways, such as coenzyme Q and dolichol biosynthesis or protein prenylation, were diversely affected by statin treatment. Statins increased the level of human HMGR protein substantially and only slightly affected the levels of Rer2 and Coq3 proteins involved in non-sterol isoprenoid biosynthesis. Statins influence the sterol pool, gene expression and protein levels of enzymes from the sterol and nonsterol isoprenoid biosynthesis branches and this effect depends on the type of statin administered. Our model system is a cheap and convenient tool for characterizing individual statins or screening for novel ones, and could also be helpful in individualized selection of the most efficient HMGR inhibitors leading to the best response and minimizing serious side effects.

  7. The effects of statins on the mevalonic acid pathway in recombinant yeast strains expressing human HMG-CoA reductase

    PubMed Central

    2013-01-01

    Background The yeast Saccharomyces cerevisiae can be a useful model for studying cellular mechanisms related to sterol synthesis in humans due to the high similarity of the mevalonate pathway between these organisms. This metabolic pathway plays a key role in multiple cellular processes by synthesizing sterol and nonsterol isoprenoids. Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the cholesterol synthesis pathway. However, the effects of statins extend beyond their cholesterol-lowering action, since inhibition of HMGR decreases the synthesis of all products downstream in the mevalonate pathway. Using transgenic yeast expressing human HMGR or either yeast HMGR isoenzyme we studied the effects of simvastatin, atorvastatin, fluvastatin and rosuvastatin on the cell metabolism. Results Statins decreased sterol pools, prominently reducing sterol precursors content while only moderately lowering ergosterol level. Expression of genes encoding enzymes involved in sterol biosynthesis was induced, while genes from nonsterol isoprenoid pathways, such as coenzyme Q and dolichol biosynthesis or protein prenylation, were diversely affected by statin treatment. Statins increased the level of human HMGR protein substantially and only slightly affected the levels of Rer2 and Coq3 proteins involved in non-sterol isoprenoid biosynthesis. Conclusion Statins influence the sterol pool, gene expression and protein levels of enzymes from the sterol and nonsterol isoprenoid biosynthesis branches and this effect depends on the type of statin administered. Our model system is a cheap and convenient tool for characterizing individual statins or screening for novel ones, and could also be helpful in individualized selection of the most efficient HMGR inhibitors leading to the best response and minimizing serious side effects. PMID:24128347

  8. Molecular cloning and characterization of a geranyl diphosphate-specific aromatic prenyltransferase from lemon.

    PubMed

    Munakata, Ryosuke; Inoue, Tsuyoshi; Koeduka, Takao; Karamat, Fazeelat; Olry, Alexandre; Sugiyama, Akifumi; Takanashi, Kojiro; Dugrand, Audray; Froelicher, Yann; Tanaka, Ryo; Uto, Yoshihiro; Hori, Hitoshi; Azuma, Jun-Ichi; Hehn, Alain; Bourgaud, Frédéric; Yazaki, Kazufumi

    2014-09-01

    Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.

  9. Identification and subcellular localization of two solanesyl diphosphate synthases from Arabidopsis thaliana.

    PubMed

    Jun, Luo; Saiki, Ryoichi; Tatsumi, Kei; Nakagawa, Tsuyoshi; Kawamukai, Makoto

    2004-12-01

    Two solanesyl diphosphate synthases, designated SPS1 and SPS2, which are responsible for the synthesis of the isoprenoid side chain of either plastoquinone or ubiquinone in Arabidopsis thaliana, were identified. Heterologous expression of either SPS1 or SPS2 allowed the generation of UQ-9 in a decaprenyl diphosphate synthase-defective strain of fission yeast and also in wild-type Escherichia coli. SPS1-GFP was found to localize in the ER while SPS2-GFP localized in the plastid of tobacco BY-2 cells. These two different subcellular localizations are thought to be the reflection of their roles in solanesyl diphosphate synthesis in two different parts: presumably SPS1 and SPS2 for the side chains of ubiquinone and plastoquinone, respectively.

  10. X-ray analysis of azido-thymidine diphosphate binding to nucleoside diphosphate kinase

    PubMed Central

    Xu, Yingwu; Sellam, Olivier; Moréra, Solange; Sarfati, Simon; Biondi, Ricardo; Véron, Michel; Janin, Joël

    1997-01-01

    To be effective as antiviral agent, AZT (3′-azido-3′-deoxythymidine) must be converted to a triphosphate derivative by cellular kinases. The conversion is inefficient and, to understand why AZT diphosphate is a poor substrate of nucleoside diphosphate (NDP) kinase, we determined a 2.3-Å x-ray structure of a complex with the N119A point mutant of Dictyostelium NDP kinase. It shows that the analog binds at the same site and, except for the sugar ring pucker which is different, binds in the same way as the natural substrate thymidine diphosphate. However, the azido group that replaces the 3′OH of the deoxyribose in AZT displaces a lysine side chain involved in catalysis. Moreover, it is unable to make an internal hydrogen bond to the oxygen bridging the β- and γ-phosphate, which plays an important part in phosphate transfer. PMID:9207061

  11. Cyclohexane-1,2-Dione Hydrolase from Denitrifying Azoarcus sp. Strain 22Lin, a Novel Member of the Thiamine Diphosphate Enzyme Family▿†

    PubMed Central

    Steinbach, Alma K.; Fraas, Sonja; Harder, Jens; Tabbert, Anja; Brinkmann, Henner; Meyer, Axel; Ermler, Ulrich; Kroneck, Peter M. H.

    2011-01-01

    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 Mg2+, 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 for

  12. Cyclohexane-1,2-dione hydrolase from denitrifying Azoarcus sp. strain 22Lin, a novel member of the thiamine diphosphate enzyme family.

    PubMed

    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

  13. Mouse ornithine decarboxylase gene: cloning, structure, and expression.

    PubMed Central

    Brabant, M; McConlogue, L; van Daalen Wetters, T; Coffino, P

    1988-01-01

    We used molecular cloning to isolate a functional gene for mouse ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) from a cell line in which that gene had been selectively amplified. The position of the 5' terminus of the mRNA was identified, and the coding sequence was shown to be preceded by a 312- or 313-nucleotide (nt) untranslated leader. The latter is highly G + C rich, particularly in its 5'-most portion. The leader can be anticipated to have extensive and stable secondary structure. The transcription unit of the gene is of relatively small size, approximately equal to 6.2 kilobases (kb) from the start site to the proximal site of polyadenylylation. Sequence analysis of DNA near the transcription start position demonstrated the presence of a "TATA" box, but no "CAAT" box. Functional properties of the cloned gene were tested by transfecting it into cultured cells. Expression of the putative full-length gene efficiently conferred ornithine decarboxylase activity on recipient mutant cells deficient in that activity. To assess the function and strength of the OrnDCase promoter region and to delimit its boundaries, we used a transient expression assay. Upstream of a bacterial chloramphenicol acetyltransferase gene was placed a portion of the OrnDCase gene, including the presumed promoter region, spanning a region from approximately equal to 3.0 kb 5' of the site of transcription initiation to the first 250 nt of the transcript. When expressed in mouse NIH 3T3 cells, this OrnDCase genomic element was comparable in strength to the Rous sarcoma virus long terminal repeat promoter. A similar construct, truncated so as to retain only 264 base pairs of the OrnDCase gene 5' to the site of transcription start, yielded undiminished levels of expression. Images PMID:3353375

  14. On the Relationship between Ribulose Diphosphate Carboxylase and Protochlorophyllide Holochrome of Phaseolus vulgaris Leaves 1

    PubMed Central

    Akoyunoglou, G.; Argyroudi-Akoyunoglou, J. H.; Guiali, A.; Dassiou, C.

    1970-01-01

    The relationship between ribulose diphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase [dimerizing], EC 4.1.1.39, formerly known as carboxydismutase) and protochlorophyllide holochrome of etiolated Phaseolus vulgaris leaves has been studied. A procedure for partially selective extraction of the two proteins was devised using tris-HCl buffer first without and then with Triton X-100. Ribulose diphosphate carboxylase was readily extracted from etiolated bean leaves without Triton X-100, and protochlorophyllide holochrome was extracted on the addition of Triton X-100. Optimal extraction conditions for protochlorophyllide holochrome have been found to be different for tissues of different ages. PMID:5427114

  15. A novel rearrangement in ESI-MSn of spirocyclic pentaerythritol di(phosphate monoamides)

    NASA Astrophysics Data System (ADS)

    Ju, Zhi-Yu; Ye, Yong; Zhong, Shang-Bin; Zou, Ru-Yi; Liao, Xin-Cheng; Zhao, Yu-Fen

    2008-06-01

    Several spirocyclic pentaerythritol di(phosphate monoamides) as intumescent flame retardants were synthesized and analyzed by electrospray ionization multistage tandem mass spectrometry. A novel amino group migration from the phosphoryl group to the two methylenes was observed. This migration is believed to be a general pathway for ions with the small size and electronic donating alkyl groups of spirocyclic pentaerythritol di(phosphate monoamides), which is assisted by twice nucleophilic substitutions. Steric and electronic effects of alkyl groups might be a key factor responsible for this migration.

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

    SciTech Connect

    Compagno, C.; Tura, A.; Ranzi, B.M.; Martegani, E. )

    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.

  17. Histidine decarboxylase, DOPA decarboxylase, and vesicular monoamine transporter 2 expression in neuroendocrine tumors: immunohistochemical study and gene expression analysis.

    PubMed

    Uccella, Silvia; Cerutti, Roberta; Vigetti, Davide; Furlan, Daniela; Oldrini, Rita; Carnevali, Ileana; Pelosi, Giuseppe; La Rosa, Stefano; Passi, Alberto; Capella, Carlo

    2006-08-01

    Histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (v-MAT2) are involved in the biosynthesis and storage of histamine. DOPA decarboxylase (DDC) is involved in the biosynthesis of a variety of amines and shares a high degree of homology with HDC. HDC and v-MAT2 immunoreactivities (IR) have recently been detected in well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroendocrine carcinomas (PDNECs) of various sites and have been proposed as general endocrine markers. We evaluated HDC and v-MAT2 IR in a series of 117 WDNETs and PDNECs from different sites. Western blotting analysis was performed to verify the specificity of anti-DDC and anti-HDC antibodies. Real-time RT-PCR was performed using specific probes for HDC and DDC on 42 cases, examined also for DDC IR. HDC and v-MAT2 IR were observed in the majority of WDNETs and PDNECs of all sites and HDC-IR cases were always also DDC-IR. In contrast, high levels of HDC mRNA were detected only in the gastroenteropancreatic WDNETs, which did not show increased DDC mRNA levels. On the other hand, bronchial carcinoids and lung PDNECs showed high DDC mRNA levels, but nearly undetectable HDC mRNA levels. Western blotting analysis showed a cross-reaction between anti-HDC and anti-DDC antibodies. HDC should not be considered as a general endocrine marker and HDC IR in bronchial carcinoids and PDNECs of the lung can probably be attributed to a cross-reaction with DDC.

  18. The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta[W

    PubMed Central

    Orlova, Irina; Nagegowda, Dinesh A.; Kish, Christine M.; Gutensohn, Michael; Maeda, Hiroshi; Varbanova, Marina; Fridman, Eyal; Yamaguchi, Shinjiro; Hanada, Atsushi; Kamiya, Yuji; Krichevsky, Alexander; Citovsky, Vitaly; Pichersky, Eran; Dudareva, Natalia

    2009-01-01

    Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta. PMID:20028839

  19. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase.

    PubMed

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian

    2017-01-01

    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5'-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase.

  20. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase

    PubMed Central

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian

    2017-01-01

    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5′-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase. PMID:28232911

  1. A Liquid-Based Colorimetric Assay of Lysine Decarboxylase and Its Application to Enzymatic Assay.

    PubMed

    Kim, Yong Hyun; Sathiyanarayanan, Ganesan; Kim, Hyun Joong; Bhatia, Shashi Kant; Seo, Hyung-Min; Kim, Jung-Ho; Song, Hun-Seok; Kim, Yun-Gon; Park, Kyungmoon; Yang, Yung-Hun

    2015-12-28

    A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

  2. Coenzyme A biosynthesis: steric course of 4'-phosphopantothenoyl-L-cysteine decarboxylase.

    PubMed

    Aberhart, D J; Ghoshal, P K; Cotting, J A; Russell, D J

    1985-12-03

    4'-Phosphopantothenoyl-L-cysteine decarboxylase (PPC decarboxylase) was partially purified from rat liver. 4'-Phosphopantothenoyl[2-2H1]-L-cysteine was synthesized and converted by PPC decarboxylase to 4'-phosphol[1-2H1]pantetheine. The product was degraded by reduction with Raney nickel followed by acidic hydrolysis to [1-2H1]ethylamine. The latter was converted to the (-)-camphanamide derivative, NMR studies of which revealed that the deuterium was located in the pro-1S position. Also, unlabeled 4'-phosphopantothenoyl-L-cysteine was incubated with PPC decarboxylase in D2O, giving, after degradation, the (-)-camphanamide of (1R)-[1-2H1]ethylamine. The results show that the decarboxylation takes place with retention of configuration. These results are discussed in terms of possible mechanisms for the decarboxylation.

  3. Regulation of glutamic acid decarboxylase mRNA expression in rat brain after sertraline treatment.

    PubMed

    Giardino, L; Zanni, M; Bettelli, C; Savina, M A; Calzà, L

    1996-09-26

    We now investigated the effect of chronic treatment with sertraline on glutamic acid decarboxylase mRNA expression in different rat brain areas by means of in situ hybridization. We found a reduced glutamic acid decarboxylase mRNA expression in the prefrontal cortex, accumbens nucleus, olfactory tubercle and reticular nucleus of the thalamus. The involvement of presynaptic modulation of gamma-amino-butyric acid transmission in the anxiolytic effect of sertraline is discussed.

  4. Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

    PubMed

    Verimli, Ural; Sehirli, Umit S

    2016-09-01

    The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p < 0.0001 for LS, p < 0.01 for MS). This study is the first to reveal the dominance of glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

  5. Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes.

    PubMed

    Bassez, T; Paris, J; Omilli, F; Dorel, C; Osborne, H B

    1990-11-01

    The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

  6. Structure and Function of 4-Hydroxyphenylacetate Decarboxylase and Its Cognate Activating Enzyme.

    PubMed

    Selvaraj, Brinda; Buckel, Wolfgang; Golding, Bernard T; Ullmann, G Matthias; Martins, Berta M

    2016-01-01

    4-Hydroxyphenylacetate decarboxylase (4Hpad) is the prototype of a new class of Fe-S cluster-dependent glycyl radical enzymes (Fe-S GREs) acting on aromatic compounds. The two-enzyme component system comprises a decarboxylase responsible for substrate conversion and a dedicated activating enzyme (4Hpad-AE). The decarboxylase uses a glycyl/thiyl radical dyad to convert 4-hydroxyphenylacetate into p-cresol (4-methylphenol) by a biologically unprecedented Kolbe-type decarboxylation. In addition to the radical dyad prosthetic group, the decarboxylase unit contains two [4Fe-4S] clusters coordinated by an extra small subunit of unknown function. 4Hpad-AE reductively cleaves S-adenosylmethionine (SAM or AdoMet) at a site-differentiated [4Fe-4S]2+/+ cluster (RS cluster) generating a transient 5'-deoxyadenosyl radical that produces a stable glycyl radical in the decarboxylase by the abstraction of a hydrogen atom. 4Hpad-AE binds up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert that is C-terminal to the RS cluster-binding motif. The ferredoxin-like domain with its two auxiliary clusters is not vital for SAM-dependent glycyl radical formation in the decarboxylase, but facilitates a longer lifetime for the radical. This review describes the 4Hpad and cognate AE families and focuses on the recent advances and open questions concerning the structure, function and mechanism of this novel Fe-S-dependent class of GREs.

  7. Purification and Some Properties of Chlorella fusca Ribulose 1,5-Diphosphate Carboxylase

    PubMed Central

    Lord, J. Michael; Brown, Richard H.

    1975-01-01

    Ribulose 1,5-diphosphate carboxylase has been purified from extracts of autotrophically grown Chlorella fusca by ammonium sulfate precipitation and centrifugation on a linear sucrose density gradient. The enzyme was homogeneous by the criterion of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 530,000, and it was composed of two types of subunit of molecular weight 53,000 and 14,000. Ribulose 1,5-diphosphate, CO2, and Mg2+ had Michaelis constant values of 15 μm, 0.3 mm, and 0.37 mm, respectively. At high bicarbonate concentration (17 mm and 50 mm), 6-phosphogluconate inhibited the enzyme, the inhibition being noncompetitive with respect to ribulose 1,5-diphosphate (Ki 0.065 mm), whereas at low bicarbonate concentration (1 mm), 6-phosphogluconate activated the enzyme. Oxygen was a competitive inhibitor with respect to CO2, suggesting the enzyme also functions as an oxygenase. This was confirmed by direct assay, a 1: 1 stoichiometry between ribulose 1,5-diphosphate consumed and O2 uptake being observed. PMID:16659083

  8. Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity*

    PubMed Central

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

    2014-01-01

    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. PMID:25378387

  9. Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain)

    PubMed Central

    Stribling, Donald; Perham, Richard N.

    1973-01-01

    Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca2+, Zn2+. It is a dimer with a molecular weight of approx. 70000, and the Km value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH4 in the presence of substrate. The Km value for fructose diphosphate is about 20μm (although the Lineweaver–Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed. PMID:4198624

  10. Dolichyl-phosphate phosphatase and dolichyl-diphosphate phosphatase in rat-liver microsomes.

    PubMed

    Belocopitow, E; Boscoboinik, D

    1982-06-15

    Dolichyl-phosphate phosphatase and dolichyl-diphosphate phosphatase activities of a liver-cell microsomal preparation were solubilized by treatment with Triton X-100. The 100,000 X g supernatant was then passed through a column of Sepharose-4B--concanavalin A. Both enzyme activities were found in the percolate. This treatment eliminated inhibition by ATP and glucose 6-phosphate in both phosphatase activities. In each case the activities were inhibited by higher concentrations of enzyme preparation due to the presence of phospholipids. The inhibitory effects of either phosphatidylcholine or phosphatidylethanolamine were due to competition for detergent. On the other hand, the effect produced by phosphatidic acid appeared to be different, since it did not change the optimal concentration of Triton X-100 for the two enzymes. Dolichyl-phosphate phosphatase was strongly inhibited by both Pi and PPi, whereas dolichyl-diphosphate phosphatase was only slightly inhibited by Pi and not at all by PPi. Dolichyl-diphosphate phosphatase was more inhibited by divalent cations than dolichyl-phosphate phosphatase. The apparent Km of dolichyl-phosphate phosphatase for dolichyl phosphate was 0.15 mM. Dolichol also inhibited dolichyl-phosphate phosphatase, but it produced a stronger inhibition on dolichyl-diphosphate phosphatase. The inhibitory effect of dolichol was not entirely due to detergent competition.

  11. Guanosine Diphosphate-l-Fucose Glycopeptide Fucosyltransferase Activity in Corynebacterium insidiosum1

    PubMed Central

    Sadowski, Peter L.; Strobel, Gary A.

    1973-01-01

    The biosynthesis of a phytotoxic glycopeptide of Corynebacterium insidiosum involves guanosine diphosphate-l-fucosyltransferase activity. This enzyme activity is most consistently associated with the cellular membranes fraction. The optimal pH for the transfer reaction is 7.5. The partially hydrolyzed toxin serves as an acceptor (primer) of l-fucose. PMID:4199136

  12. Cloning, expression and characterization of an insect geranylgeranyl diphosphate synthase from Choristoneura fumiferana.

    PubMed

    Barbar, Aline; Couture, Manon; Sen, Stephanie E; Béliveau, Catherine; Nisole, Audrey; Bipfubusa, Marie; Cusson, Michel

    2013-10-01

    Geranylgeranyl diphosphate synthase (GGPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C5), with allylic diphosphates to generate the C20 prenyl chain (GGPP) used for protein prenylation and diterpenoid biosynthesis. Here, we cloned the cDNA of a GGPPS from the spruce budworm, Choristoneura fumiferana, and characterized the corresponding recombinant protein (rCfGGPPS). As shown for other type-III GGPPSs, rCfGGPPS preferred farnesyl diphosphate (FPP; C15) over other allylic substrates for coupling with IPP. Unexpectedly, rCfGGPPS displayed inhibition by its FPP substrate at low IPP concentration, suggesting the existence of a mechanism that may regulate intracellular FPP pools. rCfGGPPS was also inhibited by its product, GGPP, in a competitive manner with respect to FPP, as reported for human and bovine brain GGPPSs. A homology model of CfGGPPS was prepared and compared to human and yeast GGPPSs. Consistent with its enzymological properties, CfGGPPS displayed a larger active site cavity that can accommodate the binding of FPP and GGPP in the region normally occupied by IPP and the allylic isoprenoid tail, and the binding of GGPP in an alternate orientation seen for GGPP binding to the human protein. To begin exploring the role of CfGGPPS in protein prenylation, its transcripts were quantified by qPCR in whole insects, along with those of other genes involved in this pathway. CfGGPPS was expressed throughout insect development and the abundance of its transcripts covaried with that of other prenylation-related genes. Our qPCR results suggest that geranylgeranylation is the predominant form of prenylation in whole C. fumiferana. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  13. Characterization of an isopentenyl diphosphate isomerase involved in the juvenile hormone pathway in Aedes aegypti.

    PubMed

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

    2012-10-01

    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); afterward 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 Mg(2+) or Mn(2+) but not Zn(2+) 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.

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

    PubMed

    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.

  15. Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis.

    PubMed Central

    Candy, J M; Duggleby, R G

    1994-01-01

    Several enzymes require thiamin diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas

  16. Biosynthesis of monoterpenes and norisoprenoids in raspberry fruits (Rubus idaeus L.): the role of cytosolic mevalonate and plastidial methylerythritol phosphate pathway.

    PubMed

    Hampel, Daniela; Swatski, Anna; Mosandl, Armin; Wüst, Matthias

    2007-10-31

    The biosynthesis of the monoterpenes (-)-alpha-pinene, linalool, and the norisoprenoids alpha- and beta-ionone in raspberry fruits (rubus idaeus L.) was investigated by in vivo feeding experiments with [5,5-(2)H2]-mevalonic acid lactone and [5,5-(2)H2]-1-deoxy-D-xylulose. The volatile compounds were extracted by stirbar sorptive extraction and analyzed using thermal desorption-multidimensional gas chromatography-mass spectrometry (TD-enantio-MDGC-MS). The feeding experiments demonstrate that (-)-alpha-pinene and (S)-linalool are exclusively synthesized via the cytosolic mevalonic acid pathway. In contrast, (2)H-labeled (R)-(E)-alpha-ionone and (2)H-labeled (E)-beta-ionone are detectable after application of d2-1-deoxy-D-xylulose and d2-mevalonic acid lactone, respectively. However, (R)-linalool reveals no incorporation of either one of the fed precursors, even though this enantiomer is detectable in the fruit tissue.

  17. Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation.

