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Sample records for pseudouridine synthases derivatives

  1. Purification, Structure and Properties of Escherichia coli tRNA Pseudouridine Synthase 1.

    Science.gov (United States)

    1987-01-01

    enzymes which are reactive at C5 of uracil ( thymidylate synthase and aminoacyl synthetases). The deduced amino acid sequence of PSUI was also compared with...localize the sites of tRNA interaction with PSUI. The mechanism elucidated by Santi and others for thymidylate synthase (34-38) provides a conceptual...aminoacyl tRNA synthetases with residue U8 of their cognate tRNA substrates (39,40). In the case of thymidylate synthase , I the catalytic nucleophile is

  2. Pseudouridine synthase 1 deficient mice, a model for Mitochondrial Myopathy with Sideroblastic Anemia, exhibit muscle morphology and physiology alterations.

    Science.gov (United States)

    Mangum, Joshua E; Hardee, Justin P; Fix, Dennis K; Puppa, Melissa J; Elkes, Johnathon; Altomare, Diego; Bykhovskaya, Yelena; Campagna, Dean R; Schmidt, Paul J; Sendamarai, Anoop K; Lidov, Hart G W; Barlow, Shayne C; Fischel-Ghodsian, Nathan; Fleming, Mark D; Carson, James A; Patton, Jeffrey R

    2016-05-20

    Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1(-/-) animals. Pus1(-/-) mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1(-/-) mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1(-/-) mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1(-/-) mice.

  3. Studies on identifying the binding sites of folate and its derivatives in Lactobacillus casei thymidylate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Maley, F.; Maley, G.F.

    1983-01-01

    It was shown that folate and its derivatives have a profound effect on stabilizing thymidylate synthase in vitro and in vivo, as a consequence of ternary formation between the folate, dUMP, or FdUMP, and the synthase. The degree to which complex formation is affected can be revealed qualitatively by circular dichroism and quantitatively by equilibrium dialysis using the Lactobacillus casei synthase. In contrast to the pteroylmonoglutamates, the pteroylpolyglutamates bind to thymidylate synthase in the absence of dUMP, but even their binding affinity is increased greatly by this nucleotide or its analogues. Similarly, treatment of the synthase with carboxypeptidase A prevents the binding of the pteroylmonoglutamates and reduces the binding of the polyglutamates without affecting dUMP binding. The latter does not protect against carboxypeptidase inactivation but does potentiate the protective effect of the pteroylpolyglutamates. To determine the region of the synthase involved in the binding of the glutamate residues, Pte(/sup 14/C)GluGlu6 was activated by a water soluble carbodiimide in the presence and absence of dUMP. This folate derivative behaved as a competitive inhibitor of 5,10-CH/sub 2/H/sub 4/PteGlu, in contrast to methotrexate which was non-competitive. Separation of the five cyanogen bromide peptides from the L. casei synthase revealed 80% of the radioactivity to be associated with CNBr-2 and about 15% with CNBr-4. Chymotrypsin treatment of CNBr-2 yielded two /sup 14/C-labeled peaks on high performance liquid chromatography, with the slower migrating one being separated further into two peaks by Bio-gel P2 chromatography. All three peptides came from the same region of CNBr-2, encompassing residues 47-61 of the enzyme. From these studies it would appear that the residues most probably involved in the fixation of PteGlu7 are lysines 50 and 58. In contrast, methotrexate appeared to bind to another region of CNBr-2.

  4. HDHD1, which is often deleted in X-linked ichthyosis, encodes a pseudouridine-5'-phosphatase.

    Science.gov (United States)