    PubMed Central

    Konrad, Zvia; Eichler, Jerry

    2002-01-01

    Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg(2+)-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein. To test whether lipid modification of the S-layer glycoprotein could explain these observations, H. volcanii cells were incubated with a radiolabelled precursor of isoprene, [(3)H]mevalonic acid. In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification. In the present study we report that the H. volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis. These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e. following protein translocation across the membrane. PMID:12069685

  18. Glucose elevates ornithine decarboxylase expression in Vero cells.

    PubMed

    Lundgren, D W; Prokay, S L

    1988-12-01

    The addition of Earle's balanced salt solution (EBSS) of amino acids that are transported by a Na+-dependent cotransport system was not required by Vero cells for ornithine decarboxylase (ODC:EC 4.1.1.17) amplification. Vero cell ODC activity was elevated tenfold above basal levels when confluent cells were incubated for 5 hr in EBSS alone. ODC activity increased as a function of the incubation time in EBSS and was not elevated above basal enzyme levels when cells were incubated in EBSS minus glucose. ODC expression increased as a function of the glucose concentration in EBSS, with 20 mM glucose producing a 90-fold increase in ODC activity. ODC expression is more responsive to glucose in high-density quiescent cultures than in low-density growing cultures. Enhanced ODC expression by glucose depended on Na+ and K+ concentrations. The specific activity of ODC was also elevated above basal levels when mannose or fructose replaced glucose in EBSS. The addition of alanine or asparagine to EBSS enhanced ODC activity above levels obtained with EBSS containing standard (5.5 mM) glucose concentrations. In the absence of glucose, alanine was more effective than asparagine in enhancing ODC expression. These results suggest that the transport of amino acids is not an absolute requirement for Vero cell ODC expression and that ODC expression is linked to changes in cellular energetics and/or ion fluxes.

  19. Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

    SciTech Connect

    Kanerva, Kristiina; Maekitie, Laura T.; Baeck, Nils; Andersson, Leif C.

    2010-07-01

    Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

  20. Expression of arginine decarboxylase in brain regions and neuronal cells

    PubMed Central

    Iyo, Abiye H.; Zhu, Meng-Yang; Ordway, Gregory A.; Regunathan, Soundar

    2010-01-01

    After our initial report of a mammalian gene for arginine decarboxylase, an enzyme for the synthesis of agmatine from arginine, we have determined the regional expression of ADC in rat. We have analyzed the expression of ADC in rat brain regions by activity, protein and mRNA levels, and the regulation of expression in neuronal cells by RNA interference. In rat brain, ADC was widely expressed in major brain regions, with a substantial amount in hypothalamus, followed by cortex, and with least amounts in locus coeruleus and medulla. ADC mRNA was detected in primary astrocytes and C6 glioma cells. While no ADC message was detected in fresh neurons (3 days old), significant message appeared in differentiated neurons (3 weeks old). PC12 cells, treated with nerve growth factor, had higher ADC mRNA compared with naive cells. The siRNA mixture directed towards the N-terminal regions of ADC cDNA down-regulated the levels of mRNA and protein in cultured neurons/C6 glioma cells and these cells produced lower agmatine. Thus, this study demonstrates that ADC message is expressed in rat brain regions, that it is regulated in neuronal cells and that the down-regulation of ADC activity by specific siRNA leads to lower agmatine production. PMID:16445852

  1. Interaction of NAP-22 with brain glutamic acid decarboxylase (GAD).

    PubMed

    Maekawa, Shohei; Kobayashi, Yuumi; Odagaki, Sin-Ichi; Makino, Midori; Kumanogoh, Haruko; Nakamura, Shun; Morita, Mitsuhiro; Hayashi, Fumio

    2013-03-14

    NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched protein localized mainly in the synaptic vesicles and the synaptic plasma membrane. Biochemically, it is recovered in the lipid raft fraction. In order to understand the physiological function of the neuronal lipid raft, NAP-22 binding proteins were screened with a pull-down assay. Glutamic acid decarboxylase (GAD) was detected through LC-MS/MS, and Western blotting using a specific antibody confirmed the result. Two isoforms of GAD, GAD65 and GAD67, were expressed in bacteria as GST-fusion forms and the interaction with NAP-22 was confirmed in vitro. Partial co-localization of NAP-22 with GAD65 and GAD67 was also observed in cultured neurons. The binding showed no effect on the enzymatic activity of GAD65 and GAD67. These results hence suggest that NAP-22 could participate in the transport of GAD65 and GAD67 to the presynaptic termini and their retention on the synaptic vesicles as an anchoring protein.

  2. Reactivation of substrate-inactivated brain glutamate decarboxylase.

    PubMed

    Meeley, M P; Martin, D L

    1983-03-01

    The effects of ATP and inorganic phosphate (Pi) on the reactivation of glutamate apodecarboxylase by its cofactor pyridoxal-5'-phosphate (pyridoxal-P) was studied. Apoenzyme was prepared by preincubation with glutamate. Apoenzyme prepared with glutamate alone was reactivated slowly and incompletely by adding a saturating concentration of pyridoxal-P (20 microM). Reactivation was slightly enhanced by 1-10 mM Pi. Reactivation by pyridoxal-P plus Pi was greatly enhanced by the presence of low concentrations (less than 100 microM) of ATP during the preparation of apoenzyme with glutamate. Reactivation was much lower if Pi was omitted. Enhancement of reactivation by ATP was due to its effect during apoenzyme formation, since ATP did not enhance reactivation if added only during reactivation and since the enhancing effect persisted after the removal of free ATP by chromatography on Sephadex G-25 after apoenzyme preparation and before reactivation. Reactivation was inhibited by high concentrations of ATP (greater than 100 microM), possibly by competition of ATP for the cofactor binding site. Four factors (glutamate, pyridoxal-P, ATP, and Pi) control a cycle of inactivation and reactivation that appears to be important in the regulation of brain glutamate decarboxylase.

  3. Anti-glutamic acid decarboxylase antibody positive neurological syndromes

    PubMed Central

    Tohid, Hassaan

    2016-01-01

    A rare kind of antibody, known as anti-glutamic acid decarboxylase (GAD) autoantibody, is found in some patients. The antibody works against the GAD enzyme, which is essential in the formation of gamma aminobutyric acid (GABA), an inhibitory neurotransmitter found in the brain. Patients found with this antibody present with motor and cognitive problems due to low levels or lack of GABA, because in the absence or low levels of GABA patients exhibit motor and cognitive symptoms. The anti-GAD antibody is found in some neurological syndromes, including stiff-person syndrome, paraneoplastic stiff-person syndrome, Miller Fisher syndrome (MFS), limbic encephalopathy, cerebellar ataxia, eye movement disorders, and epilepsy. Previously, excluding MFS, these conditions were called ‘hyperexcitability disorders’. However, collectively, these syndromes should be known as “anti-GAD positive neurological syndromes.” An important limitation of this study is that the literature is lacking on the subject, and why patients with the above mentioned neurological problems present with different symptoms has not been studied in detail. Therefore, it is recommended that more research is conducted on this subject to obtain a better and deeper understanding of these anti-GAD antibody induced neurological syndromes. PMID:27356651

  4. Chemical modification of oxalate decarboxylase to improve adsorption capacity.

    PubMed

    Lin, Rihui; He, Junbin; Wu, Jia; Cai, Xinghua; Long, Han; Chen, Shengfeng; Liu, Haiqian

    2017-05-01

    In order to enhance the adsorption capacity of oxalate decarboxylase (Oxdc) on calcium oxalate monohydrate crystals and improve the application performance of Oxdc, chemical modification of Oxdc with ethylenediaminetetraacetic dianhydride (EDTAD) was investigated in this work. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography tandem mass spectrometry (LC/MS) analysis results demonstrated that Oxdc and EDTAD have been covalently bound, and suggested that the chemical modification occurred at the free amino of the side chain and the α-amine of the N-terminus of Oxdc. Fluorescene and circular dichroic measurement showed that the structure and conformation of Oxdc were tinily altered after modification by EDTAD. The optimum pH of EDTAD-modified Oxdc was shifted to the alkaline side about 1.5 unit and it has a higher thermostability. The analysis of kinetic parameters indicated that the EDTAD-modified Oxdc showed a higher affinity towards the substrate. Through modification the adsorption capacity of Oxdc onto CaOx monohydrate crystals was increased by 42.42%. Copyright © 2017. Published by Elsevier B.V.

  5. Histidine Decarboxylase Deficiency Prevents Autoimmune Diabetes in NOD Mice

    PubMed Central

    Alkan, Manal; Machavoine, François; Rignault, Rachel; Dam, Julie; Dy, Michel; Thieblemont, Nathalie

    2015-01-01

    Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the nonobese diabetic (NOD) mouse model. To this end, we used mice (inactivated) knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC−/− mice decreased the incidence of diabetes in relation to their wild-type counterpart. Whereas the proportion of regulatory T and myeloid-derived suppressive cells was similar in both strains, histamine deficiency was associated with increased levels of immature macrophages, as compared with wild-type NOD mice. Concerning the cytokine pattern, we found a decrease in circulating IL-12 and IFN-γ in HDC−/− mice, while IL-6 or leptin remained unchanged, suggesting that histamine primarily modulates the inflammatory environment. Paradoxically, exogenous histamine given to NOD HDC−/− mice provided also protection against T1D. Our study supports the notion that histamine is involved in the pathogenesis of diabetes, thus providing additional evidence for its role in the regulation of the immune response. PMID:26090474

  6. Nitrogen isotope effects on glutamate decarboxylase from Escherichia coli

    SciTech Connect

    Abell, L.M.; O'Leary, M.H.

    1988-05-03

    The nitrogen isotope effect on the decarboxylation of glutamic acid by glutamate decarboxylase from Escherichia coli has been measured by comparison of the isotopic composition of the amino nitrogen of the product ..gamma..-aminobutyric acid isolated after 10-20% reaction with that of the starting glutamic acid. At pH 4.7, 37 /sup 0/C, the isotope effect is k/sup 14//k/sup 15/ = 0.9855 +/- 0.0006 when compared to unprotonated glutamic acid. Interpretation of this result requires knowledge of the equilibrium nitrogen isotope effect for Schiff base formation. This equilibrium isotope effect is K/sup 14//K/sup 15/ - 0.9824 for the formation of the unprotonated Schiff base between unprotonated valine and salicylaldehyde. Analysis of the nitrogen isotope effect on decarboxylation of glutamic acid and of the previously measured carbon isotope effect on this same reaction shows that decarboxylation and Schiff base formation are jointly rate limiting. The enzyme-bound Schiff base between glutamate and pyridoxal 5'-phosphate partitions approximately 2:1 between decarboxylation and return to the starting state. The nitrogen isotope effect also reveals that the Schiff base nitrogen is protonated in this intermediate.

  7. The Origin of the electrostatic Perturbation in Acetoacetate Decarboxylase

    SciTech Connect

    Ho, M.; Menetret, J; Tsuruta, H; Allen, K

    2009-01-01

    Acetoacetate decarboxylase (AADase) has long been cited as the prototypical example of the marked shifts in the pKa values of ionizable groups that can occur in an enzyme active site. In 1966, it was hypothesized that in AADase the origin of the large pKa perturbation (-4.5 log units) observed in the nucleophilic Lys 115 results from the proximity of Lys 116, marking the first proposal of microenvironment effects in enzymology. The electrostatic perturbation hypothesis has been demonstrated in a number of enzymes, but never for the enzyme that inspired its conception, owing to the lack of a three-dimensional structure. Here we present the X-ray crystal structures of AADase and of the enamine adduct with the substrate analogue 2,4-pentanedione. Surprisingly, the shift of the pKa of Lys 115 is not due to the proximity of Lys 116, the side chain of which is oriented away from the active site. Instead, Lys 116 participates in the structural anchoring of Lys 115 in a long, hydrophobic funnel provided by the novel fold of the enzyme. Thus, AADase perturbs the pKa of the nucleophile by means of a desolvation effect by placement of the side chain into the protein core while enforcing the proximity of polar residues, which facilitate decarboxylation through electrostatic and steric effects.

  8. Amiloride inhibits rat mucosal ornithine decarboxylase activity and DNA synthesis

    SciTech Connect

    Ulrich-Baker, M.G.; Wang, P.; Fitzpatrick, L.; Johnson, L.R. )

    1988-03-01

    Refeeding fasted rats induces a dramatic trophic response in gastrointestinal mucosa and is associated with elevations in both rate of DNA synthesis and ornithine decarboxylase (ODC) activity. The signal for these increases is unknown. Amiloride prevents cell alkalinization by blocking Na{sup +}-H{sup +} exchange at apical epithelial cell membranes. In study 1, rats were fasted 48 h, treated with amiloride (0.5 to 500 mg/kg), and refed for 4 h. Refeeding increased ODC activities in the jejunal mucosa (X8) and liver (X19) but not in the oxyntic gland mucosa. In the jejunum, but not the liver, the activation of ODC was completely abolished by 100 mg/kg amiloride. In study 2, the rate of DNA synthesis was determine by measuring the rate of ({sup 3}H)thymidine incorporation 16 h after refeeding. Refeeding resulted in significantly increased rates of DNA synthesis over fasted levels, and amiloride at 100 mg/kg significantly reduced the elevations in the jejenum and liver. In conclusion, amiloride inhibits the postprandial increases in jejunal ODC activity and DNA synthesis in the jejunum and liver. The results indicate that (1) the Na{sup +}-H{sup +} antiport is essential to the increased ODC activity in the jejunum and liver after a meal and (2) increases in DNA synthesis and their suppression by amiloride are not necessary linked to ODC activity.

  9. Multiple roles of the active site lysine of Dopa decarboxylase.

    PubMed

    Bertoldi, Mariarita; Voltattorni, Carla Borri

    2009-08-15

    The pyridoxal 5'-phosphate dependent-enzyme Dopa decarboxylase, responsible for the irreversible conversion of l-Dopa to dopamine, is an attractive drug target. The contribution of the pyridoxal-Lys303 to the catalytic mechanisms of decarboxylation and oxidative deamination is analyzed. The K303A variant binds the coenzyme with a 100-fold decreased apparent equilibrium binding affinity with respect to the wild-type enzyme. Unlike the wild-type, K303A in the presence of l-Dopa displays a parallel progress course of formation of both dopamine and 3,4-dihydroxyphenylacetaldehyde (plus ammonia) with a burst followed by a linear phase. Moreover, the finding that the catalytic efficiencies of decarboxylation and of oxidative deamination display a decrease of 1500- and 17-fold, respectively, with respect to the wild-type, is indicative of a different impact of Lys303 mutation on these reactions. Kinetic analyses reveal that Lys303 is involved in external aldimine formation and hydrolysis as well as in product release which affects the rate-determining step of decarboxylation.

  10. Molecular cloning and expression of the mouse ornithine decarboxylase gene.

    PubMed Central

    McConlogue, L; Gupta, M; Wu, L; Coffino, P

    1984-01-01

    We used mRNA from a mutant S49 mouse lymphoma cell line that produces ornithine decarboxylase (OrnDCase) as its major protein product to synthesize and clone cDNA. Plasmids containing OrnDCase cDNA were identified by hybrid selection of OrnDCase mRNA and in vitro translation. The two of these with the largest inserts together span 2.05 kilobases of cDNA. Southern blot analysis of DNA from wild-type or mutant S49 cells, cleaved with EcoRI or with BamHI, revealed multiple bands homologous to OrnD-Case cDNA, only one of which was amplified in the mutant cells. RNA transfer blot analysis showed that the major OrnD-Case mRNA in the mouse lymphoma cells is 2.0 kilobases long. A similar size mRNA was found in mouse kidney and was more abundant in the kidneys of mice treated with testosterone, an inducer of OrnDCase activity in that tissue. Images PMID:6582509

  11. Glutamate decarboxylase from Lactobacillus brevis: activation by ammonium sulfate.

    PubMed

    Hiraga, Kazumi; Ueno, Yoshie; Oda, Kohei

    2008-05-01

    In this study, the glutamate decarboxylase (GAD) gene from Lactobacillus brevis IFO12005 (Biosci. Biotechnol. Biochem., 61, 1168-1171 (1997)), was cloned and expressed. The deduced amino acid sequence showed 99.6% and 53.1% identity with GAD of L. brevis ATCC367 and L. lactis respectively. The His-tagged recombinant GAD showed an optimum pH of 4.5-5.0, and 54 kDa on SDS-PAGE. The GAD activity and stability was significantly dependent on the ammonium sulfate concentration, as observed in authentic GAD. Gel filtration showed that the inactive form of the GAD was a dimer. In contrast, the ammonium sulfate-activated form was a tetramer. CD spectral analyses at pH 5.5 revealed that the structures of the tetramer and the dimer were similar. Treatment of the GAD with high concentrations of ammonium sulfate and subsequent dilution with sodium glutamate was essential for tetramer formation and its activation. Thus the biochemical properties of the GAD from L. brevis IFO12005 were significantly different from those from other sources.

  12. Ornithine decarboxylase as a marker for premalignancy in the stomach.

    PubMed Central

    Patchett, S E; Alstead, E M; Butruk, L; Przytulski, K; Farthing, M J

    1995-01-01

    Assessment of mucosal ornithine decarboxylase (ODC) activity in the human large bowel may be of value as a marker of potential malignant risk. Its value as a marker of premalignancy in the upper gastrointestinal tract is less clear. Using a [14C]-ornithine bioassay, gastric mucosal ODC activity was measured in 32 normal subjects and 22 patients with confirmed gastric cancer. These results were compared with 47 patients at increased risk of upper gastrointestinal malignancy, (32 patients with partial gastric resection, 15 patients with familial adenomatous polyposis). Median ODC activity in normal subjects was 371 pmol/mg protein/h, (interquartile range (IQR), 230-617). There was no variation with age or sex and no relation to Helicobacter pylori status. Normal subjects had significantly lower ODC activity than patients with a gastric resection or confirmed gastric cancer, but similar to patients with familial adenomatous polyposis. Furthermore, no difference in activity was identified between patients with a gastric resection and established gastric cancer. ODC activity was, however, significantly increased in areas of gastric atrophy or intestinal metaplasia, regardless of the clinical group from which the samples were obtained. It is concluded that measurement of mucosal ODC activity does not provide additional predictive information of malignant risk in the stomach and investigation of other potential biomarkers of malignancy is warranted. PMID:7672662

  13. Ornithine decarboxylase encoded by chlorella virus PBCV-1.

    PubMed

    Morehead, Tiara A; Gurnon, James R; Adams, Byron; Nickerson, Kenneth W; Fitzgerald, Lisa A; Van Etten, James L

    2002-09-15

    Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A207R, which encodes a protein with 37-41% amino acid identity to ornithine decarboxylase (ODC) from many eukaryotic organisms. The a207r gene was cloned and the protein was expressed as a His-A207R fusion protein in Escherichia coli. The recombinant protein catalyzes pyridoxal 5'-phosphate-dependent decarboxylation of ornithine to putrescine, the first step in the polyamine biosynthetic pathway. The enzyme has a pH optimum of 9.0 and a temperature optimum of 42 degrees C, and it requires dithiothreitol for maximal activity. The enzyme has a K(m) for ornithine of 0.78 mM and a specific activity of 100 micromol/min/mg protein. PBCV-1 ODC is quite sensitive to the competitive inhibitor L-arginine and the irreversible inhibitor difluoromethylarginine but it is less sensitive to the irreversible inhibitor difluoromethylornithine. The a207r gene is expressed both early and late in PBCV-1 infection and is highly conserved among the chlorella viruses. The 42-kDa PBCV-1 ODC (372 amino acids) is the smallest ODC in the databases and, to our knowledge, is the first virus-encoded ODC.

  14. Studies on uroporphyrinogen decarboxylase from Chlorella kessleri (Trebouxiophyceae, Chlorophyta).

    PubMed

    Juárez, Angela B; Aldonatti, Carmen; Vigna, María S; Ríos de Molina, María Del C

    2007-02-01

    Uroporphyrinogen decarboxylase (UroD) (EC 4.1.1.37) is an enzyme from the tetrapyrrole biosynthetic pathway, in which chlorophyll is the main final product in algae. This is the first time that a study on UroD activity has been performed in a green alga (Chlorella). We isolated and partially purified the enzyme from a Chlorella kessleri (Trebouxiophyceae, Chlorophyta) strain (Copahue, Neuquén, Argentina), and describe for the first time some of its properties. In C. kessleri, the decarboxylation of uroporphyrinogen III occurs in two stages, via 7 COOH and then 6 and 5 COOH intermediates, with the decarboxylation of the 7 COOH compound being the rate-limiting step for the reaction. Cultures in the exponential growth phase showed the highest specific activity values. The most suitable conditions to measure UroD activity in C. kessleri were as follows: 0.23-0.3 mg protein/mL, approximately 6-8 micromol/L uroporphyrinogen III, and 20 min incubation time. Gel filtration chromatography and Western blot assays indicated that UroD from C. kessleri is a dimer of approximately 90 kDa formed by species of lower molecular mass, which conserves enzymatic activity.

  15. Expression of ornithine decarboxylase in precancerous and cancerous gastric lesions

    PubMed Central

    Miao, Xin-Pu; Li, Jian-Sheng; Li, Hui-Yan; Zeng, Shi-Ping; Zhao, Ye; Zeng, Jiang-Zheng

    2007-01-01

    AIM: To investigate the expression of ornithine decarboxylase (ODC) in precancerous and cancerous gastric lesions. METHODS: We studied the expression of ODC in gastric mucosa from patients with chronic superficial gastritis (CSG, n = 32), chronic atrophic gastritis [CAG, n = 43; 15 with and 28 without intestinal metaplasia (IM)], gastric dysplasia (DYS, n = 11) and gastric cancer (GC, n = 48) tissues using immunohistochemical staining. All 134 biopsy specimens of gastric mucosa were collected by gastroscopy. METHODS: The positive rate of ODC expression was 34.4%, 42.9%, 73.3%, 81.8% and 91.7% in cases with CSG, CAG without IM, CAG with IM, DYS and GC, respectively (P < 0.01), The positive rate of ODC expression increased in the order of CSG < CAG (without IM) < CAG (with IM) < DYS and finally, GC. In addition, ODC positive immunostaining rate was lower in well-differentiated GC than in poorly-differentiated GC (P < 0.05). CONCLUSION: The expression of ODC is positively correlated with the degree of malignity of gastric mucosa and development of gastric lesions. This finding indicates that ODC may be used as a good biomarker in the screening and diagnosis of precancerous lesions. PMID:17569126

  16. Ornithine Decarboxylase Inhibition: A strategy to combat various diseases.

    PubMed

    Rai, Priyanshu R; Somani, Rakesh R; Kandpile, Pooja S

    2017-09-27

    Ornithine decarboxylase is the first enzyme in the polyamine biosynthetic pathway. It is the rate-limiting enzyme which is included in the change of ornithine to putrescine which is the first polyamine. Polyamines (putrescine, spermidine, spermine) are natural and synthetic compounds which contains two or more amino group. Polyamines are highly implicated in cellular functions such as cell-growth & multiplication, DNA stabilization, gene transcription and translation, ion-channel activity, etc. Elevated levels of polyamines were found in highly proliferating tumour cells. Hence inhibition of this enzyme was found useful in cancer. α-DL-difluoromethylornithine(DFMO) (Eflornithine) an enzyme-activated irreversible inhibitor was the first of this type. However its use as an anticancer agent did not continue for long due to various reasons. Polyamines were also found to play important role in other infectious microorganism. Eflornithine is successfully used in diseases such as African sleeping sickness and are being researched against number of tropical diseases. It is widely used against hirsutism in women. Various other product (putrscine) based analogues and transition state or PLP (cofactor) based analagoues are being synthesized against diseases such as Leishmaniasis, malaria and others discussed in the article. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. Arginase, Arginine Decarboxylase, Ornithine Decarboxylase, and Polyamines in Tomato Ovaries (Changes in Unpollinated Ovaries and Parthenocarpic Fruits Induced by Auxin or Gibberellin).

    PubMed Central

    Alabadi, D.; Aguero, M. S.; Perez-Amador, M. A.; Carbonell, J.

    1996-01-01

    Arginase (EC 3.5.3.1) activity has been found in the ovaries and Young fruits of tomato (Lycopersicon esculentum Mill. cv Rutgers).Changes in arginase, arginine decarboxylase (EC 4.1.1.19), and ornithine decarboxylase activity (EC 4.1.1.17) and levels of free and conjugated putrescine, spermidine, and spermine were determined in unpollinated ovaries and in parthenocarpic fruits during the early stages of development induced by 2,4-dichlorophenoxyacetic acid (2,4-D) or gibberellic acid (GA3). Levels of arginase, free spermine, and conjugates of the three polyamines were constant in unpollinated ovaries and characteristic of a presenescent step. A marked decrease in arginase activity, free spermine, and polyamine conjugates was associated with the initiation of fruit growth due to cell division, and when cell expansion was initiated, the absence of arginase indicated a redirection of nitrogen metabolism to the synthesis of arginine. A transient increase in arginine decarboxylase and ornithine decarboxylase was also observed in 2,4-D-induced fruits. In general, 2,4-D treatments produced faster changes than GA3, and without treatment, unpollinated ovaries developed only slightly and senescence was hardly visible. Sensitivity to 2,4-D and GA3 treatment remained for at least 2 weeks postanthesis. PMID:12226441

  18. Differential stimulation of S-adenosylmethionine decarboxylase by difluoromethylornithine in the rat colon and small intestine.

    PubMed Central

    Halline, A G; Dudeja, P K; Brasitus, T A

    1989-01-01

    The effects of chronic inhibition of ornithine decarboxylase (ODC) by the specific inhibitor difluoromethylornithine (DFMO) in the rat colon and small intestine on mucosal contents of polyamines, decarboxylated S-adenosylmethionine (decarboxylated AdoMet) and S-adenosylmethionine decarboxylase (AdoMet decarboxylase) activity were studied. Administration of 1% DFMO in the drinking water for 10 or 15 weeks resulted in inhibition of ODC and decreases in intracellular putrescine and spermidine contents in both proximal and distal segments of small intestine and colon. At both time points DFMO administration resulted in a dramatic stimulation of AdoMet decarboxylase activity and a rise in decarboxylated AdoMet content in the proximal and distal small-intestinal segments compared with controls, which was not seen in either colonic segment of DFMO-treated animals. This differential stimulation of AdoMet decarboxylase by DFMO in the small intestine and colon could not be entirely explained on the basis of differences in polyamine contents, which are known to regulate this enzyme activity. Kinetic and inhibition studies of AdoMet decarboxylase in control small and large intestine revealed that: (1) there was no difference in Vmax. values between the tissues; (2) the Km for AdoMet was higher in the small intestine than in the colon; and (3) the Ki for product inhibition by decarboxylated AdoMet was higher in the small intestine than in the colon. These results suggest that the differential stimulation of AdoMet decarboxylase by DFMO in the small intestine and colon may be due to different isoenzymes and could play a significant role in the regulation of polyamine contents throughout the gut. PMID:2497738

  19. Unique behavior of Trypanosoma cruzi mevalonate kinase: A conserved glycosomal enzyme involved in host cell invasion and signaling

    PubMed Central

    Ferreira, Éden Ramalho; Horjales, Eduardo; Bonfim-Melo, Alexis; Cortez, Cristian; da Silva, Claudio Vieira; De Groote, Michel; Sobreira, Tiago José Paschoal; Cruz, Mário Costa; Lima, Fabio Mitsuo; Cordero, Esteban Mauricio; Yoshida, Nobuko; da Silveira, José Franco; Mortara, Renato Arruda; Bahia, Diana

    2016-01-01

    Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosynthesis, such as cholesterol in humans or ergosterol in trypanosomatids. MVK is conserved from bacteria to mammals, and localizes to glycosomes in trypanosomatids. During the course of T. cruzi MVK characterization, we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion. To evaluate the role of TcMVK in parasite-host cell interactions, TcMVK recombinant protein was produced and anti-TcMVK antibodies were raised in mice. TcMVK protein was detected in the supernatant of cultures of metacyclic trypomastigotes (MTs) and extracellular amastigotes (EAs) by Western blot analysis, confirming its secretion into extracellular medium. Recombinant TcMVK bound in a non-saturable dose-dependent manner to HeLa cells and positively modulated internalization of T. cruzi EAs but inhibited invasion by MTs. In HeLa cells, TcMVK induced phosphorylation of MAPK pathway components and proteins related to actin cytoskeleton modifications. We hypothesized that TcMVK is a bifunctional enzyme that in addition to playing a classical role in isoprenoid synthesis in glycosomes, it is secreted and may modulate host cell signaling required for T. cruzi invasion. PMID:27113535

  20. Metabolic analysis reveals changes in the mevalonate and juvenile hormone synthesis pathways linked to the mosquito reproductive physiology.

    PubMed

    Rivera-Perez, Crisalejandra; Nouzova, Marcela; Lamboglia, Ivanna; Noriega, Fernando G

    2014-08-01

    Juvenile hormone (JH) regulates reproductive maturation in insects; therefore interruption of JH biosynthesis has been considered as a strategy for the development of target-specific insecticides. The corpora allata (CA) from mosquitoes is highly specialized to supply variable levels of JH, which are linked to ovarian developmental stages and influenced by nutritional signals. However, very little is known about how changes in JH synthesis relate to reproductive physiology and how JH synthesis regulation is translated into changes in the CA machinery. With the advent of new methods that facilitate the analysis of transcripts, enzymes and metabolites in the minuscule CA, we were able to provide comprehensive descriptions of the mevalonic (MVA) and JH synthesis pathways by integrating information on changes in the basic components of those pathways. Our results revealed remarkable dynamic changes in JH synthesis and exposed part of a complex mechanism that regulates CA activity. Principal component (PC) analyses validated that both pathways (MVAP and JH-branch) are transcriptionally co-regulated as a single unit, and catalytic activities for the enzymes of the MVAP and JH-branch also changed in a coordinate fashion. Metabolite studies showed that global fluctuations in the intermediate pool sizes in the MVAP and JH-branch were often inversely related. PC analyses suggest that in female mosquitoes, there are at least 4 developmental switches that alter JH synthesis by modulating the flux at distinctive points in both pathways.

  1. Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells

    PubMed Central

    Alizadeh, Javad; Zeki, Amir A.; Mirzaei, Nima; Tewary, Sandipan; Rezaei Moghadam, Adel; Glogowska, Aleksandra; Nagakannan, Pandian; Eftekharpour, Eftekhar; Wiechec, Emilia; Gordon, Joseph W.; Xu, Fred. Y.; Field, Jared T.; Yoneda, Ken Y.; Kenyon, Nicholas J.; Hashemi, Mohammad; Hatch, Grant M.; Hombach-Klonisch, Sabine; Klonisch, Thomas; Ghavami, Saeid

    2017-01-01

    The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP- and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB-231(breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines. PMID:28344327

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

    PubMed

    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

  3. Switching of adenosine diphosphate receptor inhibitor after hospital discharge among myocardial infarction patients: Insights from the Treatment with Adenosine Diphosphate Receptor Inhibitors: Longitudinal Assessment of Treatment Patterns and Events after Acute Coronary Syndrome (TRANSLATE-ACS) observational study.

    PubMed

    Zettler, Marjorie E; Peterson, Eric D; McCoy, Lisa A; Effron, Mark B; Anstrom, Kevin J; Henry, Timothy D; Baker, Brian A; Messenger, John C; Cohen, David J; Wang, Tracy Y

    2017-01-01

    The reasons for postdischarge adenosine diphosphate receptor inhibitor (ADPri) switching among patients with myocardial infarction (MI) are unclear. We sought to describe the incidence and patterns of postdischarge ADPri switching among patients with acute MI treated with percutaneous coronary intervention.

  4. Nucleotide sequence and expression of the Enterobacter aerogenes alpha-acetolactate decarboxylase gene in brewer's yeast.

    PubMed Central

    Sone, H; Fujii, T; Kondo, K; Shimizu, F; Tanaka, J; Inoue, T

    1988-01-01

    The nucleotide sequence of a 1.4-kilobase DNA fragment containing the alpha-acetolactate decarboxylase gene of Enterobacter aerogenes was determined. The sequence contains an entire protein-coding region of 780 nucleotides which encodes an alpha-acetolactate decarboxylase of 260 amino acids. The DNA sequence coding for alpha-acetolactate decarboxylase was placed under the control of the alcohol dehydrogenase I promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both S. cerevisiae and Escherichia coli. Brewer's yeast cells transformed by this plasmid showed alpha-acetolactate decarboxylase activity and were used in laboratory-scale fermentation experiments. These experiments revealed that the diacetyl concentration in wort fermented by the plasmid-containing yeast strain was significantly lower than that in wort fermented by the parental strain. These results indicated that the alpha-acetolactate decarboxylase activity produced by brewer's yeast cells degraded alpha-acetolactate and that this degradation caused a decrease in diacetyl production. PMID:3278689

  5. Feedback repression of ornithine decarboxylase synthesis mediated by antizyme.

    PubMed Central

    Mitchell, J L; Choe, C Y; Judd, G G

    1996-01-01

    The induction of antizyme by spermidine and the resulting enhancement of ornithine decarboxylase (ODC) degradation have been well studied; however, little is known about the mechanism whereby elevated spermidine levels decrease synthesis of the polyamine biosynthetic enzyme. To evaluate the relative contribution of inhibited synthesis, as distinct from enhanced degradation of ODC, spermidine levels were manipulated in a variant cell line that overproduces a stable form of ODC. Spermidine did not selectively inhibit ODC synthesis in these variant cells, supporting the concept that spermidine diminishes ODC synthesis in normal cells owing to enhanced degradation of the protein in the presence of elevated antizyme levels. This model was further investigated in vitro by use of rabbit reticulocyte lysate, which catalyses simultaneous ODC mRNA translation and antizyme-stimulated degradation of ODC protein. Antizyme strongly repressed the incorporation of labelled amino acids into normal rat ODC. Unexpectedly it also diminished the apparent translation of ODC mRNA species coding for enzyme forms that are not destabilized by the post-translational addition of antizyme. The effect of antizyme on ODC translation was not observed in wheatgerm extract, in which there is no antizyme-induced degradation. Further, deletion of a short segment of antizyme necessary for the destabilization of ODC (amino acid residues 113-118) resulted in a form that bound ODC but did not diminish its apparent translation. These results suggest that the co-translational addition of antizyme to ODC results in a complex that is different from, and innately less stable than, that formed when antizyme is added post-translationally. PMID:9003359

  6. Hepatic ornithine decarboxylase induction by potato glycoalkaloids in rats.

    PubMed

    Caldwell, K A; Grosjean, O K; Henika, P R; Friedman, M

    1991-08-01

    The induction of hepatic ornithine decarboxylase (ODC) activity in rat livers by the potato glycoalkaloids alpha-solanine, alpha-chaconine, and their aglycone solanidine, has been studied. Ip administration of alpha-solanine at 7.5, 15 and 30 mg/kg body weight produced markedly elevated enzyme activity at 4 hr after treatment, with a linear dose response. The increase was four-fold at the lowest dose administered to 12-fold at the highest. ODC activity was measured at 1, 2, 3, 4, 5, 6, 8, and 24hr after alpha-solanine was given. A statistically significant increase in enzyme activity was evident at 3 hr after treatment; maximal activity occurred at 5 hr and was approximately 12 times greater than the dimethylsulphoxide (DMSO) control level. Elevated activities persisted for several hours, decreasing to about one-third of the maximal level at 8 hr. The relative effects of alpha-solanine, alpha-chaconine and solanidine on ODC activities were studied at 4 hr using an equimolar dose of 17 mM/kg body weight. ODC activity induced by alpha-chaconine was higher than that induced by alpha-solanine; the latter activity was two-thirds that of the former. The aglycone solanidine did not induce any increase in activity compared with the DMSO control. ODC activity with dexamethasone, a glucocorticoid, at 4 mg/kg body weight, followed a pattern similar to that of alpha-solanine. However, maximal activity occurred slightly earlier at 4 hr after treatment. The results show that the extent of induced ODC activity depends on the structure of the potato alkaloid.

  7. Ultraviolet radiation induction of ornithine decarboxylase in rat keratinocytes

    SciTech Connect

    Rosen, C.F.; Gajic, D.; Drucker, D.J. )

    1990-05-01

    UV radiation plays an important role in the induction of cutaneous malignancy, including basal cell and squamous cell carcinomas and malignant melanoma. In addition to its effects on DNA damage and repair mechanisms, UV radiation has been shown to modulate the expression of specific genes, altering the levels of their mRNAs and the synthesis of their corresponding proteins. In order to gain further information about the molecular effects of UV radiation, we have studied the regulation of ornithine decarboxylase (ODC) gene expression in response to UVB radiation. ODC is the rate-limiting enzyme in polyamine biosynthesis, is involved in growth and differentiation, and has been implicated in carcinogenesis. Keratinocytes grown in culture were either sham-irradiated or exposed to increasing doses of UVB (1-5 mJ/cm2). Northern blot analysis of keratinocyte RNA under basal conditions demonstrated the presence of two ODC mRNA transcripts. Increasing exposure to UVB resulted in a dose-dependent increase in the levels of both ODC mRNA transcripts. The induction of ODC gene expression following UVB was noted 2 h after UVB exposure, and ODC mRNA levels continued to increase up to 24 h after UVB exposure. The UVB-induced increase in ODC gene expression was not serum dependent, despite the ability of serum alone to induce ODC gene expression. The mRNA transcripts for actin and hexosaminidase A were not induced after UVB exposure. These studies show that the UVB-induced increase in ODC activity is due, at least in part, to an increase in ODC gene expression and they provide a useful model for the analysis of the molecular effects of UVB radiation.

  8. Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation.

    PubMed

    Lohinai, Zsolt; Keremi, Beata; Szöko, Eva; Tábi, Tamás; Szabo, Csaba; Tulassay, Zsolt; DiCesare, John C; Davis, Carole A; Collins, Lindsay M; Levine, Martin

    2015-10-01

    Lysine, a nutritionally essential amino acid, enters the oral cavity in gingival crevicular fluid (GCF). During oral hygiene restriction (OHR), lysine decarboxylase (LDC) in dento-gingival biofilms converts lysine to cadaverine. Lysine depletion impairs the dental epithelial barrier to bacterial proinflammatory products. Antibodies to LDC from Eikenella corrodens (Ecor-LDC) inhibit LDC activity and retard gingival inflammation in beagle dogs. Whether E. corrodens is the major source of LDC in dental biofilms and whether the lysine analog tranexamic acid (TA) inhibits LDC activity, biofilm accumulation, and GCF exudation in a human gingivitis model were examined. Antibodies raised in goats to LDC-rich extracts from E. corrodens cell surfaces were used to inhibit Ecor-LDC and detect it in biofilm extracts using Western blots. Ecor-LDC activity was measured at pH 4.0 to 11.0 and its TA dissociation constant (Ki) at pH 7.0. Young adults used a 5% or 10% TA mouthwash three times daily during OHR for 1 week. Ecor-LDC antibodies and TA inhibited biofilm LDC. Ki of TA for Ecor-LDC was 940 μM. TA reduced plaque index (PI) by downshifting the PI correlation with biofilm lysine content after OHR without TA. GCF was correspondingly suppressed. However, greater TA retention in saliva partially relieved GCF suppression but not biofilm lysine depletion. TA slightly inhibits LDC but strongly reduces biofilm by inhibiting bacterial lysine uptake. Unfortunately, TA may impair dental epithelial attachments by also inhibiting lysine transporter uptake. Ecor-LDC inhibitors other than lysine analogs may maintain sufficient lysine levels and attachment integrity to prevent periodontal inflammation.

  9. Purification and properties of diaminopimelate decarboxylase from Escherichia coli

    PubMed Central

    White, P. J.; Kelly, Bridget

    1965-01-01

    1. Diaminopimelate decarboxylase from a soluble extract of Escherichia coli A.T.C.C. 9637 was purified 200-fold by precipitation of nucleic acids, fractionation with acetone and then with ammonium sulphate, adsorption on calcium phosphate gel and chromatography on DEAE-cellulose or DEAE-Sephadex. 2. The purified enzyme showed only one component in the ultracentrifuge, with a sedimentation coefficient of 5·4s. One major peak and three much smaller peaks were observed on electrophoresis of the enzyme at pH8·9. 3. The mol.wt. of the enzyme was approx. 200000. The catalytic constant was 2000mol. of meso-diaminopimelic acid decomposed/min./mol. of enzyme, at 37°. The relative rates of decarboxylation at 25°, 37° and 45° were 0·17:1·0:1·6. At 37° the Michaelis constant was 1·7mm and the optimum pH was 6·7–6·8. 4. There was an excess of acidic amino acids over basic amino acids in the enzyme, which was bound only on basic cellulose derivatives at pH6·8. 5. The enzyme had an absolute requirement for pyridoxal phosphate as a cofactor; no other derivative of pyridoxine had activity. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was also needed as an activator. 6. In the presence of 2,3-dimercaptopropan-1-ol (1mm), heavy-metal ions (Cu2+, Hg2+) did not inhibit the enzyme, but there was inhibition by several amino acids with analogous structures to diaminopimelate, generally at high concentrations relative to the substrate. Penicillamine was inhibitory at relatively low concentrations; its action was prevented by pyridoxal phosphate. PMID:14343156

  10. Conformational stabilization of rat s-adenosylmethionine decarboxylase by putrescine.

    PubMed

    Wada, Makiko; Shirahata, Akira

    2010-01-01

    The activity and processing of mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is stimulated by putrescine. To obtain new insights into the mechanism through which putrescine stimulates AdoMetDC, we investigated conformational changes in rat prostate AdoMetDC in the presence or absence of putrescine. We examined the reactivity of purified rat prostate AdoMetDC to the SH-reagent iodoacetic acid (IAA) and its susceptibility to proteolysis in the presence or absence of putrescine using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The activity of AdoMetDC treated with IAA in the absence of putrescine was reduced, but about 80% of its activity remained after treatment with IAA in the presence of putrescine. In the presence of putrescine, IAA incorporation was 1.9 mol IAA/mol of AdoMetDC α-subunit, while there was no incorporation of IAA in the β-subunit of AdoMetDC. In the absence of putrescine, 5.0 mol of IAA/mol of α-subunit and 0.9 mol of IAA/mol of β-subunit were incorporated. Only Cys292 and Cys310 were carboxymethylated by IAA in the presence of putrescine. In contrast, in the absence of putrescine all cysteines were carboxymethylated by IAA. In addition, putrescine slowed the rate of AdoMetDC degradation by trypsin. These results demonstrate that the conformation of AdoMetDC purified from rat prostate is stabilized by putrescine.

  11. Prevalence of glutamic acid decarboxylase antibodies amongst young Malaysian diabetics.

    PubMed

    Wan Nazaimoon, W M; Faridah, I; Singaraveloo, M; Ismail, I S; Wan Mohamad, W B; Letchuman, R; Rasat, R; Pendek, R; Hew, F L; Sheriff, I H; Khalid, B A

    1999-01-01

    This study determined the prevalence of glutamic acid decarboxylase antibodies (GAD Ab) in a group of 926 young Malaysian diabetics of three ethnic groups, Malay, Chinese, and Indian. Patients were clinically diagnosed to be Type 1 or Type 2 before the age of 40 years. The overall GAD Ab positivity was 17.4% (161/926), significantly higher in the Type 1 than the Type 2 diabetics (35.5%, 116/329 vs. 7.5%, 45/597, P=0.0001). Compared to GAD Ab negative patients, seropositive diabetics were diagnosed at younger age (21.2+/-0.9 vs. 27.4+/-0.3 y, P=0.0001), had lower fasting (289+/-27.4 vs. 640+/-17.6 pmol/l, P=0.0001) and post-glucagon C-peptide levels (527+/-51.8 vs. 1030+/-28.9 pmol/l, P=0.0001). There were no racial differences in the prevalence of GAD Ab; of the total Type 1, 30.8, 36.4, and 39.4% were Malay, Chinese, and Indian diabetics, respectively and of the total Type 2, 8.8, 8.2, and 4.4% were Malay, Chinese, and Indian diabetics respectively. There was a curvilinear relationship between GAD Ab and the post-glucagon C-peptide levels, suggesting that GAD Ab do play a role in the beta-cells destruction and could be an important immune marker for the LADA group. This study reconfirmed previous reports that the autoimmune mechanisms in the Type 1 Asian diabetics are indeed different from the Caucasians, and further investigations should be carried out to explain the differences.

  12. Biosynthesis of uridine diphosphate N-acetyl-L-fucosamine in a cell-free system from Salmonella arizonae O:59.

    PubMed

    Druzhinina, T N; Kalinchuk, N A; Shibaev, V N

    2005-01-01

    The conversion of uridine diphosphate N-acetyl-D-glucosamine into uridine diphosphate N-acetyl-L-fucosamine was demonstrated with enzymes from cytoplasmic fraction of Salmonella arizonae O:59 cells in the presence of NAD+ (NADP+) and NADPH. The reaction product was identified by ion-pair, reverse-phase HPLC with the use of synthetic nucleoside diphosphate sugar standards under conditions specially developed for separation of uridine diphosphate 2-acetamido-2,6-dideoxyhexoses. L-Fucose dehydrogenase from porcine liver was shown to be applicable for determination of N-acetyl-L-fucosamine, this enzyme being used to confirm L-configuration of the amino sugar residue in the sugar nucleotide formed.

  13. Biosynthesis of dolichyl diphosphate N-acetylglucosamine intermediates in Ascaridia galli microsomes.

    PubMed

    Kyossev, Z; Ossikovski, E

    1991-01-01

    1. N-acetylglucosamine-1-phosphate transferase was demonstrated in the microsomal fraction of Ascaridia galli. 2. The transferase reaction depends on exogenous dolichyl phosphate as lipid acceptor and was found to be inhibited by tunicamycin. 3. The enzyme activity was optimal in the presence of sodium deoxycholate as detergent and Mg cations after 10 min of incubation. 4. The product of the transferase reaction--dolichyl diphosphate N-acetylglucosamine was converted into lipid-disaccharide-dolichyl diphosphate N,N'-diacetylchitobiose. 5. The maximum level of the conversion was achieved at 5 mM concentration of unlabelled UDP-N-acetylglucosamine, while this conversion was negligible at lower UDP-N-acetylglucosamine concentrations (0.1 and 0.5 mM).

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

    PubMed

    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.