    Preumont, Alice; Rzem, Rim; Vertommen, Didier; Van Schaftingen, Emile

    2010-10-15

    Pseudouridine, the fifth-most abundant nucleoside in RNA, is not metabolized in mammals, but is excreted intact in urine. The purpose of the present work was to search for an enzyme that would dephosphorylate pseudouridine 5'-phosphate, a potential intermediate in RNA degradation. We show that human erythrocytes contain a pseudouridine-5'-phosphatase displaying a Km ≤ 1 μM for its substrate. The activity of the partially purified enzyme was dependent on Mg2+, and was inhibited by Ca2+ and vanadate, suggesting that it belonged to the 'haloacid dehalogenase' family of phosphatases. Its low molecular mass (26 kDa) suggested that this phosphatase could correspond to the protein encoded by the HDHD1 (haloacid dehalogenase-like hydrolase domain-containing 1) gene, present next to the STS (steroid sulfatase) gene on human chromosome Xp22. Purified human recombinant HDHD1 dephosphorylated pseudouridine 5'-phosphate with a kcat of 1.6 s-1, a Km of 0.3 μM and a catalytic efficiency at least 1000-fold higher than that on which it acted on other phosphate esters, including 5'-UMP. The molecular identity of pseudouridine-5'-phosphatase was confirmed by the finding that its activity was negligible (X-linked ichthyosis patients harbouring a combined deletion of the STS gene (the X-linked ichthyosis gene) and the HDHD1 gene. Furthermore, pseudouridine-5'-phosphatase activity was 1.5-fold higher in erythrocytes from women compared with men, in agreement with the HDHD1 gene undergoing only partial inactivation in females. In conclusion, HDHD1 is a phosphatase specifically involved in dephosphorylation of a modified nucleotide present in RNA.

  5. Linker-Region Modified Derivatives of the Deoxyhypusine Synthase Inhibitor CNI-1493 Suppress HIV-1 Replication.

    Science.gov (United States)

    Schröder, Marcus; Kolodzik, Adrian; Windshügel, Björn; Krepstakies, Marcel; Priyadarshini, Poornima; Hartjen, Philip; van Lunzen, Jan; Rarey, Matthias; Hauber, Joachim; Meier, Chris

    2016-02-01

    The inhibition of cellular factors that are involved in viral replication may be an important alternative to the commonly used strategy of targeting viral enzymes. The guanylhydrazone CNI-1493, a potent inhibitor of the deoxyhypusine synthase (DHS), prevents the activation of the cellular factor eIF-5A and thereby suppresses HIV replication and a number of other diseases. Here, we report on the design, synthesis and biological evaluation of a series of CNI-1493 analogues. The sebacoyl linker in CNI-1493 was replaced by different alkyl or aryl dicarboxylic acids. Most of the tested derivatives suppress HIV-1 replication efficiently in a dose-dependent manner without showing toxic side effects. The unexpected antiviral activity of the rigid derivatives point to a second binding mode as previously assumed for CNI-1493. Moreover, the chemical stability of CNI-1493 was analysed, showing a successive hydrolysis of the imino bonds. By molecular dynamics simulations, the behaviour of the parent CNI-1493 in solution and its interactions with DHS were investigated. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. An Engineered Tryptophan Synthase Opens New Enzymatic Pathways to β-Methyltryptophan and Derivatives.

    Science.gov (United States)

    Francis, Daniel; Winn, Michael; Latham, Jonathan; Greaney, Michael F; Micklefield, Jason

    2017-02-16

    β-Methyltryptophans (β-mTrp) are precursors in the biosynthesis of bioactive natural products and are used in the synthesis of peptidomimetic-based therapeutics. Currently β-mTrp is produced by inefficient multistep synthetic methods. Here we demonstrate how an engineered variant of tryptophan synthase from Salmonella (StTrpS) can catalyse the efficient condensation of l-threonine and various indoles to generate β-mTrp and derivatives in a single step. Although l-serine is the natural substrate for TrpS, targeted mutagenesis of the StTrpS active site provided a variant (βL166V) that can better accommodate l-Thr as a substrate. The condensation of l-Thr and indole proceeds with retention of configuration at both α- and β-positions to give (2S,3S)-β-mTrp. The integration of StTrpS (βL166V) with l-amino acid oxidase, halogenase enzymes and palladium chemocatalysts provides access to further d-configured and regioselectively halogenated or arylated β-mTrp derivatives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Suppression effect of Cinnamomum cassia bark-derived component on nitric oxide synthase.