  15. Farnesyl Diphosphate Synthase Localizes to the Cytoplasm of Trypanosoma cruzi and T.brucei

    PubMed Central

    Ferella, Marcela; Li, Zhu-Hong; Andersson, Björn; Docampo, Roberto

    2008-01-01

    The farnesyl diphosphate synthase (FPPS) has previously been characterized in trypanosomes as an essential enzyme for their survival and as the target for bisphosphonates, drugs that are effective both in vitro and in vivo against these parasites. Enzymes from the isoprenoid pathway have been assigned to different compartments in eukaryotes, including trypanosomatids. We here report that FPPS localizes to the cytoplasm of both Trypanosoma cruzi and T. brucei, and is not present in other organelles such as the mitochondria and glycosomes. PMID:18406406

  16. Purification and characterization of human dehydrodolychil diphosphate synthase (DHDDS) overexpressed in E. coli.

    PubMed

    Giladi, Moshe; Edri, Ilan; Goldenberg, Michal; Newman, Hadas; Strulovich, Roi; Khananshvili, Daniel; Haitin, Yoni; Loewenstein, Anat

    2017-04-01

    Protein asparagine (N)-linked glycosylation is a post-translational modification that occurs in the endoplasmic reticulum; it plays an important role in protein folding, oligomerization, quality control, sorting, and transport. Accordingly, disorders of glycosylation may affect practically every organ system. Dehydrodolichyl diphosphate synthase (DHDDS) is an eukaryotic cis prenyltransferase (cis-PT) that catalyzes chain elongation of farnesyl diphosphate via multiple condensations with isopentenyl diphosphate to form dehydrodolichyl diphosphate, a precursor for the glycosyl carrier dolichylpyrophophate involved in N-linked glycosylation. Mutations in DHDDS were shown to result in retinitis pigmentosa, ultimately leading to blindness, but the exact molecular mechanism by which the mutations affect DHDDS function remains elusive. In addition, bacterial cis-PT homologs are involved in bacterial wall synthesis and are therefore potential targets for new antibacterial agents. However, as eukaryotic cis-PT were not thoroughly characterized structurally and functionally, rational design of prokaryotic cis-PT specific drugs is currently impossible. Here, we present a simple protocol for purification of functionally active human DHDDS under non-denaturating conditions using a codon-optimized construct. The purified protein forms a stable homodimer, similar to its bacterial homologs, and shows time- and substrate-dependent activity. Purification of this protein requires the presence of a detergent for protein solubility. The protocol described here may be utilized for the overexpression of other eukaryotic cis-PT. Future structural and functional studies of the recombinant DHDDS may shed light on the mechanisms underlying DHDDS-related retinitis pigmentosa and lead to novel therapeutic approaches.

  17. Role of isopentenyl-diphosphate isomerase in heterologous cyanobacterial (Synechocystis) isoprene production.

    PubMed

    Chaves, Julie E; Romero, Paloma Rueda; Kirst, Henning; Melis, Anastasios

    2016-12-01

    Heterologous production of isoprene (C5H8) hydrocarbons in cyanobacteria, emanating from sunlight, CO2, and water, is now attracting increasing attention. The concept entails application of an isoprene synthase transgene from terrestrial plants, heterologously expressed in cyanobacteria, aiming to reprogram carbon flux in the terpenoid biosynthetic pathway toward formation and spontaneous release of this volatile chemical from the cell and liquid culture. However, flux manipulations and carbon-partitioning reactions between isoprene (the product) and native terpenoid biosynthesis for cellular needs are not yet optimized for isoprene yield. The primary reactant for isoprene biosynthesis is dimethylallyl diphosphate (DMAPP), whereas both DMAPP and its isopentenyl diphosphate (IPP) isomer are needed for cellular terpenoid biosynthesis. The present work addressed the function of an isopentenyl diphosphate (IPP) isomerase in cyanobacteria and its role in carbon partitioning between IPP and DMAPP, both of which serve, in variable ratios, as reactants for the synthesis of different cellular terpenoids. The work was approached upon the heterologous expression in Synechocystis of the "isopentenyl diphosphate isomerase" gene (FNI) from Streptococcus pneumoniae, using isoprene production as a "reporter process" for substrate partitioning between DMAPP and IPP. It is shown that transgenic expression of the FNI gene in Synechocystis resulted in a 250 % increase in the "reporter isoprene" rate and yield, suggesting that the FNI isomerase shifted the endogenous DMAPP-IPP steady-state pool size toward DMAPP, thereby enhancing rates and yield of isoprene production. The work provides insight into the significance and functional role of the IPP isomerase in these photosynthetic microorganisms.

  18. A cesium copper vanadyl-diphosphate: Synthesis, crystal structure and physical properties

    SciTech Connect

    Shvanskaya, Larisa; Yakubovich, Olga; Bychkov, Andrey; Shcherbakov, Vasiliy; Golovanov, Alexey; Zvereva, Elena; Volkova, Olga; Vasiliev, Alexander

    2015-02-15

    A non-centrosymmetric orthorhombic diphosphate, Cs{sub 2}Cu{sub 1+x}(VO){sub 2−x}(P{sub 2}O{sub 7}){sub 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, VO{sub 5} tetragonal pyramids and P{sub 2}O{sub 7} 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 Cu{sup 2+} ions and (VO){sup 2+} units disorder in the system. It is supposed that the charge and geometry of the framework are controlled by the Cu{sup 2+}/(VO){sup 2+} ratio; its variation may lead to a design of new materials. - Graphical abstract: A microporous 3D anionic framework of the first copper vanadium-diphosphate Cs{sub 2}Cu{sub 1.1}(VO){sub 1.9}(P{sub 2}O{sub 7}){sub 2}. The similarity in behaviour of Cu and V paramagnetic subsystems revealed by ESR study. - Highlights: • The first copper vanadium-diphosphate Cs{sub 2}Cu{sub 1.1}(VO){sub 1.9}(P{sub 2}O{sub 7}){sub 2} is reported. • A 3D anionic framework is characterized by disorder in distribution of Cu and V atoms. • Structural relations with topologically similar compounds are discussed. • The similarity in behaviour of Cu and V paramagnetic subsystems has been revealed.

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

    PubMed

    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.

  20. The presence of dolichol in a lipid diphosphate N-acetylglucosamine from Saccharomyces cerevisiae (baker's yeast).

    PubMed Central

    Reuvers, F; Boer, P; Hemming, F W

    1978-01-01

    The lipid moiety of a lipid diphosphate N-acetylglucosamine, an intermediate in glycosylation of proteins, was studied. Ozonolysis of the compound gave evidence for an alpha-saturated isoprene unit. Alkaline hydrolysis of the glycolipid, followed by high-pressure liquid chromatography, showed the presence of a series of polyprenol homologues identical with those isolated directly from Saccharomyces cerevisiae (baker's yeast). No particular homologue was preferred in the enzymic transfer of N-acetylglucosamine 1-phosphate to endogenous dolichol monophosphate. PMID:348196

  1. Enantioselective Inhibition of Squalene Synthase by Aziridine Analogues of Presqualene Diphosphate

    PubMed Central

    Koohang, Ali; Bailey, Jessica L.; Erickson, Hans K.; Owen, David; Poulter, C. Dale

    2013-01-01

    Squalene synthase catalyzes the conversion of two molecules of (E,E)-farnesyl diphosphate to squalene via the cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). Since this novel reaction constitutes the first committed step in sterol biosynthesis, there has been considerable interest and research on the stereochemistry and mechanism of the process and in the design of selective inhibitors of the enzyme. This paper reports the synthesis and characterization of five racemic and two enantiopure aziridine analogues of PSPP and the evaluation of their potencies as inhibitors of recombinant yeast squalene synthase. The key aziridine-2-methanol intermediates (6-OH, 7-OH, and 8-OH) were obtained by N-alkylations or by an N-acylation–reduction sequence of (±)-, (2R,3S)-, and (2S,3R)-2,3-aziridinofarnesol (9-OH) protected as tert-butyldi-methylsilyl ethers. SN2 displacements of the corresponding methanesulfonates with pyrophosphate and methanediphosphonate anions afforded aziridine 2-methyl diphosphates and methanediphosphonates bearing N-undecyl, N-bishomogeranyl, and N-(α-methylene)bishomogeranyl substituents as mimics for the 2,6,10-trimethylundeca-2,5,9-trienyl side chain of PSPP. The 2R,3S diphosphate enantiomer bearing the N-bishomogeranyl substituent corresponding in absolute stereochemistry to PSPP proved to be the most potent inhibitor (IC50 1.17 ± 0.08 μM in the presence of inorganic pyrophosphate), a value 4-fold less than that of its 2S,3R stereoisomer. The other aziridine analogues bearing the N-(α-methylene)bishomogeranyl and N-undecyl substituents, and the related methanediphosphonates, exhibited lower affinities for recombinant squalene synthase. PMID:20545375

  2. Functional analysis of type 1 isopentenyl diphosphate isomerase from Halobacterium sp. NRC-1.

    PubMed

    Hoshino, Takeshi; Eguchi, Tadashi

    2007-10-01

    Here we report the characterization of the type-1 isopentenyl diphosphate isomerase derived from Halobacterium sp. NRC-1. The expressed purified enzyme showed maximum isomerase activity in the presence of 1 M NaCl at 37 degrees C at pH 6.0. This type-1 enzyme appears to be the first for which the Co2+ ion is required for activity.

  3. Role of nucleoside diphosphate kinase in the activation of anti-HIV nucleoside analogs.

    PubMed

    Schneider, B; Sarfati, R; Deville-Bonne, D; Véron, M

    2000-06-01

    Nucleoside analogs are currently used in antiretrovirus therapies. The best known example is AZT one of the first drug to be used for the treatment of AIDS. However, only the triphosphate derivatives of these compounds act as substrates of the viral reverse transcriptase. Since they do not enter cells, nucleoside analogs are administered and phosphorylated by cellular kinases. The last step in this phosphorylation pathway is catalyzed by nucleoside diphosphate (NDP) kinase. The incorporation of the nucleoside triphosphates into nascent viral DNA chain results in termination of the elongation process. We have performed kinetics studies of the phosphorylation reaction by NDP kinase of dideoxynucleoside diphosphates such as 2',3'-dideoxy-3'-azidothymidine diphosphate (AZT-DP) and 2',3'-dideoxy-2',3'-didehydrothymidine diphosphate (d4T-DP). We show that the catalytic efficiency is strongly decreased and, therefore, that the reaction step catalyzed by NDP kinase constitutes a bottleneck in the processing pathway of anti-HIV compounds. In addition, the affinity of the analogs in the absence of catalysis was determined using a catalytically inactive NDP kinase mutant, showing a reduction of affinity by a factor of 2 to 30, depending on the analog. The structure of NDP kinase provides a structural explanation for these results. Indeed, all nucleoside analogs acting as chain terminators must lack a 3'-OH in the nucleotide deoxyribose. Unfortunately, this same substitution is detrimental for their capacity to be phosphorylated by NDP kinase. This defines the framework for the design of new nucleoside analogs with increased efficiency in antiretroviral therapies.

  4. Inhibition of isoprene biosynthesis pathway enzymes by phosphonates, bisphosphonates, and diphosphates.

    PubMed

    Cheng, Feng; Oldfield, Eric

    2004-10-07

    We have investigated the docking of a variety of inhibitors and substrates to the isoprene biosynthesis pathway enzymes farnesyl diphosphate synthase (FPPS), isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IPPI) and deoxyxylulose-5-phosphate reductoisomerase (DXR) using the Lamarckian genetic alogorithm program, AutoDock. The docked ligand structures are predicted with a approximately 0.8 A rms deviation from the structures determined crystallographically. The errors found are a function of the number of atoms in the ligand (R = 0.91, p < 0.0001) and, to a lesser extent, on the resolution of the crystallographic structure (R = 0.70, p < 0.008). The structures of three isoprenoid diphosphates docked to the FPPS enzyme reveal strong electrostatic interactions with Mg(2+), lysine and arginine active site residues. Similar results are obtained with the docking of four IPPI inhibitors to the IPPI enzyme. The DXR substrate, deoxyxylulose-5-phosphate, is found to dock to Mn(2+)-NADPH-DXR in an almost identical manner as does the inhibitor fosimdomycin to Mn(2+)-DXR (ligand heavy atom rms deviation = 0.90 A) and is poised to interact with NADPH. Bisphosphonate inhibitors are found to bind to the allylic binding sites in both eukaryotic and prokaryotic FPPSs, in good accord with recent crystallographic results (a 0.4 A rms deviation from the X-ray structure with the E. coli enzyme). Overall, these results show for the first time that the geometries of a broad variety of phosphorus-containing inhibitors and substrates of isoprene biosynthesis pathway enzymes can be well predicted by using computational methods, which can be expected to facilitate the design of novel inhibitors of these enzymes.

  5. The phase diagram of charged colloidal lipid A-diphosphate dispersions.

    PubMed

    Reichelt, Hendrik; Faunce, Chester A; Paradies, Henrich H

    2008-03-20

    Small-angle X-ray-scattering, light-scattering, and electron microscope experiments were used to determine the phase transitions of colloidal lipid A-diphosphate aqueous dispersions. The phases detected were a correlated liquid phase, a face-centered cubic (Fd3m) and a body-centered cubic (Im3m) colloidal crystal phase and a new glass phase. These experimentally determined phases were shown to be in accord with theoretically predicted equilibrium phases.

  6. Incubation of 2-methylisoborneol synthase with the intermediate analog 2-methylneryl diphosphate.

    PubMed

    Chou, Wayne Kw; Gould, Colin A; Cane, David E

    2017-03-01

    Incubation of synthetic 2-methylneryl diphosphate (2-MeNPP, 10) with 2-methylisoborneol synthase (MIBS) gave a mixture of products that differed significantly from that derived from the natural substrate (E)-2-methylgeranyl diphosphate (3, 2-MeGPP). The proportion of (-)-2-methylisoborneol (1) decreased from 89 to 17% while that of 2-methylenebornane (4) increased from 10 to 26%, with the relative yields of the isomeric homo-monoterpenes 2-methyl-2-bornene (5) and 1-methylcamphene (6) remaining essentially unchanged (<1% each), as determined by chiral GC-MS analysis. The majority of the product mixture resulting from the MIBS-catalyzed cyclization of 2-MeNPP (10) consisted of the anomalous monocyclic homo-monoterpenes (±)-2-methylllimonene (15, 39%) and 2-methyl-α-terpineol (13, 10%), as well as the acylic derivatives 2-methylnerol (11, 7%) and 2-methyllinalool (14, <1%). The steady-state kinetic parameters of the MIBS-catalyzed reaction, determined using [1-(3)H]-2-methylneryl diphosphate (2-MeNPP), were kcat 0.0046±0.0003 s(-1), Km 18±6 μm and kcat/Km 2.55 × 10(2) M(-1) s(-1). In comparison, the natural substrate 2-MeGPP had a kcat 0.105±0.007 s(-1), Km 95±49 μm and kcat/Km 1.11 × 10(3) M(-1) s(-1). Taken together with earlier X-ray crystallographic studies of MIBS, as well as previous investigations of the mechanistically related plant monoterpene cyclase, bornyl diphosphate synthase, these results provide important insights into the binding and cyclization of both native substrates and intermediates and their analogs.The Journal of Antibiotics advance online publication, 1 March 2017; doi:10.1038/ja.2017.24.

  7. Chromosomal Integration and Expression of Two Bacterial α-Acetolactate Decarboxylase Genes in Brewer's Yeast

    PubMed Central

    Blomqvist, K.; Suihko, M.-L.; Knowles, J.; Penttilä, M.

    1991-01-01

    A bacterial gene encoding α-acetolactate decarboxylase, isolated from Klebsiella terrigena or Enterobacter aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase (PGK1) or the alcohol dehydrogenase (ADH1) promoter and were integrated by gene replacement by using cotransformation into the PGK1 or ADH1 locus, respectively, of a brewer's yeast. The expression level of the α-acetolactate decarboxylase gene of the PGK1 integrant strains was higher than that of the ADH1 integrants. Under pilot-scale brewing conditions, the α-acetolactate decarboxylase activity of the PGK1 integrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and no lagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, and the quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer. Images PMID:16348559

  8. The effect of a high fat diet on pyruvate decarboxylase deficiency without central nervous system involvement.

    PubMed

    Kodama, S; Yagi, R; Ninomiya, M; Goji, K; Takahashi, T; Morishita, Y; Matsuo, T

    1983-01-01

    A nine-year-old Japanese boy with low pyruvate decarboxylase activity in fibroblasts showed no central nervous symptoms except for muscle fatigue. The pyruvate decarboxylase activities in fibroblasts of the patient and two control subjects were 0.407 +/- 0.083, 1.029 +/- 0.137 and 1.607 +/- 0.096 mumoles/g protein/30 min, respectively. The Michaelis-Menten constant (Km) was the same in the patient and controls. There was no inhibitor of pyruvate decarboxylase in the patient's fibroblasts. A high fat diet has been given to the patient for five years. At present he does not complain of any kind of muscle fatigue, except after severe exercise. Mental and physiological development of the patient are within the normal ranges. However, trials of orally administered thiamine hydrochloride or thiamine hydrochloride combined with lipoamide did not improve his muscle fatigue.

  9. Optimization of a Non-Radioactive High-Throughput Assay for Decarboxylase Enzymes

    PubMed Central

    2010-01-01

    Herein, we describe the optimization of a linked enzyme assay suitable for high-throughput screening of decarboxylases, a target family whose activity has historically been difficult to quantify. Our approach uses a commercially available bicarbonate detection reagent to measure decarboxylase activity. The assay is performed in a fully enclosed automated screening system under inert nitrogen atmosphere to minimize perturbation by exogenous CO2. Receiver operating characteristic (ROC) analysis following a pilot screen of a small library of ∼3,600 unique molecules for inhibitors of Trypanosoma brucei ornithine decarboxylase quantitatively demonstrates that the assay has excellent discriminatory power (area under the curve = 0.90 with 95% confidence interval between 0.82 and 0.97). PMID:20085486

  10. Cloning and nucleotide sequence of wild type and a mutant histidine decarboxylase from Lactobacillus 30a.

    PubMed

    Vanderslice, P; Copeland, W C; Robertus, J D

    1986-11-15

    Prohistidine decarboxylase from Lactobacillus 30a is a protein that autoactivates to histidine decarboxylase by cleaving its peptide chain between serines 81 and 82 and converting Ser-82 to a pyruvoyl moiety. The pyruvoyl group serves as the prosthetic group for the decarboxylation reaction. We have cloned and determined the nucleotide sequence of the gene for this enzyme from a wild type strain and from a mutant with altered autoactivation properties. The nucleotide sequence modifies the previously determined amino acid sequence of the protein. A tripeptide missed in the chemical sequence is inserted, and three other amino acids show conservative changes. The activation mutant shows a single change of Gly-58 to an Asp. Sequence analysis up- and downstream from the gene suggests that histidine decarboxylase is part of a polycistronic message, and that the transcriptional promotor region is strongly homologous to those of other Gram-positive organisms.

  11. Silver Vanadium Diphosphate Ag2VP2O8: Electrochemistry and Characterization of Reduced Material providing Mechanistic Insights

    PubMed Central

    Takeuchi, Esther S.; Lee, Chia-Ying; Chen, Po-Jen; Menard, Melissa C.; Marschilok, Amy C.; Takeuchi, Kenneth J.

    2013-01-01

    Silver vanadium phosphorous oxides (AgwVxPyOz) are notable battery cathode materials due to their high energy density and demonstrated ability to form in-situ Ag metal nanostructured electrically conductive networks within the cathode. While analogous silver vanadium diphosphate materials have been prepared, electrochemical evaluations of these diphosphate based materials have been limited. We report here the first electrochemical study of a silver vanadium diphosphate, Ag2VP2O8, where the structural differences associated with phosphorous oxides versus diphosphates profoundly affect the associated electrochemistry. Reminiscent of Ag2VO2PO4 reduction, in-situ formation of silver metal nanoparticles was observed with reduction of Ag2VP2O8. However, counter to Ag2VO2PO4 reduction, Ag2VP2O8 demonstrates a significant decrease in conductivity upon continued electrochemical reduction. Structural analysis contrasting the crystallography of the parent Ag2VP2O8 with that of the proposed Li2VP2O8 reduction product is employed to gain insight into the observed electrochemical reduction behavior, where the structural rigidity associated with the diphosphate anion may be associated with the observed particle fracturing upon deep electrochemical reduction. Further, the diphosphate anion structure may be associated with the high thermal stability of the partially reduced Ag2VP2O8 materials, which bodes well for enhanced safety of batteries incorporating this material. PMID:25866419

  12. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant...

  13. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant...

  14. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant...

  15. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant...

  16. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme... FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus subtilis. The food additive alpha...

  17. Inhibition of Geranylgeranyl Diphosphate Synthase by Bisphosphonates: A Crystallographic and Computational Investigation

    PubMed Central

    Chen, Cammy K.-M.; Hudock, Michael P.; Zhang, Yonghui; Guo, Rey-Ting; Cao, Rong; No, Joo Hwan; Liang, Po-Huang; Ko, Tzu-Ping; Chang, Tao-Hsin; Chang, Shiou-chi; Song, Yongcheng; Axelson, Jordan; Kumar, Anup; Wang, Andrew H.-J.; Oldfield, Eric

    2008-01-01

    We report the x-ray structures of several bisphosphonate inhibitors of geranylgeranyl diphosphate synthase, a target for anti-cancer drugs. Bisphosphonates containing unbranched sidechains bind to either the farnesyl diphosphate (FPP) substrate site, the geranylgeranyl diphosphate (GGPP) product site, and in one case, both sites, with the bisphosphonate moiety interacting with 3 Mg2+ that occupy the same position as found in FPP synthase. However, each of three “V-shaped” bisphosphonates binds to both the FPP and GGPP sites. Using the Glide program, we reproduced the binding modes of 10 bisphosphonates with an RMS error of 1.3Å. Activities of the bisphosphonates in GGPPS inhibition were predicted with an overall error of 2x, using a comparative molecular similarity analysis, based on a docked-structure alignment. These results show that some GGPPS inhibitors can occupy both substrate and product site, and that binding modes as well as activity can be accurately predicted, facilitating the further development of GGPPS inhibitors as anti-cancer agents. PMID:18800762

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

    PubMed

    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. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2007-06-22

    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.

  20. Molecular Cloning and Characterization of a Geranyl Diphosphate-Specific Aromatic Prenyltransferase from Lemon1[W

    PubMed Central

    Munakata, Ryosuke; Inoue, Tsuyoshi; Koeduka, Takao; Karamat, Fazeelat; Olry, Alexandre; Sugiyama, Akifumi; Takanashi, Kojiro; Dugrand, Audray; Froelicher, Yann; Tanaka, Ryo; Uto, Yoshihiro; Hori, Hitoshi; Azuma, Jun-Ichi; Hehn, Alain; Bourgaud, Frédéric; Yazaki, Kazufumi

    2014-01-01

    Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species. PMID:25077796

  1. Cloning and functional analysis of a cDNA encoding Ginkgo biloba farnesyl diphosphate synthase.

    PubMed

    Wang, Peng; Liao, Zhihua; Guo, Liang; Li, Wenchao; Chen, Min; Pi, Yan; Gong, Yifu; Sun, Xiaofen; Tang, Kexuan

    2004-10-31

    Farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2. 5.1.10) catalyzes the synthesis of farnesyl diphosphate, and provides precursor for biosynthesis of sesquiterpene and isoprenoids containing more than 15 isoprene units in Ginkgo biloba. Here we report the cloning, characterization and functional analysis of a new cDNA encoding FPS from G. biloba. The full-length cDNA (designated GbFPS) had 1731 bp with an open reading frame of 1170 bp encoding a polypeptide of 390 amino acids. The deduced GbFPS was similar to other known FPSs and contained all the conserved regions of trans-prenyl chain-elongating enzymes. Structural modeling showed that GbFPS had the typical structure of FPS, the most prominent feature of which is the arrangement of 13 core helices around a large central cavity. Southern blot analysis revealed a small FPS gene family in G. biloba. Expression analysis indicated that GbFPS expression was high in roots and leaves, and low in stems. Functional complementation of GbFPS in an FPS-deficient strain confirmed that GbFPS mediates farnesyl diphosphate biosynthesis.