    Science.gov (United States)

    Lee, Hoi-Seon; Kim, Byung-Su; Kim, Moo-Key

    2002-12-18

    The inhibitory effects of Cinnamomum cassia bark-derived material on nitric oxide (NO) production in RAW 264.7 cells was determined through the evaluation of NO production and expression of inducible nitric oxide and compared to the effects of three commercially available compounds, cinnamyl alcohol, cinnamic acid, and eugenol. The biologically active constituents of C. cassia extract were characterized as trans-cinnamaldehyde by spectral analysis. The inhibitory effects varied with both chemical and concentration used. Potent inhibitory effects of cinnamaldehyde against NO production were 81.5 and 71.7% at 1.0 and 0.5 microg/microL, respectively, and a 41.2% inhibitory effect was revealed at 0.1 microg/microL. However, little or no activity was observed for cinnamic acid and eugenol. Suppression effects of cinnamaldehyde on inducible nitric oxide synthase expression were revealed by Western blot analysis. As a naturally occurring therapeutic agent, trans-cinnamaldehyde could be useful for developing new types of NO inhibitors.

  8. T cell–derived inducible nitric oxide synthase switches off TH17 cell differentiation

    Science.gov (United States)

    Yang, Jianjun; Zhang, Ruihua; Lu, Geming; Shen, Yu; Peng, Liang; Zhu, Chen; Cui, Miao; Wang, Weidong; Arnaboldi, Paul; Tang, Meng; Gupta, Monica; Qi, Chen-Feng; Jayaraman, Padmini; Zhu, Hongfa; Jiang, Bo; Chen, Shu-hsia; He, John Cijiang; Ting, Adrian T.; Zhou, Ming-Ming; Kuchroo, Vijay K.; Morse, Herbert C.; Ozato, Keiko; Sikora, Andrew G.

    2013-01-01

    RORγt is necessary for the generation of TH17 cells but the molecular mechanisms for the regulation of TH17 cells are still not fully understood. We show that activation of CD4+ T cells results in the expression of inducible nitric oxide synthase (iNOS). iNOS-deficient mice displayed enhanced TH17 cell differentiation but without major effects on either TH1 or TH2 cell lineages, whereas endothelial NOS (eNOS) or neuronal NOS (nNOS) mutant mice showed comparable TH17 cell differentiation compared with wild-type control mice. The addition of N6-(1-iminoethyl)-l-lysine dihydrochloride (L-NIL), the iNOS inhibitor, significantly enhanced TH17 cell differentiation, and S-nitroso-N-acetylpenicillamine (SNAP), the NO donor, dose-dependently reduced the percentage of IL-17–producing CD4+ T cells. NO mediates nitration of tyrosine residues in RORγt, leading to the suppression of RORγt-induced IL-17 promoter activation, indicating that NO regulates IL-17 expression at the transcriptional level. Finally, studies of an experimental model of colitis showed that iNOS deficiency results in more severe inflammation with an enhanced TH17 phenotype. These results suggest that NO derived from iNOS in activated T cells plays a negative role in the regulation of TH17 cell differentiation and highlight the importance of intrinsic programs for the control of TH17 immune responses. PMID:23797094

  9. Novel anti-inflammatory chalcone derivatives inhibit the induction of nitric oxide synthase and cyclooxygenase-2 in mouse peritoneal macrophages.

    Science.gov (United States)

    Herencia, F; Ferrándiz, M L; Ubeda, A; Guillén, I; Dominguez, J N; Charris, J E; Lobo, G M; Alcaraz, M J

    1999-06-18

    In a previous work, we tested a series of chalcone derivatives as possible anti-inflammatory compounds. We now investigate the effects of three of those compounds, CHI, CH8 and CH12, on nitric oxide and prostanoid generation in mouse peritoneal macrophages stimulated with lipopolysaccharide and in the mouse air pouch injected with zymosan, where they showed a dose-dependent inhibition with inhibitory concentration 50% values in the microM range. This effect was not the consequence of a direct inhibitory action on enzyme activities. Our results demonstrated that chalcone derivatives inhibited de novo inducible nitric oxide synthase and cyclooxygenase-2 synthesis, being a novel therapeutic approach for inflammatory diseases.