  2. Optimization of primaquine diphosphate tablet formulation for controlled drug release using the mixture experimental design.

    PubMed

    Duque, Marcelo Dutra; Kreidel, Rogério Nepomuceno; Taqueda, Maria Elena Santos; Baby, André Rolim; Kaneko, Telma Mary; Velasco, Maria Valéria Robles; Consiglieri, Vladi Olga

    2013-01-01

    A tablet formulation based on hydrophilic matrix with a controlled drug release was developed, and the effect of polymer concentrations on the release of primaquine diphosphate was evaluated. To achieve this purpose, a 20-run, four-factor with multiple constraints on the proportions of the components was employed to obtain tablet compositions. Drug release was determined by an in vitro dissolution study in phosphate buffer solution at pH 6.8. The polynomial fitted functions described the behavior of the mixture on simplex coordinate systems to study the effects of each factor (polymer) on tablet characteristics. Based on the response surface methodology, a tablet composition was optimized with the purpose of obtaining a primaquine diphosphate release closer to a zero order kinetic. This formulation released 85.22% of the drug for 8 h and its kinetic was studied regarding to Korsmeyer-Peppas model, (Adj-R(2) = 0.99295) which has confirmed that both diffusion and erosion were related to the mechanism of the drug release. The data from the optimized formulation were very close to the predictions from statistical analysis, demonstrating that mixture experimental design could be used to optimize primaquine diphosphate dissolution from hidroxypropylmethyl cellulose and polyethylene glycol matrix tablets.

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

    PubMed Central

    2012-01-01

    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. PMID:23137178

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

    PubMed Central

    Wang, Jianrong; Li, Yangyuan; Liu, Danni

    2014-01-01

    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. PMID:25474088

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

    PubMed Central

    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

  6. Ornithine Decarboxylase Activity Is Required for Prostatic Budding in the Developing Mouse Prostate

    PubMed Central

    Gamat, Melissa; Malinowski, Rita L.; Parkhurst, Linnea J.; Steinke, Laura M.; Marker, Paul C.

    2015-01-01

    The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO) to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS) epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating

  7. Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis.

    PubMed

    Bunsupa, Somnuk; Hanada, Kousuke; Maruyama, Akira; Aoyagi, Kaori; Komatsu, Kana; Ueno, Hideki; Yamashita, Madoka; Sasaki, Ryosuke; Oikawa, Akira; Saito, Kazuki; Yamazaki, Mami

    2016-08-01

    Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer's disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase. © 2016 American Society of Plant Biologists. All Rights Reserved.

  8. Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis1[OPEN

    PubMed Central

    Bunsupa, Somnuk; Hanada, Kousuke; Maruyama, Akira; Aoyagi, Kaori; Komatsu, Kana; Ueno, Hideki; Yamashita, Madoka; Sasaki, Ryosuke; Oikawa, Akira; Yamazaki, Mami

    2016-01-01

    Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer’s disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata. We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase. PMID:27303024

  9. NUTRITIONAL REQUIREMENTS OF LACTOBACILLUS 30a FOR GROWTH AND HISTIDINE DECARBOXYLASE PRODUCTION

    PubMed Central

    Guirard, Beverly M.; Snell, Esmond E.

    1964-01-01

    Guirard, Beverly M. (University of California, Berkeley), and Esmond E. Snell. Nutritional requirements of Lactobacillus 30a for growth and histidine decarboxylase production. J. Bacteriol. 87:370–376. 1964.—The nutritional requirements of Lactobacillus 30a include each of the naturally occurring amino acids, several B vitamins, ascorbic acid, glucose, acetate, and oleate. The nutritional requirements for optimal histidine decarboxylase production (up to 900 μliters of CO2 per hr per mg of cells) differ to some extent from those for optimal growth. Wholly synthetic and partially defined media are described which produce high enzyme activity, together with rapid and luxuriant growth. PMID:14151059

  10. Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

    DOEpatents

    Maupin-Furlow, Julie A [Gainesville, FL; Talarico, Lee Ann [Gainesville, FL; Raj, Krishnan Chandra [Tamil Nadu, IN; Ingram, Lonnie O [Gainesville, FL

    2008-02-05

    The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells.

  11. Observational Study of a French and Belgian Multicenter Cohort of 23 Patients Diagnosed in Adulthood With Mevalonate Kinase Deficiency

    PubMed Central

    Durel, Cécile-Audrey; Aouba, Achille; Bienvenu, Boris; Deshayes, Samuel; Coppéré, Brigitte; Gombert, Bruno; Acquaviva-Bourdain, Cécile; Hachulla, Eric; Lecomte, Frédéric; Touitou, Isabelle; Ninet, Jacques; Philit, Jean-Baptiste; Messer, Laurent; Brouillard, Marc; Girard-Madoux, Marie-Hélène; Moutschen, Michel; Raison-Peyron, Nadia; Hutin, Pascal; Duffau, Pierre; Trolliet, Pierre; Hatron, Pierre-Yves; Heudier, Philippe; Cevallos, Ramiro; Lequerré, Thierry; Brousse, Valentine; Lesire, Vincent; Audia, Sylvain; Maucort-Boulch, Delphine; Cuisset, Laurence; Hot, Arnaud

    2016-01-01

    Abstract The aim of this study was to describe the clinical and biological features of Mevalonate kinase deficiency (MKD) in patients diagnosed in adulthood. This is a French and Belgian observational retrospective study from 2000 to 2014. To constitute the cohort, we cross-check the genetic and biochemical databases. The clinical, enzymatic, and genetic data were gathered from medical records. Twenty-three patients were analyzed. The mean age at diagnosis was 40 years, with a mean age at onset of symptoms of 3 years. All symptomatic patients had fever. Febrile attacks were mostly associated with arthralgia (90.9%); lymphadenopathy, abdominal pain, and skin lesions (86.4%); pharyngitis (63.6%); cough (59.1%); diarrhea, and hepatosplenomegaly (50.0%). Seven patients had psychiatric symptoms (31.8%). One patient developed recurrent seizures. Three patients experienced renal involvement (13.6%). Two patients had angiomyolipoma (9.1%). All but one tested patients had elevated serum immunoglobulin (Ig) D level. Twenty-one patients had genetic diagnosis; most of them were compound heterozygote (76.2%). p.Val377Ile was the most prevalent mutation. Structural articular damages and systemic AA amyloidosis were the 2 most serious complications. More than 65% of patients displayed decrease in severity and frequency of attacks with increasing age, but only 35% achieved remission. MKD diagnosed in adulthood shared clinical and genetic features with classical pediatric disease. An elevated IgD concentration is a good marker for MKD in adults. Despite a decrease of severity and frequency of attacks with age, only one-third of patients achieved spontaneous remission. PMID:26986117

  12. Paraneoplastic Neurological Syndromes and Glutamic Acid Decarboxylase Antibodies

    PubMed Central

    Ariño, Helena; Höftberger, Romana; Gresa-Arribas, Nuria; Martínez-Hernandez, Eugenia; Armangue, Thaís; Kruer, Michael C.; Arpa, Javier; Domingo, Julio; Rojc, Bojan; Bataller, Luis; Saiz, Albert; Dalmau, Josep; Graus, Francesc

    2016-01-01

    IMPORTANCE Little is known of glutamic acid decarboxylase antibodies (GAD-abs) in the paraneoplastic context. Clinical recognition of such cases will lead to prompt tumor diagnosis and appropriate treatment. OBJECTIVE To report the clinical and immunological features of patients with paraneoplastic neurological syndromes (PNS) and GAD-abs. DESIGN, SETTING, AND PARTICIPANTS Retrospective case series study and immunological investigations conducted in February 2014 in a center for autoimmune neurological disorders. Fifteen cases with GAD65-abs evaluated between 1995 and 2013 who fulfilled criteria of definite or possible PNS without concomitant onconeural antibodies were included in this study. MAIN OUTCOMES AND MEASURES Analysis of the clinical records of 15 patients and review of 19 previously reported cases. Indirect immunofluorescence with rat hippocampal neuronal cultures and cell-based assays with known neuronal cell-surface antigens were used. One hundred six patients with GAD65-abs and no cancer served as control individuals. RESULTS Eight of the 15 patients with cancer presented as classic paraneoplastic syndromes (5 limbic encephalitis, 1 paraneoplastic encephalomyelitis, 1 paraneoplastic cerebellar degeneration, and 1 opsoclonus-myoclonus syndrome). When compared with the 106 non-PNS cases, those with PNS were older (median age, 60 years vs 48 years; P = .03), more frequently male (60% vs 13%; P < .001), and had more often coexisting neuronal cell-surface antibodies, mainly against γ-aminobutyric acid receptors (53%vs 11%; P < .001). The tumors more frequently involved were lung (n = 6) and thymic neoplasms (n = 4). The risk for an underlying tumor was higher if the presentation was a classic PNS, if it was different from stiff-person syndrome or cerebellar ataxia (odds ratio, 10.5; 95%CI, 3.2–34.5), or if the patient had coexisting neuronal cell-surface antibodies (odds ratio, 6.8; 95%CI, 1.1–40.5). Compared with the current series, the 19 previously

  13. Significance of highly conserved aromatic residues in Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.

    PubMed

    Kharel, Yugesh; Zhang, Yuan-Wei; Fujihashi, Masahiro; Miki, Kunio; Koyama, Tanetoshi; Khare, Yugesh

    2003-12-01

    Undecaprenyl diphosphate synthase catalyzes the sequential condensation of eight molecules of isopentenyl diphosphate (IPP) in the cis-configuration into farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of the bacterial cell wall. This cis-type prenyltransferase exhibits a quite different mode of binding of homoallylic substrate IPP from that of trans-type prenyltransferase [Kharel Y. et al. (2001) J. Biol. Chem. 276, 28459-28464]. In order to know the IPP binding mode in more detail, we selected six highly conserved residues in Regions III, IV, and V among nine conserved aromatic residues in Micrococcus luteus B-P 26 UPP synthase for substitution by site-directed mutagenesis. The mutant enzymes were expressed and purified to homogeneity, and then their effects on substrate binding and the catalytic function were examined. All of the mutant enzymes showed moderately similar far-UV CD spectra to that of the wild-type, indicating that none of the replacement of conserved aromatic residues affected the secondary structure of the enzyme. Kinetic analysis showed that the replacement of Tyr-71 with Ser in Region III, Tyr-148 with Phe in Region IV, and Trp-210 with Ala in Region V brought about 10-1,600-fold decreases in the kcat/Km values compared to that of the wild-type but the Km values for both substrates IPP and FPP resulted in only moderate changes. Substitution of Phe-207 with Ser in Region V resulted in a 13-fold increase in the Km value for IPP and a 1,000-2,000-fold lower kcat/Km value than those of the wild-type, although the Km values for FPP showed about no significant changes. In addition, the W224A mutant as to Region V showed 6-fold and 14-fold increased Km values for IPP and FPP, respectively, and 100-250-fold decreased kcat/Km values as compared to those of the wild-type. These results suggested that these conserved aromatic residues play important roles in the binding with both substrates

  14. Mevalonolactone disrupts mitochondrial functions and induces permeability transition pore opening in rat brain mitochondria: Implications for the pathogenesis of mevalonic aciduria.

    PubMed

    Cecatto, Cristiane; Amaral, Alexandre Umpierrez; da Silva, Janaína Camacho; Wajner, Alessandro; Godoy, Kálita Dos Santos; Ribeiro, Rafael Teixeira; Gonçalves, Aline de Mello; Vargas, Carmen Regla; Wajner, Moacir

    2017-03-09

    Mevalonic aciduria (MVA) is caused by severe deficiency of mevalonic kinase activity leading to tissue accumulation and high urinary excretion of mevalonic acid (MA) and mevalonolactone (ML). Patients usually present severe neurologic symptoms whose pathophysiology is poorly known. Here, we tested the hypothesis that the major accumulating metabolites are toxic by investigating the in vitro effects of MA and ML on important mitochondrial functions in rat brain and liver mitochondria. ML, but not MA, markedly decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and the capacity to retain Ca(2+) in the brain, besides inducing mitochondrial swelling. These biochemical alterations were totally prevented by the classical inhibitors of mitochondrial permeability transition (MPT) cyclosporine A and ADP, as well as by ruthenium red in Ca(2+)-loaded mitochondria, indicating the involvement of MPT and an important role for mitochondrial Ca(2+) in these effects. ML also induced lipid peroxidation and markedly inhibited aconitase activity, an enzyme that is highly susceptible to free radical attack, in brain mitochondrial fractions, indicating that lipid and protein oxidative damage may underlie some of ML-induced deleterious effects including MTP induction. In contrast, ML and MA did not compromise oxidative phosphorylation in the brain and all mitochondrial functions evaluated in the liver, evidencing a selective toxicity of ML towards the central nervous system. Our present study provides for the first time evidence that ML impairs essential brain mitochondrial functions with the involvement of MPT pore opening. It is therefore presumed that disturbance of brain mitochondrial homeostasis possibly contributes to the neurologic symptoms in MVA.

  15. Inhibition of mevalonate pathway is involved in alendronate-induced cell growth inhibition, but not in cytokine secretion from macrophages in vitro.

    PubMed

    Töyräs, Anu; Ollikainen, Jouko; Taskinen, Markku; Mönkkönen, Jukka

    2003-07-01

    Bisphosphonates are antiresorptive drugs used for the treatment of metabolic bone diseases. They can be divided into two different pharmacological classes: nitrogen-containing and non-nitrogen-containing bisphosphonates. Non-nitrogen-containing bisphosphonates, like clodronate, are metabolised to a toxic ATP-analogue preventing osteoclast mediated bone resorption. Nitrogen-containing bisphosphonates, including alendronate, prevent osteoclast function by inhibiting the mevalonate pathway. Clodronate is known to have anti-inflammatory properties while alendronate induces cytokine secretion from lipopolysaccharide- (LPS) induced macrophages. This study investigates whether the cytotoxicity and cytokine production induced by alendronate and LPS could be counteracted by clodronate or products of mevalonate pathway: oxidized low density lipoprotein (ox-LDL), farnesol and geranylgeraniol. Treatment with alendronate increased LPS-induced secretion of IL-1beta, IL-6 and TNF-alpha from RAW 264 macrophages 2.4-, 1.4- and 1.8-fold, respectively. This treatment was cytotoxic for macrophages as indicated by lowered cell viability. Clodronate and ox-LDL both counteracted the cytokine secretion and cytotoxicity of alendronate. Farnesol and geranylgeraniol did neither reverse the cytokine secretion nor reduce the cytotoxicity of alendronate. Clodronate and ox-LDL were able to counteract the effects of alendronate on macrophages in vitro, probably by their known ability to inhibit DNA binding activity of transcription factors, nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1). These findings suggest that inhibition of mevalonate pathway is not the mechanism responsible for the proinflammatory response caused by alendronate, as it is in alendronate-induced apoptosis and prevention of osteoclast function.

  16. Atorvastatin attenuates homocysteine-induced migration of smooth muscle cells through mevalonate pathway involving reactive oxygen species and p38 MAPK.

    PubMed

    Bao, Xiao-mei; Zheng, Hongchao

    2015-08-01

    Statins have been reported to have an antioxidant effect against homocysteine (Hcy)-induced endothelial dysfunction. It is unknown whether they have the same effect against migration of vascular smooth muscle cells (VSMCs) induced by Hcy. In this study, it was investigated whether and how atorvastatin could inhibit the Hcy-induced migration in cultured VSMCs and revealed the possible redox mechanism. VSMCs were isolated from the thoracic aortas of Sprague-Dawley rats. The migration of VSMCs was examined using a transwell technique and cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay. Reactive oxygen species (ROS) were measured using the fluoroprobe 2'7'-dichlorodihydrofluorescein diacetate. The activity of NADPH oxidase was assessed by lucigenin enhanced chemiluminescence. Expressions of Nox1 mRNA and p-p38MAPK protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. The results showed that atorvastatin inhibited the migration of VSMCs induced by Hcy, which was reversed by the mevalonate. In addition, pretreatment with the NADPH oxidase inhibitor DPI, the free radical scavenger NAC and the p38 MAPK inhibitor SB203580 blocked Hcy-induced VSMCs migration. Furthermore, atorvastatin suppressed Hcy-induced activation of NADPH oxidase and ROS, attenuated Hcy-induced overexpression of Nox1mRNA. Similar effects occurred with VSMCs transfected with Nox1 siRNA. Moreover, atorvastatin other than DPI, NAC, SB203580 and Nox1 siRNA transfection blocked Hcy-induced p38 MAPK phosphorylation, which was also reversed by the mevalonate. The data demonstrates that atorvastatin inhibits Hcy-induced VSMCs migration in a mevalonate pathway. Furthermore, a part of the biological effect of atorvastatin involves a decrease in the levels of Nox1-dependent ROS generation and p38 MAPK activation.

  17. Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity.

    PubMed

    Hwang, In-Wook; Makishima, Yu; Suzuki, Tomohiro; Kato, Tatsuya; Park, Sungjo; Terzic, Andre; Chung, Shin-Kyo; Park, Enoch Y

    2015-11-01

    Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is a vital catalytic reaction of lipid metabolism. While it is established that phosphorylation of MCD modulates the enzymatic activity, the specific phosphorylation sites associated with the catalytic function have not been documented due to lack of sufficient production of MCD with proper post-translational modifications. Here, we used the silkworm-based Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system to express human MCD (hMCD) and mapped phosphorylation effects on enzymatic function. Purified MCD from silkworm displayed post-translational phosphorylation and demonstrated coherent enzymatic activity with high yield (-200 μg/silkworm). Point mutations in putative phosphorylation sites, Ser-204 or Tyr-405 of hMCD, identified by bioinformatics and proteomics analyses reduced the catalytic activity, underscoring the functional significance of phosphorylation in modulating decarboxylase-based catalysis. Identified phosphorylated residues are distinct from the decarboxylation catalytic site, implicating a phosphorylation-induced global conformational change of MCD as responsible in altering catalytic function. We conclude that phosphorylation of Ser-204 and Tyr-405 regulates the decarboxylase function of hMCD leveraging the silkworm-based BmNPV bacmid expression system that offers a fail-safe eukaryotic production platform implementing proper post-translational modification such as phosphorylation.

  18. The reaction of dimethyltin(IV) dichloride with thiamine diphosphate (H2TDP): synthesis and structure of [SnMe2(HTDP)(H2O)]Cl.H2O, and possibility of a hitherto unsuspected role of the metal cofactor in the mechanism of vitamin-B1-dependent enzymes.

    PubMed

    Casas, José S; Castellano, Eduardo E; Couce, María D; Ellena, Javier; Sánchez, Agustín; Sánchez, José L; Sordo, José; Taboada, Carmen

    2004-03-22

    The complex [SnMe(2)(HTDP)(H(2)O)]Cl.H(2)O, synthesized by reaction between dimethyltin(IV) dichloride and thiamine diphosphate hydrochloride (H(3)TDPCl) in water, was characterized by X-ray diffractometry and IR and Raman spectroscopy in the solid state, and by electrospray mass spectrometry (ESMS) and NMR spectroscopy ((1)H, (13)C, (31)P, (119)Sn and inverse-detection (1)H,(15)N HMBC) in aqueous solution. In the solid state the HTDP(-) anion chelates the metal via one oxygen atom of each phosphate group [Sn-O = 2.062(3), 2.292(3) A], and another oxygen atom belonging to the terminal phosphate links the SnMe(2)(2+) cations into chains. The tin atom has distorted octahedral coordination involving the trans methyl groups, the above-mentioned diphosphate oxygen atoms, and the oxygen atom of the coordinated water molecule. The thiamine moiety has F conformation. NMR studies suggest that the interaction between the organometallic cation and the HTDP(-) ligand persists in D(2)O solution, which is in keeping with the ESMS spectrum showing a peak corresponding to [SnMe(2)(HTDP)]. Both in the solid state and in solution, the acidic HTDP(-) proton in the complex is located on the N(1') atom of the pyrimidine ring. The enzymatic behavior of native pyruvate decarboxylase (EC 4.1.1.1, PDC), obtained from baker's yeast, was compared in a coupled assay with that shown by the "SnMe(2)-holoenzyme" created by incubation of apoPDC with [SnMe(2)(HTDP)(H(2)O)]Cl.H(2)O. The SnMe(2)-holoenzyme exhibited about 34% of the activity of the native enzyme (with a Michaelis-Menten constant of 2.7 microM, as against 6.4 microM for native PDC), so confirming the very low specificity of PDC regarding the identity of its metal ion cofactor. In view of the observed protonation of N(1'), it is suggested that the role of divalent cations in the mechanism of thiamine-diphosphate-dependent enzymes may be not only to anchor the cofactor in its binding site but also to shift the acidic proton of HTDP

  19. The ornithine decarboxylase gene of Caenorhabditis elegans: Cloning, mapping and mutagenesis

    SciTech Connect

    Macrae, M.; Coffino, P.; Plasterk, R.H.A.

    1995-06-01

    The gene (odc-1) encoding ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was cloned and characterized. Two introns interrupt the coding sequence of the gene. The deduced protein contains 442 amino acids and is homologous to ornithine decarboxylases of other eukaryotic species. In vitro translation of a transcript of the cDNA yielded an enzymatically active product. The mRNA is 1.5 kb in size and is formed by trans-splicing to SL1, a common 5{prime} RNA segment. odc-1 maps to the middle of LG V, between dpy-11 and unc-42 and near a breakpoint of the nDf32 deficiency strain. Enzymatic activity is low in starved 1 (L1) larva and, after feeding, rises progressively as the worms develop. Targeted gene disruption was used to create a null allele. Homozygous mutants are normally viable and show no apparent defects, with the exception of a somewhat reduced brood size. In vitro assays for ornithine decarboxylase activity, however, show no detectable enzymatic activity, suggesting that ornithine decarboxylase is dispensible for nematode growth in the laboratory. 37 refs., 6 figs., 1 tab.

  20. Membrane Inlet for Mass Spectrometric Measurement of Catalysis by Enzymatic Decarboxylases

    PubMed Central

    Moral, Mario E. G.; Tu, Chingkuang; Richards, Nigel G. J.; Silverman, David N.