  10. Bone marrow-derived versus parenchymal sources of inducible nitric oxide synthase in experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Zehntner, Simone P; Bourbonniere, Lyne; Hassan-Zahraee, Mina

    2004-01-01

    The role of nitric oxide (NO) in central nervous system (CNS) inflammation is uncertain. Whereas experimental autoimmune encephalomyelitis (EAE) is exacerbated in mice deficient in inducible nitric oxide synthase (iNOS), inhibitor studies have suggested a pro-inflammatory role for NO. These discr...

  11. Brain-derived neurotrophic factor and nitric oxide synthase inhibitor protect the vestibular organ against gentamicin ototoxicity.

    Science.gov (United States)

    Takumida, Masaya; Anniko, Matti

    2002-01-01

    In order to find a way to develop a new treatment for inner ear disorders, the effects of a nitric oxide synthase (NOS) inhibitor [N-nitro-L-arginine methylester (L-NAME)] and a neurotrophin [brain-derived neurotrophic factor (BDNF)] were investigated. The effect of L-NAME and BDNF on gentamicin-induced vestibular hair cell damage was investigated by using the in vitro LIVE/DEAD system. Both L-NAME and BDNF individually reduced the vestibular hair cell damage caused by gentamicin but the combination of L-NAME and BDNF was more successful in preventing damage. It is therefore suggested that treatment with a combination of an NOS inhibitor and a neurotrophin will help us to treat inner ear disorders.

  12. Identification and characterization of a novel trehalose synthase gene derived from saline-alkali soil metagenomes.

    Directory of Open Access Journals (Sweden)

    Ling Jiang

    Full Text Available A novel trehalose synthase (TreS gene was identified from a metagenomic library of saline-alkali soil by a simple activity-based screening system. Sequence analysis revealed that TreS encodes a protein of 552 amino acids, with a deduced molecular weight of 63.3 kDa. After being overexpressed in Escherichia coli and purified, the enzymatic properties of TreS were investigated. The recombinant TreS displayed its optimal activity at pH 9.0 and 45 °C, and the addition of most common metal ions (1 or 30 mM had no inhibition effect on the enzymatic activity evidently, except for the divalent metal ions Zn(2+ and Hg(2+. Kinetic analysis showed that the recombinant TreS had a 4.1-fold higher catalytic efficiency (Kcat/K m for maltose than for trehalose. The maximum conversion rate of maltose into trehalose by the TreS was reached more than 78% at a relatively high maltose concentration (30%, making it a good candidate in the large-scale production of trehalsoe after further study. In addition, five amino acid residues, His172, Asp201, Glu251, His318 and Asp319, were shown to be conserved in the TreS, which were also important for glycosyl hydrolase family 13 enzyme catalysis.

  13. Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in ...

    Indian Academy of Sciences (India)

    that eNOS-derived NO may not be involved in the injuries occurring during H/R in the heart. We conclude that L-. NIO would not be useful in alleviating the adverse effects of cardiac H/R. 1. Introduction. Hypoxia resulting from ischaemia, haemorrhage and other cardiovascular problems is common in clinical medicine and.

  14. Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in ...

    Indian Academy of Sciences (India)

    The results showed that L-NIO administration lowered NOx levels in all the experimental groups. However, no change was found in the lipid peroxidation level, the percentage of apoptotic cells or nitrated protein expression, implying that eNOS-derived NO may not be involved in the injuries occurring during H/R in the heart.

  15. Development of urea and thiourea kynurenamine derivatives: synthesis, molecular modeling, and biological evaluation as nitric oxide synthase inhibitors.

    Science.gov (United States)

    Chayah, Mariem; Carrión, M Dora; Gallo, Miguel A; Jiménez, Rosario; Duarte, Juan; Camacho, M Encarnación

    2015-05-01

    Herein we describe the synthesis of a new family of kynurenamine derivatives with a urea or thiourea moiety, together with their in vitro biological evaluation as inhibitors of both neuronal and inducible nitric oxide synthases (nNOS and iNOS, respectively), enzymes responsible for the biogenesis of NO. These compounds were synthesized from a 5-substituted-2-nitrophenyl vinyl ketone scaffold in a five-step procedure with moderate to high chemical yields. In general, the assayed compounds show greater inhibition of iNOS than of nNOS, with 1-[3-(2-amino-5-chlorophenyl)-3-oxopropyl]-3-ethylurea (compound 5 n) being the most potent iNOS inhibitor in the series and the most iNOS/nNOS-selective compound. In this regard, we performed molecular modeling studies to propose a binding mode for this family of compounds to both enzymes and, thereby, to elucidate the differential molecular features that could explain the observed selectivity between iNOS and nNOS. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Platelet-derived growth factor-DD targeting arrests pathological angiogenesis by modulating glycogen synthase kinase-3beta phosphorylation.

    Science.gov (United States)

    Kumar, Anil; Hou, Xu; Lee, Chunsik; Li, Yang; Maminishkis, Arvydas; Tang, Zhongshu; Zhang, Fan; Langer, Harald F; Arjunan, Pachiappan; Dong, Lijin; Wu, Zhijian; Zhu, Linda Y; Wang, Lianchun; Min, Wang; Colosi, Peter; Chavakis, Triantafyllos; Li, Xuri

    2010-05-14

    Platelet-derived growth factor-DD (PDGF-DD) is a recently discovered member of the PDGF family. The role of PDGF-DD in pathological angiogenesis and the underlying cellular and molecular mechanisms remain largely unexplored. In this study, using different animal models, we showed that PDGF-DD expression was up-regulated during pathological angiogenesis, and inhibition of PDGF-DD suppressed both choroidal and retinal neovascularization. We also demonstrated a novel mechanism mediating the function of PDGF-DD. PDGF-DD induced glycogen synthase kinase-3beta (GSK3beta) Ser(9) phosphorylation and Tyr(216) dephosphorylation in vitro and in vivo, leading to increased cell survival. Consistently, GSK3beta activity was required for the antiangiogenic effect of PDGF-DD targeting. Moreover, PDGF-DD regulated the expression of GSK3beta and many other genes important for angiogenesis and apoptosis. Thus, we identified PDGF-DD as an important target gene for antiangiogenic therapy due to its pleiotropic effects on vascular and non-vascular cells. PDGF-DD inhibition may offer new therapeutic options to treat neovascular diseases.

  17. Endothelial dysfunction in DOCA-salt-hypertensive mice: role of neuronal nitric oxide synthase-derived hydrogen peroxide.

    Science.gov (United States)

    Silva, Grazielle C; Silva, Josiane F; Diniz, Thiago F; Lemos, Virginia S; Cortes, Steyner F

    2016-06-01

    Endothelial dysfunction is a common problem associated with hypertension and is considered a precursor to the development of micro- and macro-vascular complications. The present study investigated the involvement of nNOS (neuronal nitric oxide synthase) and H2O2 (hydrogen peroxide) in the impaired endothelium-dependent vasodilation of the mesenteric arteries of DOCA (deoxycorticosterone acetate)-salt-hypertensive mice. Myograph studies were used to investigate the endothelium-dependent vasodilator effect of ACh (acetylcholine). The expression and phosphorylation of nNOS and eNOS (endothelial nitric oxide synthase) were studied by Western blot analysis. Immunofluorescence was used to examine the localization of nNOS and eNOS in the endothelial layer of the mesenteric artery. The vasodilator effect of ACh is strongly impaired in mesenteric arteries of DOCA-salt-hypertensive mice. Non-selective inhibition of NOS sharply reduced the effect of ACh in both DOCA-salt-hypertensive and sham mice. Selective inhibition of nNOS and catalase led to a higher reduction in the effect of ACh in sham than in DOCA-salt-hypertensive mice. Production of H2O2 induced by ACh was significantly reduced in vessels from DOCA-salt-hypertensive mice, and it was blunted after nNOS inhibition. The expression of both eNOS and nNOS was considerably lower in DOCA-salt-hypertensive mice, whereas phosphorylation of their inhibitory sites was increased. The presence of nNOS was confirmed in the endothelial layer of mesenteric arteries from both sham and DOCA-salt-hypertensive mice. These results demonstrate that endothelial dysfunction in the mesenteric arteries of DOCA-salt-hypertensive mice is associated with reduced expression and functioning of nNOS and impaired production of nNOS-derived H2O2 Such findings offer a new perspective for the understanding of endothelial dysfunction in hypertension. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  18. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

    Directory of Open Access Journals (Sweden)

    Grigoris D. Amoutzias

    2016-04-01

    Full Text Available Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs and polyketides (PKs are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes and type-I polyketide synthases (PKSes-I, respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.