    2011-01-01

    Membrane inlet mass spectrometry (MIMS) uses diffusion across a permeable membrane to detect in solution uncharged molecules of small molecular weight. We point out here the application of MIMS to determine catalytic properties of decarboxylases using as an example catalysis by oxalate decarboxylase (OxDC) from Bacillus subtilis. The decarboxylase activity generates carbon dioxide and formate from the non-oxidative reaction, but is accompanied by a concomitant oxidase activity that consumes oxalate and oxygen and generates CO2 and hydrogen peroxide. The application of MIMS in measuring catalysis by OxDC involves the real-time and continuous detection of oxygen and product CO2 from the ion currents of their respective mass peaks. Steady-state catalytic constants for the decarboxylase activity obtained by measuring product CO2 using MIMS are comparable to those acquired by the traditional endpoint assay based on the coupled reaction with formate dehydrogenase, and measuring consumption of O2 using MIMS also estimates the oxidase activity. Use of isotope-labeled substrate (13C2-enriched oxalate) in MIMS provides a method to characterize the catalytic reaction in cell suspensions by detecting the mass peak for product 13CO2 (m/z 45), avoiding inaccuracies due to endogenous 12CO2. PMID:21782782

  1. Membrane inlet for mass spectrometric measurement of catalysis by enzymatic decarboxylases.

    PubMed

    Moral, Mario E G; Tu, Chingkuang; Richards, Nigel G J; Silverman, David N

    2011-11-01

    Membrane inlet mass spectrometry (MIMS) uses diffusion across a permeable membrane to detect in solution uncharged molecules of small molecular weight. We point out here the application of MIMS to determine catalytic properties of decarboxylases using as an example catalysis by oxalate decarboxylase (OxDC) from Bacillus subtilis. The decarboxylase activity generates carbon dioxide and formate from the nonoxidative reaction but is accompanied by a concomitant oxidase activity that consumes oxalate and oxygen and generates CO(2) and hydrogen peroxide. The application of MIMS in measuring catalysis by OxDC involves the real-time and continuous detection of oxygen and product CO(2) from the ion currents of their respective mass peaks. Steady-state catalytic constants for the decarboxylase activity obtained by measuring product CO(2) using MIMS are comparable to those acquired by the traditional endpoint assay based on the coupled reaction with formate dehydrogenase, and measuring consumption of O(2) using MIMS also estimates the oxidase activity. The use of isotope-labeled substrate ((13)C(2)-enriched oxalate) in MIMS provides a method to characterize the catalytic reaction in cell suspensions by detecting the mass peak for product (13)CO(2) (m/z 45), avoiding inaccuracies due to endogenous (12)CO(2). Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Structural and Mechanistic Studies on Klebsiella pneumoniae 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2010-11-12

    The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. Here we present the structures of Klebsiella pneumoniae OHCU decarboxylase in unliganded form and with bound allantoin. These structures provide evidence that ligand binding organizes the active site residues for catalysis. Modeling of the substrate and intermediates provides additional support for this hypothesis. In addition we characterize the steady state kinetics of this enzyme and report the first OHCU decarboxylase inhibitor, allopurinol, a structural isomer of hypoxanthine. This molecule is a competitive inhibitor of K. pneumoniae OHCU decarboxylase with a K{sub i} of 30 {+-} 2 {micro}m. Circular dichroism measurements confirm structural observations that this inhibitor disrupts the necessary organization of the active site. Our structural and biochemical studies also provide further insights into the mechanism of catalysis of OHCU decarboxylation.

  3. Presentation of opsoclonus myoclonus ataxia syndrome with glutamic acid decarboxylase antibodies.

    PubMed

    Bhandari, Hanul Srinivas

    2012-08-08

    In this rare case, the patient presented with opsoclonus, myoclonus and ataxia. Serological and imaging studies revealed high glutamic acid decarboxylase antibody (GAD-Ab) levels. High-dose corticosteroids were of no benefit and subsequent intravenous immunoglobulin (IVIg) administration proved resolution of the condition. Levetiracetam proved useful in symptomatically controlling the myoclonus. Follow-up GAD-Ab levels were within normal limits.

  4. Inhibition of pyruvate decarboxylase from Z. mobilis by novel analogues of thiamine pyrophosphate: investigating pyrophosphate mimics.

    PubMed

    Erixon, Karl M; Dabalos, Chester L; Leeper, Finian J

    2007-03-07

    Replacement of the thiazolium ring of thiamine pyrophosphate with a triazole gives extremely potent inhibitors of pyruvate decarboxylase from Z. mobilis, with K(I) values down to 20 pM; this system was used to explore pyrophosphate mimics and several effective analogues were discovered.

  5. Detection and transfer of the glutamate decarboxylase gene in Streptococcus thermophilus

    USDA-ARS?s Scientific Manuscript database

    GABA (gamma-aminobutyric acid) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermen...

  6. Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110

    USDA-ARS?s Scientific Manuscript database

    GABA ('-aminobutyric acid) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermented da...

  7. DPD epitope-specific glutamic acid decarboxylase GAD)65 autoantibodies in children with Type 1 diabetes

    USDA-ARS?s Scientific Manuscript database

    To study whether DPD epitope-specific glutamate decarboxylase autoantibodies are found more frequently in children with milder forms of Type 1 diabetes. We prospectively evaluated 75 children with new-onset autoimmune Type 1 diabetes, in whom we collected demographic, anthropometric and clinical dat...

  8. The Degradation of 14C-Glutamic Acid by L-Glutamic Acid Decarboxylase.

    ERIC Educational Resources Information Center

    Dougherty, Charles M; Dayan, Jean

    1982-01-01

    Describes procedures and semi-micro reaction apparatus (carbon dioxide trap) to demonstrate how a particular enzyme (L-Glutamic acid decarboxylase) may be used to determine the site or sites of labeling in its substrate (carbon-14 labeled glutamic acid). Includes calculations, solutions, and reagents used. (Author/SK)

  9. 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.

    PubMed

    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.

  10. Competence of Thiamin Diphosphate-Dependent Enzymes with 2'-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin.

    PubMed

    Nemeria, Natalia S; Shome, Brateen; DeColli, Alicia A; Heflin, Kathryn; Begley, Tadhg P; Meyers, Caren Freel; Jordan, Frank

    2016-02-23

    Bacimethrin (4-amino-5-hydroxymethyl-2-methoxypyrimidine), a natural product isolated from some bacteria, has been implicated as an inhibitor of bacterial and yeast growth, as well as in inhibition of thiamin biosynthesis. Given that thiamin biosynthetic enzymes could convert bacimethrin to 2'-methoxythiamin diphosphate (MeOThDP), it is important to evaluate the effect of this coenzyme analogue on thiamin diphosphate (ThDP)-dependent enzymes. The potential functions of MeOThDP were explored on five ThDP-dependent enzymes: the human and Escherichia coli pyruvate dehydrogenase complexes (PDHc-h and PDHc-ec, respectively), the E. coli 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), and the human and E. coli 2-oxoglutarate dehydrogenase complexes (OGDHc-h and OGDHc-ec, respectively). Using several mechanistic tools (fluorescence, circular dichroism, kinetics, and mass spectrometry), it was demonstrated that MeOThDP binds in the active centers of ThDP-dependent enzymes, however, with a binding mode different from that of ThDP. While modest activities resulted from addition of MeOThDP to E. coli PDHc (6-11%) and DXPS (9-14%), suggesting that MeOThDP-derived covalent intermediates are converted to the corresponding products (albeit with rates slower than that with ThDP), remarkably strong activity (up to 75%) resulted upon addition of the coenzyme analogue to PDHc-h. With PDHc-ec and PDHc-h, the coenzyme analogue could support all reactions, including communication between components in the complex. No functional substitution of MeOThDP for ThDP was in evidence with either OGDH-h or OGDH-ec, shown to be due to tight binding of ThDP.

  11. Geranylgeranyl diphosphate synthase in fission yeast is a heteromer of farnesyl diphosphate synthase (FPS), Fps1, and an FPS-like protein, Spo9, essential for sporulation.

    PubMed

    Ye, Yanfang; Fujii, Makoto; Hirata, Aiko; Kawamukai, Makoto; Shimoda, Chikashi; Nakamura, Taro

    2007-09-01

    Both farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. Here, we describe two novel Schizosaccharomyces pombe genes, fps1(+) and spo9(+), whose products are similar to FPS in primary structure, but whose functions differ from one another. Fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. Expression of fps1(+), but not spo9(+), suppresses the lethality of a Saccharomyces cerevisiae FPS-deficient mutant and also restores ubiquinone synthesis in an Escherichia coli ispA mutant, which lacks FPS activity, indicating that S. pombe Fps1 in fact functions as an FPS. In contrast to a typical FPS gene, no apparent GGPS homologues have been found in the S. pombe genome. Interestingly, although neither fps1(+) nor spo9(+) expression alone in E. coli confers clear GGPS activity, coexpression of both genes induces such activity. Moreover, the GGPS activity is significantly reduced in the spo9 mutant. In addition, the spo9 mutation perturbs the membrane association of a geranylgeranylated protein, but not that of a farnesylated protein. Yeast two-hybrid and coimmunoprecipitation analyses indicate that Fps1 and Spo9 physically interact. Thus, neither Fps1 nor Spo9 alone functions as a GGPS, but the two proteins together form a complex with GGPS activity. Because spo9 was originally identified as a sporulation-deficient mutant, we show here that expansion of the forespore membrane is severely inhibited in spo9Delta cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9Delta cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation.

  12. The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition.

    PubMed

    Kanjee, Usheer; Gutsche, Irina; Ramachandran, Shaliny; Houry, Walid A

    2011-11-01

    The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a "zone of inhibition" between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH.

  13. Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

    PubMed Central

    Romagnoli, Gabriele; Luttik, Marijke A. H.; Kötter, Peter; Pronk, Jack T.

    2012-01-01

    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols. PMID:22904058

  14. Substrate specificity of thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases in Saccharomyces cerevisiae.

    PubMed

    Romagnoli, Gabriele; Luttik, Marijke A H; Kötter, Peter; Pronk, Jack T; Daran, Jean-Marc

    2012-11-01

    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols.

  15. Monomeric S-adenosylmethionine decarboxylase from plants provides an alternative to putrescine stimulation.

    PubMed

    Bennett, Eric M; Ekstrom, Jennifer L; Pegg, Anthony E; Ealick, Steven E

    2002-12-10

    S-Adenosylmethionine decarboxylase has been implicated in cell growth and differentiation and is synthesized as a proenzyme, which undergoes autocatalytic cleavage to generate an active site pyruvoyl group. In mammals, S-adenosylmethionine decarboxylase is active as a dimer in which each protomer contains one alpha subunit and one beta subunit. In many higher organisms, autocatalysis and decarboxylation are stimulated by putrescine, which binds in a buried site containing numerous negatively charged residues. In contrast, plant S-adenosylmethionine decarboxylases are fully active in the absence of putrescine, with rapid autocatalysis that is not stimulated by putrescine. We have determined the structure of the S-adenosylmethionine decarboxylase from potato, Solanum tuberosum, to 2.3 A resolution. Unlike the previously determined human enzyme structure, the potato enzyme is a monomer in the crystal structure. Ultracentrifugation studies show that the potato enzyme is also a monomer under physiological conditions, with a weak self-association constant of 6.5 x 10(4) M(-)(1) for the monomer-dimer association. Although the potato enzyme contains most of the buried charged residues that make up the putrescine binding site in the human enzyme, there is no evidence for a putrescine binding site in the potato enzyme. Instead, several amino acid substitutions, including Leu13/Arg18, Phe111/Arg114, Asp174/Val181, and Phe285/His294 (human/potato), provide side chains that mimic the role of putrescine in the human enzyme. In the potato enzyme, the positively charged residues form an extensive network of hydrogen bonds bridging a cluster of highly conserved negatively charged residues and the active site, including interactions with the catalytic residues Glu16 and His249. The results explain the constitutively high activity of plant S-adenosylmethionine decarboxylases in the absence of putrescine and are consistent with previously proposed models for how putrescine together

  16. Platelet adenosine diphosphate receptor inhibition provides no advantage in predicting need for platelet transfusion or massive transfusion.

    PubMed

    Stettler, Gregory R; Moore, Ernest E; Moore, Hunter B; Nunns, Geoffrey R; Huebner, Benjamin R; Einersen, Peter; Ghasabyan, Arsen; Silliman, Christopher C; Banerjee, Anirban; Sauaia, Angela

    2017-09-27

    Thrombelastography platelet mapping is a useful assay to assess antiplatelet therapy. Inhibited response to the adenosine diphosphate receptor on platelets occurs early after injury, but recent work suggests this alteration occurs even with minor trauma. However, the utility of thrombelastography platelet mapping, specifically the percent of adenosine diphosphate receptor inhibition, in predicting outcomes and guiding platelet transfusion in trauma-induced coagulopathy remains unknown We assessed the role of percent of adenosine diphosphate-inhibition in predicting survival, requirement for massive transfusion or platelet transfusion in patients at risk for trauma-induced coagulopathy. Thrombelastography platelet mapping was assessed in 303 trauma activation patients from 2014-2016 and in 89 healthy volunteers. Percent of adenosine diphosphate-inhibition is presented as median and interquartile range. We compared the area under the receiver operating characteristic curve of percent of adenosine diphosphate-inhibition, platelet count, and rapid thrombelastography maximum amplitude for in-hospital mortality, massive transfusion (>10 red blood cells or death/6 hours), and platelet transfusion (>0 platelet units or death/6 hour). Overall, 35 (11.5%) patient died, 27 (8.9%) required massive transfusion and 46, platelet transfusions (15.2%). Median percent of adenosine diphosphate-inhibition was 42.5% (interquartile range: 22.4-69.1%), compared with 4.3 % (interquartile range: 0-13.5%) in healthy volunteers (P < .0001). Patients that died, had a massive transfusion, or platelet transfusion had higher percent of adenosine diphosphate-inhibition than those that did not (P < .05 for all). However, percent of adenosine diphosphate-inhibition did not add significantly to the predictive performance of maximum amplitude or platelet count for any of the 3 outcomes, after adjustment for confounders. Subgroup analyses by severe traumatic brain injury, severe injury and

  17. Overexpression of an isoprenyl diphosphate synthase in spruce leads to unexpected terpene diversion products that function in plant defense.

    PubMed

    Nagel, Raimund; Berasategui, Aileen; Paetz, Christian; Gershenzon, Jonathan; Schmidt, Axel

    2014-02-01

    Spruce (Picea spp.) and other conifers employ terpenoid-based oleoresin as part of their defense against herbivores and pathogens. The short-chain isoprenyl diphosphate synthases (IDS) are situated at critical branch points in terpene biosynthesis, producing the precursors of the different terpenoid classes. To determine the role of IDS and to create altered terpene phenotypes for assessing the defensive role of terpenoids, we overexpressed a bifunctional spruce IDS, a geranyl diphosphate and geranylgeranyl diphosphate synthase in white spruce (Picea glauca) saplings. While transcript level (350-fold), enzyme activity level (7-fold), and in planta geranyl diphosphate and geranylgeranyl diphosphate levels (4- to 8-fold) were significantly increased in the needles of transgenic plants, there was no increase in the major monoterpenes and diterpene acids of the resin and no change in primary isoprenoids, such as sterols, chlorophylls, and carotenoids. Instead, large amounts of geranylgeranyl fatty acid esters, known from various gymnosperm and angiosperm plant species, accumulated in needles and were shown to act defensively in reducing the performance of larvae of the nun moth (Lymantria monacha), a conifer pest in Eurasia. These results show the impact of overexpression of an IDS and the defensive role of an unexpected accumulation product of terpenoid biosynthesis with the potential for a broader function in plant protection.

  18. Structure of geranyl diphosphate C-methyltransferase from Streptomyces coelicolor and implications for the mechanism of isoprenoid modification†

    PubMed Central

    Köksal, Mustafa; Chou, Wayne K. W.; Cane, David E.; Christianson, David W.

    2012-01-01

    Geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is the first methyltransferase discovered that modifies an acyclic isoprenoid diphosphate, geranyl diphosphate (GPP), to yield a non-canonical acyclic allylic diphosphate product, 2-methylgeranyl diphosphate, which serves as the substrate for a subsequent cyclization reaction catalyzed by a terpenoid cyclase, methylisoborneol synthase. Here, we report the crystal structures of GPPMT in complex with GPP or the substrate analogue geranyl-S-thiolodiphosphate (GSPP) along with S-adenosyl-l-homocysteine in the cofactor binding site, resulting from in situ demethylation of S-adenosyl-l-methionine, at 2.05 Å and 1.82 Å resolution, respectively. These structures suggest that both GPP and GSPP can undergo catalytic methylation in crystalline GPPMT, followed by dissociation of the isoprenoid product. S-adenosyl-l-homocysteine remains bound in the active site, however, and does not exchange with a fresh molecule of cofactor S-adenosyl-l-methionine. These structures provide important clues regarding the molecular mechanism of the reaction, especially with regard to the face of the 2,3 double bond of GPP that is methylated as well as the stabilization of the resulting carbocation intermediate through cation-π interactions. PMID:22455498

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

    SciTech Connect

    Misra, Gauri; Aggarwal, Anita; Mittal, Sonia; Singh, Yogendra; Ramachandran, Ravishankar

    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%.

  20. Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle.

    PubMed

    Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G; Köllner, Tobias G

    2016-03-15

    Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene-producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon-intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors.

  1. Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle

    PubMed Central

    Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G.; Köllner, Tobias G.

    2016-01-01

    Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon–intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors. PMID:26936952

  2. Relationship of isopentenyl diphosphate (IDP) isomerase activity to isoprene emission of oak leaves.

    PubMed

    Brüggemann, Nicolas; Schnitzler, Jörg-Peter

    2002-10-01

    Oaks emit large amounts of isoprene, a compound that plays an important role in tropospheric chemistry. Isopentenyl diphosphate isomerase (IDI, E.C. 5.3.3.2) catalyzes the isomerization of isopentenyl diphosphate (IDP) to dimethylallyl diphosphate (DMADP), and in isoprene-emitting plants, isoprene synthase (IS) converts the DMADP to isoprene. To study the role of IDI in isoprene biosynthesis of oak leaves, we compared IDI and IS activities in pedunculate oak (Quercus robur L.) and pubescent oak (Quercus pubescens Willd.) with the isoprene emission rates of these species. We developed a non-radioactive enzyme assay to detect IDI activity in crude leaf extracts of Q. robur. The substrate dependency of IDI activity showed biphasic kinetics with Michaelis constants (K(m)(IDP)) of 0.7 +/- 0.2 micro M for a high-affinity phase and 39.5 +/- 6.9 micro M for a low-affinity phase, potentially attributable to different IDI isoforms. Under standard assay conditions, the temperature optimum for IDI activity was about 42 degrees C, but IDI activity was detectable up to 60 degrees C. A sharp pH optimum appeared around pH 7, with 20 mM Mg(2+) also required for IDI activity. Neither IDI activity nor IS activity showed diurnal variation in Q. robur leaves. The sum of IDI activities showed a significant linear correlation with IS activity in both Q. robur and Q. pubescens leaves, and both enzyme activities showed a linear relationship to isoprene emission factors in leaves of these oak species, indicating the possible involvement of IDI in isoprene biosynthesis by oak leaves.

  3. Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator

    SciTech Connect

    Flentke, G.R.; Frey, P.A. )

    1990-03-06

    UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5{prime}-diphosphate chloroacetol (UDC) and uridine 5{prime}-diphosphate bromoacetol (UCB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a K{sub D} of 0.110 mM and k{sub inact} of 0.84 min{sup {minus}1} at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent binding of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD{sup +}. The inactivation of epimerase by uridine 5{prime}-diphosphate ({sup 2}H{sub 2})chloroacetol proceeds with a primary kinetic isotope effect (k{sub H}/k{sub D}) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD{sup +} at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD{sup +} is proposed to be the chromophore with {lambda}{sub max} at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.

  4. A 31P-NMR study of the interaction of Mg2+ ions with nucleoside diphosphates.

    PubMed Central

    Tran-Dinh, S; Neumann, J M

    1977-01-01

    The interaction of Mg2+ with nucleoside disphosphates : ADP, GDP, CDP and UDP has been studied by phosphorus magnetic resonance spectroscopy in aqueous solution. The results show that these four nucleotides behave similarly, the Mg2+ ion binds to the alpha but not to the beta phosphate moiety. The strength of the interaction of Mg2+ ions with nucleoside diphosphates is weaker than with nucleoside triphosphates. The association of Mg2+ on the phosphate chain is stronger in a neutral than in an acid medium. PMID:14328

  5. Strength Characteristics of Resorbable Osteoconductive Ceramics Based on Diphosphates of Calcium and Alkali Metals

    NASA Astrophysics Data System (ADS)

    Putlayev, V. I.; Evdokimov, P. V.; Garshev, A. V.; Prosvirin, D. V.; Klimashina, E. S.; Safronova, T. V.; Ivanov, V. K.

    2014-02-01

    An investigation into the strength characteristics of ceramics based on diphosphates Ca(3- x)М2 x (PO4)2 ( x = 0-1 and М = Na, K) provides evidence of composition strengthening in the range х = 0.6-0.8 containing the greatest amount of the supercooled high-temperature modification α-СаМРО4. The method of high-temperature x-ray diffractometry is used to examine thermal expansion of rhenanite phases of СаМРО4.

  6. A kinetic study of pig liver pyruvate kinase activated by fructose diphosphate

    PubMed Central

    Macfarlane, Neil; Ainsworth, Stanley

    1974-01-01

    The paper reports a study of the reaction between phosphoenolpyruvate, ADP and Mg2+ catalysed by pig liver pyruvate kinase when activated by fructose diphosphate and K+. The experimental results are consistent with two non-sequential mechanisms in which the substrates and products of the reaction are phosphoenolpyruvate, ADP, Mg2+, pyruvate and MgATP. Pyruvate release occurs before ADP binding. Two Mg2+ ions are involved, though the two Mg2+-binding sites cannot be occupied simultaneously. An isomerized enzyme complex forms before release of MgATP. Values were determined for the Michaelis constants of the reaction. Apparent MgATP inhibition constants are also given. PMID:4850216

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

    PubMed

    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.

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

    PubMed

    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.

  9. The binding mode of human nucleoside diphosphate kinase B to single-strand DNA.

    PubMed

    Agou, F; Raveh, S; Véron, M

    2000-06-01

    In this paper, we studied the interaction of the human isoform B of nucleoside diphosphate kinase (NDP kinase B) with the nuclease hypersensitive element (NHE) present in the promoter element of the c-myc oncogene. The DNA-binding properties of NDP kinase B and other NDP kinases are compared and the nucleotide requirement for binding are discussed. Using quantitative methods, we identified the DNA-binding sites on the protein and we proposed a structural model for a complex of one hexameric NDP kinase B with an oligonucleotide.

  10. Immunohistochemical evidence for the coexistence of histidine decarboxylase-like and glutamate decarboxylase-like immunoreactivities in nerve cells of the magnocellular nucleus of the posterior hypothalamus of rats.

    PubMed Central

    Takeda, N; Inagaki, S; Shiosaka, S; Taguchi, Y; Oertel, W H; Tohyama, M; Watanabe, T; Wada, H

    1984-01-01

    Immunohistochemical staining of alternate consecutive sections revealed numerous histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22)-like immunoreactive neurons that also contained glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15)-like immunoreactive structures in the tuberal magnocellular nucleus, the caudal magnocellular nucleus, and the postmammillary caudal magnocellular nucleus of the posterior hypothalamus of rats. Furthermore, in immunohistochemical double-staining procedures, almost all neurons in the magnocellular nuclei had both histidine decarboxylase-like and glutamate decarboxylase-like immunoreactivities. These results suggest the coexistence of histamine and gamma-aminobutyric acid in single neurons in these nuclei. Images PMID:6594708

  11. Oritavancin Diphosphate

    PubMed Central

    Cada, Dennis J.; Baker, Danial E.

    2014-01-01

    Each month, subscribers to The Formulary Monograph Service receive 5 to 6 well-documented monographs on drugs that are newly released or are in late phase 3 trials. The monographs are targeted to Pharmacy & Therapeutics Committees. Subscribers also receive monthly 1-page summary monographs on agents that are useful for agendas and pharmacy/nursing in-services. A comprehensive target drug utilization evaluation/medication use evaluation (DUE/MUE) is also provided each month. With a subscription, the monographs are sent in print and are also available on-line. Monographs can be customized to meet the needs of a facility. A drug class review is now published monthly with The Formulary Monograph Service. Through the cooperation of The Formulary, Hospital Pharmacy publishes selected reviews in this column. For more information about The Formulary Monograph Service, call The Formulary at 800-322-4349. The December 2014 monograph topics are olodaterol, peginterferon beta-1a, testosterone nasal gel, ferric citrate corredination complex, and safinamide. The Safety MUE is on olodaterol. PMID:25673895

  12. Farnesyl Diphosphate Analogues with Aryl Moieties are Efficient Alternate Substrates for Protein Farnesyltransferase

    PubMed Central

    Subramanian, Thangaiah; Pais, June E.; Liu, Suxia; Troutman, Jerry M.; Suzuki, Yuta; Subramanian, Karunai Leela; Fierke, Carol; Andres, Douglas A.; Spielmann, H. Peter

    2012-01-01

    Farnesylation is an important post-translational modification essential for proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of analogue transfer to peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple turnover activity depends on the analogue structure. Analogues where the first isoprene is replaced by a benzyl group and an analogue where each isoprene is replaced by an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single turnover rate constant for alkylation does depend on the Ca1a2X peptide sequence. These results suggest that the isoprenoid transition state conformation is preferred over the inactive E•FPP• Ca1a2X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for design of novel inhibitors. PMID:22989235

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

    PubMed

    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.

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

    SciTech Connect

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

    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.

  15. Reversed-phase ion-pair high-performance liquid chromatography assay of polyprenyl diphosphate oligomer homologues.

    PubMed

    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.

  16. Induction of isoprenyl diphosphate synthases, plant hormones and defense signalling genes correlates with traumatic resin duct formation in Norway spruce (Picea abies).

    PubMed

    Schmidt, Axel; Nagel, Raimund; Krekling, Trygve; Christiansen, Erik; Gershenzon, Jonathan; Krokene, Paal

    2011-12-01

    Norway spruce (Picea abies) defends itself against herbivores and pathogens by formation of traumatic resin ducts filled with terpenoid-based oleoresin. An important group of enzymes in terpenoid biosynthesis are the short-chain isoprenyl diphosphate synthases which produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of monoterpenes, sesquiterpenes, and diterpene resin acids, respectively. After treatment with methyl jasmonate (MJ) we investigated the expression of all isoprenyl diphosphate synthase genes characterized to date from Norway spruce and correlated this with formation of traumatic resin ducts and terpene accumulation. Formation of traumatic resin ducts correlated with higher amounts of monoterpenes, sesquiterpenes and diterpene resin acids and an upregulation of isoprenyl diphosphate synthase genes producing geranyl diphosphate or geranylgeranyl diphosphate. Among defense hormones, jasmonate and jasmonate-isoleucine conjugate accumulated to higher levels in trees with extensive traumatic resin duct formation, whereas salicylate did not. Jasmonate and ethylene are likely to both be involved in formation of traumatic resin ducts based on elevated transcripts of genes encoding lipoxygenase and 1-aminocyclopropane-1-carboxylic acid oxidase associated with resin duct formation. Other genes involved in defense signalling in other systems, mitogen-activated protein kinase3 and nonexpressor of pathogenesis-related gene1, were also associated with traumatic resin duct formation. These responses were detected not only at the site of MJ treatment, but also systemically up to 60 cm above the site of treatment on the trunk.

  17. Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus†

    PubMed Central

    Thibodeaux, Christopher J.; Mansoorabadi, Steven O.; Kittleman, William; Chang, Wei-chen; Liu, Hung-wen

    2011-01-01

    The type II isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IDI-2) is a flavin mononucleotide (FMN)-dependent enzyme that catalyzes the reversible isomerization of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP), a reaction with no net change in redox state of the coenzyme or substrate. Here, UV-vis spectral analysis of the IDI-2 reaction revealed the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme was incubated with IPP or DMAPP. When IDI-2 was reconstituted with 1-deazaFMN and 5-deazaFMN, similar reduced neutral forms of the deazaflavin analogues were observed in the presence of IPP. Single turnover stopped-flow absorbance experiments indicated that this flavin intermediate formed and decayed at kinetically competent rates in the pre-steady-state and, thus, most likely represents a true intermediate in the catalytic cycle. UV-vis spectra of the reaction mixtures reveal trace amounts of a neutral semiquinone, but evidence for the presence of IPP-based radicals could not be obtained by EPR spectroscopy. Rapid-mix chemical quench experiments show no burst in DMAPP formation, suggesting that the rate determining step in the forward direction (IPP to DMAPP) occurs prior to DMAPP formation. A solvent deuterium kinetic isotope effect (D2OVmax = 1.5) was measured on vo in steady-state kinetic experiments at saturating substrate concentrations. A substrate deuterium kinetic isotope effect was also measured on the initital velocity (DVmax = 1.8) and on the decay rate of the flavin intermediate (Dks = 2.3) in single-turnover stopped-flow experiments using (R)-[2-2H]-IPP. Taken together, these data suggest that the C2–H bond of IPP is cleaved in the rate determining step and that general acid/base catalysis may be involved during turnover. Possible mechanisms for the IDI-2 catalyzed reaction are presented and discussed in terms of the available X-ray crystal structures. PMID:18229948

  18. Genetic regulation of glycogen biosynthesis in Escherichia coli: in vitro effects of cyclic AMP and guanosine 5'-diphosphate 3'-diphosphate and analysis of in vivo transcripts.

    PubMed Central

    Romeo, T; Preiss, J

    1989-01-01

    Glycogen accumulation in Escherichia coli is inversely related to the growth rate and occurs most actively when cells enter the stationary phase. The levels of the three biosynthetic enzymes undergo corresponding changes under these conditions, suggesting that genetic control of enzyme biosynthesis may account for at least part of the regulation (J. Preiss, Annu. Rev. Microbiol. 38:419-458, 1984). We have begun to explore the molecular basis of this control by identifying factors which affect the expression of the glycogen genes and by determining the 5'-flanking regions required to mediate the regulatory effects. The in vitro coupled transcription-translation of two of the biosynthetic genes, glgC (ADPglucose pyrophosphorylase) and glgA (glycogen synthase), was enhanced up to 26- and 10-fold, respectively, by cyclic AMP (cAMP) and cAMP receptor protein (CRP). Guanosine 5'-diphosphate 3'-diphosphate stimulated the expression of these genes 3.6- and 1.8-fold, respectively. The expression of glgB (glycogen branching enzyme) was affected weakly or negligibly by the above-mentioned compounds. Assays which measured the in vitro formation of the first dipeptide of glgC showed that a restriction fragment which contained 0.5 kilobases of DNA upstream from the initiation codon supported cAMP-CRP-activated expression. Sequence-specific binding of cAMP-CRP to a 243-base-pair restriction fragment from the region upstream from glgC was observed by virtue of the altered electrophoretic mobility of the bound DNA. S1 nuclease protection analysis identified 5' termini of four in vivo transcripts within 0.5 kilobases of the glgC coding region. The relative concentrations of transcripts were higher in the early stationary phase than in the exponential phase. Two mutants which overproduced the biosynthesis enzymes accumulated elevated levels of specific transcripts. The 5' termini of three of the transcripts were mapped to a high resolution. Their upstream sequences showed weak

  19. [Properties of 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate--an intermediate in the non-mevalonate isoprenoid biosynthesis].

    PubMed

    Ostrovskiĭ, D N; Demina, G P; Deriabina, Iu I; Goncharenko, A V; Eberl, M; Shumaev, K B; Shashkov, A S

    2003-01-01

    Extraction and purification from the biomass of Corynebacterium ammoniagenes of 2-C-methyl-D-erhythritol 2,4-cyclopyrophosphate (MEC) was associated with its spontaneous transformation into a number of derivatives (which was due to pyrophosphate bond lability and the formation of complexes with metals). These derivatives included 1,2-cyclophospho-4-phosphate, 2,4-diphosphate, 2,3-cyclophosphate, 1,4-diphosphate, and 3,5-diphosphate (identified by 1H, 31P, and 13C NMR spectroscopy) and accounted for about 10% MEC. When added to a solution of DNA in the presence of the Fenton reagent, MEC prevented DNA decomposition. In addition, MEC slowed down the interaction of the reagent with tempol radicals, which indicates that complexation of ferrous ions by MEC attenuates their ability to catalyze the formation of hydroxyl radicals from hydrogen peroxide. In the presence of 0.23 mM MEC, the rate of respiration of rat liver mitochondria increased 1.8 times. At 0.1-1.0 mM, MEC activated in vitro proliferation of human Vgamma9 T-cells. It is suggested that MEC acts as an endogenous stabilizing agent for bacterial cells subjected to oxidative stress and as an immunomodulator for eukaryotic hosts.

  20. Effect of the hexapeptide dalargin on ornithine decarboxylase activity in the duodenal mucosa of rats with experimental duodenal ulcer

    SciTech Connect

    Yarygin, K.N.; Shitin, A.G.; Polonskii, V.M.; Vinogradov, V.A.

    1987-08-01

    The authors study the effect of dalargin on ornithine decarboxylase in homogenates of the duodenal ulcer from rats with experimental duodenal ulcer induced by cysteamine. Activity of the enzyme was expressed in pmoles /sup 14/CO/sub 2//mg protein/h. Protein was determined by Lowry's method. The findings indicate that stimulation of ornithine decarboxylase and the antiulcerative effect of dalargin may be due to direct interaction of the peptide with cells of the intestinal mucosa and with enterocytes.

  1. HemQ: An iron-coproporphyrin oxidative decarboxylase for protoheme synthesis in Firmicutes and Actinobacteria

    SciTech Connect

    Dailey, Harry A.; Gerdes, Svetlana

    2015-02-21

    Genes for chlorite dismutase-like proteins are found widely among heme-synthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named HemQ, are ironcoproporphyrin (coproheme) decarboxylases that catalyze the oxidative decarboxylation of coproheme III into protoheme IX. As purified, HemQs do not contain bound heme, but readily bind exogeneously supplied heme with low micromolar affinity. We find that the heme-bound form of HemQ has low peroxidase activity and in the presence of peroxide the bound heme may be destroyed. Furthermore, it is possible that HemQ may serve a dual role as a decarboxylase in heme biosynthesis and a regulatory protein in heme homeostasis.

  2. HemQ: An iron-coproporphyrin oxidative decarboxylase for protoheme synthesis in Firmicutes and Actinobacteria

    DOE PAGES

    Dailey, Harry A.; Gerdes, Svetlana

    2015-02-21

    Genes for chlorite dismutase-like proteins are found widely among heme-synthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named HemQ, are ironcoproporphyrin (coproheme) decarboxylases that catalyze the oxidative decarboxylation of coproheme III into protoheme IX. As purified, HemQs do not contain bound heme, but readily bind exogeneously supplied heme with low micromolar affinity. We find that the heme-bound form of HemQ has low peroxidase activity and in the presence of peroxide the bound heme may be destroyed.more » Furthermore, it is possible that HemQ may serve a dual role as a decarboxylase in heme biosynthesis and a regulatory protein in heme homeostasis.« less

  3. Increase of histidine decarboxylase activity in mice hypothalamus after intracerebroventricular administration of lipopolysaccharide.

    PubMed

    Niimi, M; Mochizuki, T; Cacabelos, R; Yamatodani, A

    1993-10-01

    The effect of intracerebroventricular (icv) administration of lipopolysaccharide on histidine decarboxylase activity and histamine content in the hypothalamus were investigated in male mice of ddY strain in vivo. Two-fold increase in histidine decarboxylase activity (HDC) was observed 4 h after administration of 50 mcg lipopolysaccharide, and HDC activity returned to the basal level within 12 h after injection. Furthermore, histamine contents showed a slight decrease at 1 and 2 h and a mild increase at 12 h after administration. However, changes in histamine content were not statistically significant. These results suggest that the increase of HDC activity in the hypothalamus by lipopolysaccharide may be involved in the central neuroimmune responses.

  4. Identification of FAH Domain-containing Protein 1 (FAHD1) as Oxaloacetate Decarboxylase*

    PubMed Central

    Pircher, Haymo; von Grafenstein, Susanne; Diener, Thomas; Metzger, Christina; Albertini, Eva; Taferner, Andrea; Unterluggauer, Hermann; Kramer, Christian; Liedl, Klaus R.; Jansen-Dürr, Pidder

    2015-01-01

    Fumarylacetoacetate hydrolase (FAH) domain-containing proteins occur in both prokaryotes and eukaryotes, where they carry out diverse enzymatic reactions, probably related to structural differences in their respective FAH domains; however, the precise relationship between structure of the FAH domain and the associated enzyme function remains elusive. In mammals, three FAH domain-containing proteins, FAHD1, FAHD2A, and FAHD2B, are known; however, their enzymatic function, if any, remains to be demonstrated. In bacteria, oxaloacetate is subject to enzymatic decarboxylation; however, oxaloacetate decarboxylases (ODx) were so far not identified in eukaryotes. Based on molecular modeling and subsequent biochemical investigations, we identified FAHD1 as a eukaryotic ODx enzyme. The results presented here indicate that dedicated oxaloacetate decarboxylases exist in eukaryotes. PMID:25575590

  5. Observation of Superoxide Production During Catalysis of Bacillus subtilis Oxalate Decarboxylase at pH4

    PubMed Central

    Twahir, Umar T.; Stedwell, Corey N.; Lee, Cory T.; Richards, Nigel G. J.; Polfer, Nicolas C.; Angerhofer, Alexander

    2015-01-01

    This contribution describes the trapping of the hydroperoxyl radical at a pH of 4 during turnover of wild-type oxalate decarboxylase and its T165V mutant using the spin trap BMPO. Radicals were detected and identified by a combination of EPR and mass spectrometry. Superoxide, or its conjugate acid, the hydroperoxyl radical, is expected as an intermediate in the decarboxylation and oxidation reactions of the oxalate monoanion both of which are promoted by oxalate decarboxylase. Another intermediate, the carbon dioxide radical anion was also observed. The quantitative yields of superoxide trapping is similar in the wild type and the mutant while it is significantly different for the trapping of the carbon dioxide radical anion. This suggests that the two radicals are released from different sites of the protein. PMID:25526893

  6. Unusual space-group pseudo symmetry in crystals of human phosphopantothenoylcysteine decarboxylase

    SciTech Connect

    Manoj, N.; Ealick, S.E.

    2010-12-01

    Phosphopantothenoylcysteine (PPC) decarboxylase is an essential enzyme in the biosynthesis of coenzyme A and catalyzes the decarboxylation of PPC to phosphopantetheine. Human PPC decarboxylase has been expressed in Escherichia coli, purified and crystallized. The Laue class of the diffraction data appears to be {bar 3}m, suggesting space group R32 with two monomers per asymmetric unit. However, the crystals belong to the space group R3 and the asymmetric unit contains four monomers. The structure has been solved using molecular replacement and refined to a current R factor of 29%. The crystal packing can be considered as two interlaced lattices, each consistent with space group R32 and with the corresponding twofold axes parallel to each other but separated along the threefold axis. Thus, the true space group is R3 with four monomers per asymmetric unit.

  7. A coenzyme-independent decarboxylase/oxygenase cascade for the efficient synthesis of vanillin.

    PubMed

    Furuya, Toshiki; Miura, Misa; Kino, Kuniki

    2014-10-13

    Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant-derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme-independent decarboxylase and a coenzyme-independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM, 1.2 g L(-1) ) via 4-vinylguaiacol in one pot, without the generation of any detectable aromatic by-products. The efficient method described here might be applicable to the synthesis of other high-value chemicals from plant-derived aromatics.

  8. HemQ: an iron-coproporphyrin oxidative decarboxylase for protoheme synthesis in Firmicutes and Actinobacteria

    PubMed Central

    Dailey, Harry A.; Gerdes, Svetlana

    2015-01-01

    Genes for chlorite dismutase-like proteins are found widely among hemesynthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named HemQ, are ironcoproporphyrin (coproheme) decarboxylases that catalyze the oxidative decarboxylation of coproheme III into protoheme IX. As purified, HemQs do not contain bound heme, but readily bind exogeneously supplied heme with low micromolar affinity. The heme-bound form of HemQ has low peroxidase activity and in the presence of peroxide the bound heme may be destroyed. Thus, it is possible that HemQ may serve a dual role as a decarboxylase in heme biosynthesis and a regulatory protein in heme homeostasis. PMID:25711532

  9. Effect of guanosine 5'-diphosphate 3'-diphosphate and related nucleoside polyphosphates on induction of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli.

    PubMed

    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.

  10. Arginine and lysine decarboxylases and the acid tolerance response of Salmonella Typhimurium.

    PubMed

    Alvarez-Ordóñez, Avelino; Fernández, Ana; Bernardo, Ana; López, Mercedes

    2010-01-01

    Salmonella Typhimurium CECT 443 inactivation at pH 2.5 in Mineral Medium (MM) and MM supplemented with 0.01% (w/v) arginine, lysine or glutamic acid was studied using stationary-phase cells grown in buffered BHI pH 7.0 (non-acid adapted cells) and acidified BHI up to pH 4.5 with acetic, citric, lactic and hydrochloric acids (acid adapted cells). In all cases, acid adapted cells, with D-values ranging from 23.34 to 86.90 min, showed a significantly higher acid resistance than non-acid adapted cells, with D-values between 8.90 and 10.29 min. Whereas the conditions used for acid adaptation did not exert a significant effect on the acid resistance of the S. Typhimurium CECT 443 resulting cells, the inclusion of lysine and arginine in the challenge medium protected them against acid inactivation, reaching D-values of about 2 and 3 times higher, respectively, than those found in MM or MM supplemented with glutamic acid. None of these three amino acids significantly modified the acid resistance of non-acid adapted cells. The relative expression level of adiA (encoding the arginine decarboxylase), adiY (encoding the transcriptional activator of adiA), cadA (encoding the lysine decarboxylase) and cadB (encoding the lysine/cadaverine transport protein) was examined by quantitative PCR. Acid adapted cells showed higher relative expression levels for both systems, arginine decarboxylase and lysine decarboxylase, which demonstrates that the induction of specialized pH-homeostatic systems plays an important role in S. Typhimurium CECT 443 protection against acid stress. However, the increased acid resistance showed by acid adapted cells challenged in MM arginine or lysine free suggests the existence of other microbial survival strategies.

  11. Cell density-correlated induction of pyruvate decarboxylase under aerobic conditions in the yeast Pichia stipitis.

    PubMed

    Mergler, M; Klinner, U

    2001-01-01

    During the aerobic batch cultivation of P. stipitis CBS 5776 with glucose, pyruvate decarboxylase was activated in a cell number-correlated manner. Activation started when a cell number between 7 x 10(7) and x 10(8) cells ml(-1) was reached and the enzyme activity increased during further cultivation. This induction might have been triggered either by an unknown quorum sensing system or by a shortage of cytoplasmic acetyl-CoA.

  12. Gene controlling L-glutamic acid decarboxylase synthesis in Escherichia coli K-12.

    PubMed

    Lupo, M; Halpern, Y S

    1970-08-01

    Genetically related Escherichia coli K-12 strains were found to differ widely in their l-glutamic acid decarboxylase (GAD) activity. This variation is due to differences in the amount of GAD produced by the different cultures, rather than to the appearance of altered enzymes differing in catalytic activity. A regulatory gene, gadR, which controls the amount of GAD was mapped on the E. coli K-12 chromosome. A strain with a lesion in the structural gene for GAD is described.

  13. Autoradiographic measurement of relative changes in ornithine decarboxylase in axotomized superior cervical ganglion neurons

    SciTech Connect

    Wells, M.R.

    1986-05-01

    An autoradiographic method is described for detecting changes in ornithine decarboxylase in axotomized superior cervical ganglion neurons of rats using (3H)difluoromethylornithine. An increase in binding to neurons was seen at 12 h and 1 day after crushing the postganglionic nerves. Binding returned to control values between 3 and 5 days postoperation. The patterns found using this method were in general agreement with prior reports of enzymatic changes in whole ganglia.

  14. Overexpression, purification, crystallization and preliminary structural studies of p-coumaric acid decarboxylase from Lactobacillus plantarum

    SciTech Connect

    Rodríguez, Héctor; Rivas, Blanca de las; Muñoz, Rosario; Mancheño, José M.

    2007-04-01

    The enzyme p-coumaric acid decarboxylase (PDC) from L. plantarum has been recombinantly expressed, purified and crystallized. The structure has been solved at 2.04 Å resolution by the molecular-replacement method. The substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His{sub 6}-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris–HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 Å resolution. Crystals belonged to the tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 43.15, c = 231.86 Å. The estimated Matthews coefficient was 2.36 Å{sup 3} Da{sup −1}, corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism.

  15. Novel protein–protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani

    SciTech Connect

    Mishra, Arjun K.; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J. Venkatesh

    2015-01-09

    Highlights: • L. donovani spermidine synthase and S-adenosylmethionine decarboxylase have been cloned and purified. • S-adenosylmethionine decarboxylase has autocatalytic property. • GST pull down assay shows the two proteins to form a metabolon. • Isothermal titration calorimetry shows that binding was exothermic having K{sub d} value of 0.4 μM. • Interaction confirmed by fluorescence spectroscopy and size exclusion chromatography. - Abstract: Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein–protein interaction between SpdSyn and AdoMetDc. The protein–protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes.

  16. Different mRNAs code for dopa decarboxylase in tissues of neuronal and nonneuronal origin

    SciTech Connect

    Krieger, M.; Coge, F.; Gros, F.; Thibault, J. )

    1991-03-15

    A cDNA clone for dopa decarboxylase has been isolated from a rat pheochromocytoma cDNA library and the cDNA sequence has been determined. It corresponds to an mRNA of 2094 nucleotides. The length of the mRNA was measured by primer-extension of rat pheochromocytoma RNA and the 5{prime} end of the sequence of the mRNA was confirmed by the PCR. A probe spanning the translation initiation site of the mRNA was used to hybridize with mRNAs from various organs of the rat. S1 nuclease digestion of the mRNAs annealed with this probe revealed two classes of mRNAs. The comparison of the cDNA sequence and published sequences for rat liver, human pheochromocytoma, and Droxophila dopa decarboxylase supported the conclusion that two mRNAs are produced: one is specific for tissue of neuronal origin and the other is specific for tissues of nonneuronal (mesodermal or endodermal) origin. The neuronal mRNA contains a 5{prime} untranslated sequence that is highly conserved between human and rat pheochromocytoma including a GA stretch. The coding sequence and the 3{prime} untranslated sequence of mRNAs from rat liver and pheochromocytoma are identical. The rat mRNA differs only in the 5{prime} untranslated region. Thus a unique gene codes for dopa decarboxylase and this gene gives rise to at least two transcripts presumably in response to different signals during development.