  19. Endothelial Nitric Oxide Synthase-Derived Nitric Oxide Prevents Dihydrofolate Reductase Degradation via Promoting S-Nitrosylation.

    Science.gov (United States)

    Cai, Zhejun; Lu, Qiulun; Ding, Ye; Wang, Qilong; Xiao, Lei; Song, Ping; Zou, Ming-Hui

    2015-11-01

    Dihydrofolate reductase (DHFR) is a key protein involved in tetrahydrobiopterin (BH4) regeneration from 7,8-dihydrobiopterin (BH2). Dysfunctional DHFR may induce endothelial nitric oxide (NO) synthase (eNOS) uncoupling resulting in enzyme production of superoxide anions instead of NO. The mechanism by which DHFR is regulated is unknown. Here, we investigate whether eNOS-derived NO maintains DHFR stability. DHFR activity, BH4 content, eNOS activity, and S-nitrosylation were assessed in human umbilical vein endothelial cells and in aortas isolated from wild-type and eNOS knockout mice. In human umbilical vein endothelial cells, depletion of intracellular NO by transfection with eNOS-specific siRNA or by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-both of which had no effect on DHFR mRNA levels-markedly reduced DHFR protein levels in parallel with increased DHFR polyubiquitination. Supplementation of S-nitroso-l-glutathione (GSNO), a NO donor, or MG132, a potent inhibitor of the 26S proteasome, prevented eNOS silencing and PTIO-induced DHFR reduction in human umbilical vein endothelial cells. PTIO suppressed S-nitrosylation of DHFR, whereas GSNO promoted DHFR S-nitrosylation. Mutational analysis confirmed that cysteine 7 of DHFR was S-nitrosylated. Cysteine 7 S-nitrosylation stabilized DHFR from ubiquitination and degradation. Experiments performed in aortas confirmed that PTIO or eNOS deficiency reduces endothelial DHFR, which can be abolished by MG132 supplementation. We conclude that S-nitrosylation of DHFR at cysteine 7 by eNOS-derived NO is crucial for DHFR stability. We also conclude that NO-induced stabilization of DHFR prevents eNOS uncoupling via regeneration of BH4, an essential eNOS cofactor. © 2015 American Heart Association, Inc.

  20. Pseudouridine and N6-methyladenosine modifications weaken PUF protein/RNA interactions.

    Science.gov (United States)

    Vaidyanathan, Pavanapuresan P; AlSadhan, Ishraq; Merriman, Dawn K; Al-Hashimi, Hashim M; Herschlag, Daniel

    2017-05-01

    RNA modifications are ubiquitous in biology, with over 100 distinct modifications. While the vast majority were identified and characterized on abundant noncoding RNA such as tRNA and rRNA, the advent of sensitive sequencing-based approaches has led to the discovery of extensive and regulated modification of eukaryotic messenger RNAs as well. The two most abundant mRNA modifications-pseudouridine (Ψ) and N6-methyladenosine (m6A)-affect diverse cellular processes including mRNA splicing, localization, translation, and decay and modulate RNA structure. Here, we test the hypothesis that RNA modifications directly affect interactions between RNA-binding proteins and target RNA. We show that Ψ and m6A weaken the binding of the human single-stranded RNA binding protein Pumilio 2 (hPUM2) to its consensus motif, with individual modifications having effects up to approximately threefold and multiple modifications giving larger effects. While there are likely to be some cases where RNA modifications essentially fully ablate protein binding, here we see modest responses that may be more common. Such modest effects could nevertheless profoundly alter the complex landscape of RNA:protein interactions, and the quantitative rather than qualitative nature of these effects underscores the need for quantitative, systems-level accounting of RNA:protein interactions to understand post-transcriptional regulation. © 2017 Vaidyanathan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  1. Co-transplantation with Myeloid-Derived Suppressor Cells Protects Cell Transplants: A Crucial Role of Inducible Nitric Oxide Synthase

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    Arakawa, Yusuke; Qin, Jie; Chou, Hong-Shuie; Bhatt, Sumantha; Wang, Lianfu; Stuehr, Dennis; Ghosh, Arnab; Fung, John J.; Lu, Lina; Qian, Shiguang

    2014-01-01

    Background Islet transplantation is an alternative to pancreas transplantation to cure type 1 diabetes, but both require chronic immunosuppression, which is often accompanied by deleterious side effects. The purpose of this study was to explore prolongation of islet allograft survival by co-transplantation with myeloid-derived suppressor cells (MDSCs) without requirement of immunosuppression, and determine the role of inducible nitric oxide synthase (iNOS) produced by MDSCs in immune regulation. Methods Bone marrow cells were isolated from wild type (WT) or iNOS−/− mice and cultured in the presence of GM-CSF and hepatic stellate cells (HSCs), resulting in the generation of MDSCs. WT or iNOS−/− MDSCs were co-transplanted with islet allografts under the renal capsule of diabetic recipient mice. Results Addition of HSCs into DC culture promoted generation of MDSCs (instead of DCs). MDSCs had elevated expression of iNOS upon exposure to IFN-γ and inhibited T cell responses in an MLR culture. Co-transplantation with WT MDSCs markedly prolonged survival of islet allografts, which was associated with reduced infiltration of CD8+ T cells due to inhibited proliferative response. These effects were significantly attenuated when MDSCs were deficient in iNOS. Furthermore, iNOS−/− MDSCs largely lost their ability to protect islet allografts. Conclusions Co-transplantation with HSC-induced MDSCs significantly extends islet allograft survival through iNOS-mediated T cell inhibition. The results demonstrate the potential use of in vitro generated MDSCs as a novel adjunctive immunotherapy for islet transplantation. PMID:24642686

  2. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans.

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    Bito, Tomohiro; Yabuta, Yukinori; Ichiyanagi, Tsuyoshi; Kawano, Tsuyoshi; Watanabe, Fumio

    2014-01-01

    In this study, we showed that cyanocobalamin dodecylamine, a ribose 5'-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), methylmalonic aciduria cblC type (cblc-1), and methionine synthase reductase (mtrr-1). In contrast, the level of the mRNAs encoding cob(I)alamin adenosyltransferase (mmab-1) was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  3. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Tomohiro Bito

    2014-01-01

    Full Text Available In this study, we showed that cyanocobalamin dodecylamine, a ribose 5′-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1, methylmalonic acidemia cobalamin A complementation group (mmaa-1, methylmalonic aciduria cblC type (cblc-1, and methionine synthase reductase (mtrr-1. In contrast, the level of the mRNAs encoding cob(Ialamin adenosyltransferase (mmab-1 was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  4. Ferulic acid and its water-soluble derivatives inhibit nitric oxide production and inducible nitric oxide synthase expression in rat primary astrocytes.

    Science.gov (United States)

    Kikugawa, Masaki; Ida, Tomoaki; Ihara, Hideshi; Sakamoto, Tatsuji

    2017-08-01

    We recently reported that two water-soluble derivatives of ferulic acid (1-feruloyl glycerol, 1-feruloyl diglycerol) previously developed by our group exhibited protective effects against amyloid-β-induced neurodegeneration in vitro and in vivo. In the current study, we aimed to further understand this process by examining the derivatives' ability to suppress abnormal activation of astrocytes, the key event of neurodegeneration. We investigated the effects of ferulic acid (FA) derivatives on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in rat primary astrocytes. The results showed that these compounds inhibited NO production and iNOS expression in a concentration-dependent manner and that the mechanism underlying these effects was the suppression of the nuclear factor-κB pathway. This evidence suggests that FA and its derivatives may be effective neuroprotective agents and could be useful in the treatment of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