  17. Experimental Evidence and In Silico Identification of Tryptophan Decarboxylase in Citrus Genus.

    PubMed

    De Masi, Luigi; Castaldo, Domenico; Pignone, Domenico; Servillo, Luigi; Facchiano, Angelo

    2017-02-11

    Plant tryptophan decarboxylase (TDC) converts tryptophan into tryptamine, precursor of indolealkylamine alkaloids. The recent finding of tryptamine metabolites in Citrus plants leads to hypothesize the existence of TDC activity in this genus. Here, we report for the first time that, in Citrus x limon seedlings, deuterium labeled tryptophan is decarboxylated into tryptamine, from which successively deuterated N,N,N-trimethyltryptamine is formed. These results give an evidence of the occurrence of the TDC activity and the successive methylation pathway of the tryptamine produced from the tryptophan decarboxylation. In addition, with the aim to identify the genetic basis for the presence of TDC, we carried out a sequence similarity search for TDC in the Citrus genomes using as a probe the TDC sequence reported for the plant Catharanthus roseus. We analyzed the genomes of both Citrus clementina and Citrus sinensis, available in public database, and identified putative protein sequences of aromatic l-amino acid decarboxylase. Similarly, 42 aromatic l-amino acid decarboxylase sequences from 23 plant species were extracted from public databases. Potential sequence signatures for functional TDC were then identified. With this research, we propose for the first time a putative protein sequence for TDC in the genus Citrus.

  18. EPR Spin Trapping of an Oxalate-Derived Free Radical in the Oxalate Decarboxylase Reaction

    PubMed Central

    Imaram, Witcha; Saylor, Benjamin T.; Centonze, Christopher P.; Richards, Nigel G. J.; Angerhofer, Alexander

    2011-01-01

    EPR spin trapping experiments on bacterial oxalate decarboxylase from Bacillus subtilis under turn-over conditions are described. The use of doubly 13C-labeled oxalate leads to a characteristic splitting of the observed radical adducts using the spin trap N-tert-butyl-α-phenylnitrone linking them directly to the substrate. The radical was identified as the carbon dioxide radical anion which is a key intermediate in the hypothetical reaction mechanism of both decarboxylase and oxidase activities. X-ray crystallography had identified a flexible loop, SENS161-4, which acts as a lid to the putative active site. Site directed mutagenesis of the hinge amino acids, S161 and T165 was explored and showed increased radical trapping yields compared to the wild type. In particular, T165V shows approximately ten times higher radical yields while at the same time its decarboxylase activity was reduced by about a factor of ten. This mutant lacks a critical H-bond between T165 and R92 resulting in compromised control over its radical chemistry allowing the radical intermediate to leak into the surrounding solution. PMID:21277974

  19. Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA.

    PubMed

    Michael, A J; Furze, J M; Rhodes, M J; Burtin, D

    1996-02-15

    A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription-PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC routine to putrescine is present.

  20. 3,4-Dihydroxyphenylalanine (dopa) decarboxylase activity in the arthropod nervous system.

    PubMed

    Murdock, L L; Wirtz, R A; Köhler, G

    1973-04-01

    1. When homogenates of brains from mature adult locusts (Locusta migratoria) were incubated with l-3-(3,4-dihydroxyphenyl)[3-(14)C]alanine the major radioactive metabolite was dopamine, suggesting the presence of a dopa (3,4-dihydroxyphenylalanine) decarboxylase. 2. Decarboxylation of l-dopa by this tissue, measured under optimum conditions by a radiochemical method, was 21mumol of CO(2)/h per g wet wt. Apparent decarboxylation of l-tyrosine proceeded at 0.34mumol of CO(2)/h per g wet wt. There was no detectable decarboxylation of l-tryptophan, l-histidine or l-phenylalanine. 3. Dopa decarboxylase activity was found in all major regions of the ventral nerve cord of the mature locust (range: 4-7mumol of CO(2)/h per g wet wt.) but was low or absent in thoracic peripheral nerve. 4. Marked decarboxylation of l-dopa was found in homogenates of brains of four other species of insects, and in brain and ventral nerve cord, but not in the claw nerve, of the crayfish. 5. The activity of the locust brain enzyme may be slightly lower at the time of imaginal ecdysis than during the mature period. By contrast, the dopa decarboxylase that produces dopamine as an intermediate in cuticle biosynthesis is known to be high in activity at the time of ecdysis and low in activity during the intermoult stages.

  1. Mevalonate-derived quinonemethide triterpenoid from in vitro roots of Peritassa laevigata and their localization in root tissue by MALDI imaging.

    PubMed

    Pina, Edieidia S; Silva, Denise B; Teixeira, Simone P; Coppede, Juliana S; Furlan, Maysa; França, Suzelei C; Lopes, Norberto P; Pereira, Ana Maria S; Lopes, Adriana A

    2016-03-04

    Biosynthetic investigation of quinonemethide triterpenoid 22β-hydroxy-maytenin (2) from in vitro root cultures of Peritassa laevigata (Celastraceae) was conducted using (13)C-precursor. The mevalonate pathway in P. laevigata is responsible for the synthesis of the quinonemethide triterpenoid scaffold. Moreover, anatomical analysis of P. laevigata roots cultured in vitro and in situ showed the presence of 22β-hydroxy-maytenin (2) and maytenin (1) in the tissues from transverse or longitudinal sections with an intense orange color. MALDI-MS imaging confirmed the distribution of (2) and (1) in the more distal portions of the root cap, the outer cell layers, and near the vascular cylinder of P. laevigata in vitro roots suggesting a role in plant defense against infection by microorganisms as well as in the root exudation processes.

  2. Mevalonate-derived quinonemethide triterpenoid from in vitro roots of Peritassa laevigata and their localization in root tissue by MALDI imaging

    PubMed Central

    Pina, Edieidia S.; Silva, Denise B.; Teixeira, Simone P.; Coppede, Juliana S.; Furlan, Maysa; França, Suzelei C.; Lopes, Norberto P.; Pereira, Ana Maria S.; Lopes, Adriana A.

    2016-01-01

    Biosynthetic investigation of quinonemethide triterpenoid 22β-hydroxy-maytenin (2) from in vitro root cultures of Peritassa laevigata (Celastraceae) was conducted using 13C-precursor. The mevalonate pathway in P. laevigata is responsible for the synthesis of the quinonemethide triterpenoid scaffold. Moreover, anatomical analysis of P. laevigata roots cultured in vitro and in situ showed the presence of 22β-hydroxy-maytenin (2) and maytenin (1) in the tissues from transverse or longitudinal sections with an intense orange color. MALDI-MS imaging confirmed the distribution of (2) and (1) in the more distal portions of the root cap, the outer cell layers, and near the vascular cylinder of P. laevigata in vitro roots suggesting a role in plant defense against infection by microorganisms as well as in the root exudation processes. PMID:26943243

  3. Mevalonate-derived quinonemethide triterpenoid from in vitro roots of Peritassa laevigata and their localization in root tissue by MALDI imaging

    NASA Astrophysics Data System (ADS)

    Pina, Edieidia S.; Silva, Denise B.; Teixeira, Simone P.; Coppede, Juliana S.; Furlan, Maysa; França, Suzelei C.; Lopes, Norberto P.; Pereira, Ana Maria S.; Lopes, Adriana A.

    2016-03-01

    Biosynthetic investigation of quinonemethide triterpenoid 22β-hydroxy-maytenin (2) from in vitro root cultures of Peritassa laevigata (Celastraceae) was conducted using 13C-precursor. The mevalonate pathway in P. laevigata is responsible for the synthesis of the quinonemethide triterpenoid scaffold. Moreover, anatomical analysis of P. laevigata roots cultured in vitro and in situ showed the presence of 22β-hydroxy-maytenin (2) and maytenin (1) in the tissues from transverse or longitudinal sections with an intense orange color. MALDI-MS imaging confirmed the distribution of (2) and (1) in the more distal portions of the root cap, the outer cell layers, and near the vascular cylinder of P. laevigata in vitro roots suggesting a role in plant defense against infection by microorganisms as well as in the root exudation processes.

  4. Evidence for a different metabolic behaviour of cytidine diphosphate choline after oral and intravenous administration to rats.

    PubMed

    Paroni, R; Cighetti, G; Del Puppo, M; Kienle, M G

    1985-09-01

    Radioactivity plasma decay was studied in rats after intravenous and oral administration of cytidine diphosphate [methyl-14C]choline at doses of 25 and 300 mg/kg. The kinetics fitted well with a two compartment open model and showed a long lasting elimination phase with a half-life ranging from 2.0 to 2.6 days for the two doses and the two administration routes. Absorption of cytidine diphosphate choline radioactivity was complete after oral treatment with the low dose and accounted for 94.5% of the dose when 300 mg/kg of cytidine diphosphate [methyl-14C]choline were administered. However the distribution of radioactivity in tissues, urine and expired air suggest metabolic differences, at least from a quantitative point of view, between the oral and intravenous treatments. In particular, the higher excretion of radioactivity associated with trimethylamine in urine found when cytidine diphosphate [methyl-14C]choline was given orally, suggest that the compound may be metabolized, at least in part, previous to its gastrointestinal absorption.

  5. Dependence of the product chain-length on detergents for long-chain E-polyprenyl diphosphate synthases.

    PubMed

    Pan, Jian-Jung; Ramamoorthy, Gurusankar; Poulter, C Dale

    2013-07-23

    Long-chain E-polyprenyl diphosphate synthases (E-PDS) catalyze repetitive addition of isopentenyl diphosphate (IPP) to the growing prenyl chain of an allylic diphosphate. The polyprenyl diphosphate products are required for the biosynthesis of ubiquinones and menaquinones required for electron transport during oxidative phosphorylation to generate ATP. In vitro, the long-chain PDSs require addition of phospholipids or detergents to the assay buffer to enhance product release and maintain efficient turnover. During preliminary assays of product chain-length with anionic, zwitterionic, and nonionic detergents, we discovered considerable variability. Examination of a series of nonionic PEG detergents with several long-chain E-PDSs from different organisms revealed that in vitro incubations with nonaethylene glycol monododecyl ether or Triton X-100 typically gave chain-lengths that corresponded to those of the isoprenoid moieties in respiratory quinones synthesized in vivo. In contrast, incubations in buffer with n-butanol, CHAPS, DMSO, n-octyl-β-glucopyranoside, or β-cyclodextrin or in buffer without detergent typically proceeded more slowly and gave a broad range of chain-lengths.

  6. Dependence of the product chain-length on detergents for long-chain E-polyprenyl diphosphate synthases

    PubMed Central

    Pan, Jian-Jung; Ramamoorthy, Gurusankar; Poulter, C. Dale

    2013-01-01

    Long-chain E-polyprenyl diphosphate synthases (E-PDS) catalyze repetitive addition of isopentenyl diphosphate (IPP) to the growing prenyl chain of an allylic diphosphate. The polyprenyl diphosphate products are required for the biosynthesis of ubiquinones and menaquinones required for electron transport during oxidative phosphorylation to generate ATP. In vitro, the long-chain PDSs require addition of phospholipids or detergents to the assay buffer to enhance product release and maintain efficient turnover. During preliminary assays of product chain-length with anionic, zwitterionic, and non-ionic detergents, we discovered considerable variability. Examination of a series of non-ionic PEG detergents with several long-chain E-PDSs from different organisms revealed that in vitro incubations with nonaethylene glycol monododecyl ether or Triton X-100 typically gave chain lengths that corresponded to those of the isoprenoid moieties in respiratory quinones synthesized in vivo. In contrast incubations in buffer with n-butanol, CHAPS, DMSO, n-octyl-β-glucopyranoside, or β-cyclodextrin or in buffer without detergent typically proceeded more slowly and gave a broad range of chain lengths. PMID:23802587

  7. A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni).

    PubMed

    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 ara<>ispH 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.

  8. Tracer studies on the incorporation of [2-14C]-DL-mevalonate into chlorophylls a and b, alpha-chaconine, and alpha-solanine of potato sprouts.

    PubMed

    Kozukue, N; Tsuchida, H; Friedman, M

    2001-01-01

    Chlorophyll and glycoalkaloids are synthesized in different parts of the potato plant including leaves, tubers, and sprouts. Although light stimulates the biosynthesis of both constituents, the question of whether the two biosynthetic pathways are under the same genetic control has not been resolved. This study investigated the dynamics of incorporation of labeled [2-(14)C]-DL-mavalonate into chlorophyll a, chlorophyll b, and the glycoalkaloids alpha-chaconine and alpha-solanine in potato sprouts after 7 and 14 days of storage in the light and in the dark. No chlorophyll synthesis occurred in the dark. Fractionation of the "glycoalkaloid" extract followed by high-performance liquid chromatography produced four peaks. The fractions were collected and analyzed for radioactivity. About 80% of the radioactivity resided in fraction 1, the composition of which is unknown. Two of the fractions, with 1-14% of the original label, were alpha-chaconine and alpha-solanine. The radioactivity derived from mevalonate largely resides in unidentified compound(s) eluting as a single peak on the HPLC column before the peaks associated with the glycoalkaloids. The specific radioactivity of alpha-chaconine and alpha-solanine increased approximately 2-fold in going from 7 to 14 days of exposure in the light and in the dark. These and additional observations point to the near identity of the dynamics of biosynthesis of the two glycoalkaloids. These data also implicate a non-mevalonate pathway for the synthesis of both chlorophylls and the glycoalkaloids and are consistent with independent genetic control of the concurrent formation of the two classes of compounds during greening of potatoes.

  9. Isolation and sequence of the gene encoding ornithine decarboxylase, SPE1, from Candida albicans by complementation of a spe1 delta strain of Saccharomyces cerevisiae.

    PubMed

    McNemar, M D; Gorman, J A; Buckley, H R

    1997-11-01

    The gene encoding ornithine decarboxylase, SPE1, from the pathogenic yeast Candida albicans has been isolated by complementation of an ornithine decarboxylase-negative (spe1 delta) strain of Saccharomyces cerevisiae. Four transformants, three of which contain plasmids with the SPE1 gene, were isolated by selection on polyamine-free medium. The C. albicans ornithine decarboxylase (ODC) showed high homology with other eukaryotic ODCs at both the amino acid and nucleic acid levels.

  10. Effects of irradiation on the thorium phosphate diphosphate ceramics and consequences on its dissolution

    NASA Astrophysics Data System (ADS)

    Tamain, C.; Özgümüs, A.; Dacheux, N.; Garrido, F.; Thomé, L.

    2006-06-01

    Thorium phosphate diphosphate samples (β-TPD), proposed as a ceramic for the long term immobilization of actinides, were externally irradiated with ions at several energies in order to simulate the self-irradiation as well as with γ-rays. The influence of the electronic energy loss was first investigated. XRD measurements showed a complete amorphization of the material at 1013 cm-2 of 840 MeV krypton while no significant structural change occurred at 5 × 1013 cm-2 of 410 MeV sulfur. The dissolution of raw and irradiated pellets was studied versus several parameters such as the amorphized fraction, the radiolytic species (as instance hydrogen peroxide) produced in situ in the leachate during irradiation, the temperature and the leachate acidity. The results obtained reveal a significant increase of the kinetics of dissolution for amorphized pellets compared to the raw ceramic.

  11. Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1.

    PubMed

    Gómez Barroso, J A; Pereira, H; Miranda, M; Pereira, C; Garratt, R C; Aguilar, C F

    2010-07-01

    The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 A resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 A. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

  12. Geranyl and Neryl Triazole Bisphosphonates as Inhibitors of Geranylgeranyl Diphosphate Synthase

    PubMed Central

    Zhou, Xiang; Ferree, Sarah D.; Wills, Veronica S.; Born, Ella J.; Tong, Huaxiang; Holstein, Sarah A.

    2014-01-01

    When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase. PMID:24726306

  13. Structure of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Entamoeba histolytica.

    PubMed

    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. Interaction of thiamin diphosphate with phosphorylated and dephosphorylated mammalian pyruvate dehydrogenase complex.

    PubMed

    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.

  15. Synthetic Pathway for Production of Five-Carbon Alcohols from Isopentenyl Diphosphate

    PubMed Central

    Chou, Howard H.

    2012-01-01

    Synthetic biological pathways could enhance the development of novel processes to produce chemicals from renewable resources. On the basis of models that describe the evolution of metabolic pathways and enzymes in nature, we developed a framework to rationally identify enzymes able to catalyze reactions on new substrates that overcomes one of the major bottlenecks in the assembly of a synthetic biological pathway. We verified the framework by implementing a pathway with two novel enzymatic reactions to convert isopentenyl diphosphate into 3-methyl-3-butenol, 3-methyl-2-butenol, and 3-methylbutanol. To overcome competition with native pathways that share the same substrate, we engineered two bifunctional enzymes that redirect metabolic flux toward the synthetic pathway. Taken together, our work demonstrates a new approach to the engineering of novel synthetic pathways in the cell. PMID:22941086

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

    PubMed

    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.

  17. Structure Conservation and Differential Expression of Farnesyl Diphosphate Synthase Genes in Euphorbiaceous Plants

    PubMed Central

    Guo, Dong; Li, Hui-Liang; Peng, Shi-Qing

    2015-01-01

    Farnesyl diphosphate synthase (FPS) is a key enzyme of isoprenoids biosynthesis. However, knowledge of the FPSs of euphorbiaceous species is limited. In this study, ten FPSs were identified in four euphorbiaceous plants. These FPSs exhibited similar exon/intron structure. The deduced FPS proteins showed close identities and exhibited the typical structure of plant FPS. The members of the FPS family exhibit tissue expression patterns that vary among several euphorbiaceous plant species under normal growth conditions. The expression profiles reveal spatial and temporal variations in the expression of FPSs of different tissues from Euphorbiaceous plants. Our results revealed wide conservation of FPSs and diverse expression in euphorbiaceous plants during growth and development. PMID:26389894

  18. Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif.

    PubMed

    Hurd, Matthew C; Kwon, Moonhyuk; Ro, Dae-Kyun

    2017-08-26

    Lippia dulcis (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of L. dulcis leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (LdTPS3 and LdTPS9) were isolated from L. dulcis leaf cDNA. To elucidate their catalytic functions, E. coli-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). In vitro enzyme assays showed that LdTPS3 encodes bornyl diphosphate synthase (thus named LdBPPS) while LdTPS9 encodes linalool synthase. Interestingly, the N-terminus of LdBPPS possesses two arginine-rich (RRX8W) motifs, and enzyme assays showed that the presence of both RRX8W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX8W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and L. dulcis. The discovery of LdBPPS opens up the possibility of engineering L. dulcis to remove the undesirable product, camphor. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate.

    PubMed

    Samoudi, Mojtaba; Omid Yeganeh, Negar; Shahbani Zahiri, Hossein; Shariati, Parvin; Hajhosseini, Reza

    2015-01-01

    Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters. Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 .

  20. Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate

    PubMed Central

    Samoudi, Mojtaba; Omid Yeganeh, Negar; Shahbani Zahiri, Hossein; Shariati, Parvin; Hajhosseini, Reza

    2015-01-01

    Background: Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). Methods: Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters. Results: Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. Conclusion: The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 . PMID:26306151

  1. Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue.

    PubMed

    Tong, Huaxiang; Wiemer, Andrew J; Neighbors, Jeffrey D; Hohl, Raymond J

    2008-07-15

    Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are branch point intermediates of isoprenoid biosynthesis. Inhibitors of isoprenoid biosynthesis, such as the statins and bisphosphonates, are widely used therapeutic agents. However, little is known about the degree to which they alter levels of upstream and downstream isoprenoids, including FPP and GGPP. Therefore, we developed a method to isolate and quantify FPP and GGPP from mammalian tissues. Tissues from mice were collected, snap frozen in liquid nitrogen, and stored at -80 degrees C. FPP and GGPP were isolated by a combined homogenization and extraction procedure and were purified with a C18 solid phase extraction column. Farnesyl protein transferase (FTase) or geranylgeranyl protein transferase I (GGTase I) were used to conjugate FPP and GGPP with fluorescent dansylated peptides. FPP and GGPP were quantified by high-performance liquid chromatography (HPLC). The respective concentrations of FPP and GGPP are as follows: 0.355+/-0.030 and 0.827+/-0.082 units of nmol/g wet tissues in brain, 0.320+/-0.019 and 0.293+/-0.035 units of nmol/g wet tissues in kidney, 0.326+/-0.064 and 0.213+/-0.029 units of nmol/g wet tissues in liver, and 0.364+/-0.015 and 0.349+/-0.023 units of nmol/g wet tissues in heart (means+/-SEM). This method allows for determination of FPP and GGPP concentrations in any tissue type and is sensitive enough to detect changes following treatment with inhibitors of isoprenoid biosynthesis.

  2. An intersubunit disulfide bridge stabilizes the tetrameric nucleoside diphosphate kinase of Aquifex aeolicus.

    PubMed

    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.

  3. Bornyl-diphosphate synthase from Lavandula angustifolia: A major monoterpene synthase involved in essential oil quality.

    PubMed

    Despinasse, Yolande; Fiorucci, Sébastien; Antonczak, Serge; Moja, Sandrine; Bony, Aurélie; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis; Jullien, Frédéric

    2017-05-01

    Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221.

  4. Incubation of 2-Methylisoborneol Synthase with the Intermediate Analogue 2-Methylneryl Diphosphate

    PubMed Central

    Chou, Wayne K. W.; Gould, Colin A.; Cane, David E.

    2017-01-01

    Incubation of synthetic 2-methylneryl diphosphate (2-MeNPP, 10) with 2-methylisoborneol synthase (MIBS) gave a mixture of products that differed significantly from that derived from the natural substrate (E)-2-methylgeranyl disphosphate (3, 2-MeGPP. The proportion of (−)-2-methylisoborneol (1) decreased from 89% to 17% while that of 2-methylenebornane (4) increased from 10% to 26%, with the relative yields of the isomeric homo-monoterpenes 2-methyl-2-bornene (5) and 1-methylcamphene (6) remaining essentially unchanged (<1% each), as determined by chiral gas chromatographic-mass spectrometric (GC-MS) analysis. The majority of the product mixture resulting from the MIBS-catalyzed cyclization of 2-MeNPP (10) consisted of the anomalous monocyclic homo-monoterpenes (±)-2-methylllimonene (15, 39%) and 2-methyl-α-terpineol (13, 10%), as well as the acylic derivatives 2-methylnerol (11, 7%) and 2-methyllinalool (14, <1%). The steady state kinetic parameters of the MIBS-catalyzed reaction, determined using [1-3H]-2-MeNPP, were kcat 0.0046 ± 0.0003 s−1, Km 18 ± 6 μM, and kcat/Km 2.55 × 102 M−1s−1. By comparison, the natural substrate 2-MeGPP had a kcat 0.105 ± 0.007 s−1, Km 95 ± 49 μM, and kcat/Km 1.11 × 103 M−1s−1. Taken together with earlier X-ray crystallographic studies of MIBS, as well as previous investigations of the mechanistically related plant monoterpene cyclase, bornyl diphosphate synthase, these results provide important insights into the binding and cyclization of both native substrates and intermediates and their analogues. PMID:28246382

  5. Stathmin slows down guanosine diphosphate dissociation from tubulin in a phosphorylation-controlled fashion.

    PubMed

    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.

  6. 3'-Phosphorylated nucleotides are tight binding inhibitors of nucleoside diphosphate kinase activity.

    PubMed

    Schneider, B; Xu, Y W; Janin, J; Véron, M; De