  5. Pathogenic cycle between the endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine and the leukocyte-derived hemoprotein myeloperoxidase

    Czech Academy of Sciences Publication Activity Database

    von Leitner, E.C.; Klinke, A.; Atzler, D.; Slocum, J.L.; Lund, N.; Kielstein, J.T.; Maas, R.; Schmidt-Haupt, R.; Pekarová, Michaela; Hellwinkel, O.; Tsikas, D.; D'Alecy, L.G.; Lau, D.; Willems, S.; Kubala, Lukáš; Ehmke, H.; Meinertz, T.; Blankenberg, S.; Schwedhelm, E.; Gadegbeku, C.A.; Boger, R.H.; Baldus, S.; Sydow, K.

    2011-01-01

    Roč. 124, č. 4 (2011), s. 2735-U342 ISSN 0009-7322 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : arteriosclerosis * leukocytes * nitric oxide synthase Subject RIV: BO - Biophysics Impact factor: 14.739, year: 2011

  6. Insufficient Astrocyte-Derived Brain-Derived Neurotrophic Factor Contributes to Propofol-Induced Neuron Death Through Akt/Glycogen Synthase Kinase 3β/Mitochondrial Fission Pathway.

    Science.gov (United States)

    Liu, Yanan; Yan, Yasheng; Inagaki, Yasuyoshi; Logan, Sarah; Bosnjak, Zeljko J; Bai, Xiaowen

    2017-07-01

    Growing animal evidence demonstrates that prolonged exposure to propofol during brain development induces widespread neuronal cell death, but there is little information on the role of astrocytes. Astrocytes can release neurotrophic growth factors such as brain-derived neurotrophic factor (BDNF), which can exert the protective effect on neurons in paracrine fashion. We hypothesize that during propofol anesthesia, BDNF released from developing astrocytes may not be sufficient to prevent propofol-induced neurotoxicity. Hippocampal astrocytes and neurons isolated from neonatal Sprague Dawley rats were exposed to propofol at a clinically relevant dose of 30 μM or dimethyl sulfoxide as control for 6 hours. Propofol-induced cell death was determined by propidium iodide (PI) staining in astrocyte-alone cultures, neuron-alone cultures, or cocultures containing either low or high density of astrocytes (1:9 or 1:1 ratio of astrocytes to neurons ratio [ANR], respectively). The astrocyte-conditioned medium was collected 12 hours after propofol exposure and measured by protein array assay. BDNF concentration in astrocyte-conditioned medium was quantified using enzyme-linked immunosorbent assay. Neuron-alone cultures were treated with BDNF, tyrosine receptor kinase B inhibitor cyclotraxin-B, glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021, or mitochondrial fission inhibitor Mdivi-1 before propofol exposure. Western blot was performed for quantification of the level of protein kinase B and GSK3β. Mitochondrial shape was visualized through translocase of the outer membrane 20 staining. Propofol increased cell death in neurons by 1.8-fold (% of PI-positive cells [PI%] = 18.6; 95% confidence interval [CI], 15.2-21.9, P .05]). Astrocytes secreted BDNF in a cell density-dependent way and propofol decreased BDNF secretion from astrocytes. Administration of BDNF, CHIR99021, or Mdivi-1 significantly attenuated the propofol-induced neuronal death and aberrant mitochondria in

  7. Prenyldiphosphate synthases and gibberellin biosynthesis

    NARCIS (Netherlands)

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

    2013-01-01

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

  8. Phenotype commitment in vascular smooth muscle cells derived from coronary atherosclerotic plaques: differential gene expression of endothelial Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    ML Rossi

    2009-06-01

    Full Text Available Unstable angina and myocardial infarction are the clinical manifestations of the abrupt thrombotic occlusion of an epicardial coronary artery as a result of spontaneous atherosclerotic plaque rupture or fissuring, and the exposure of highly thrombogenic material to blood. It has been demonstrated that the proliferation of vascular smooth muscle cells (VSMCs and impaired bioavailabilty of nitric oxide (NO are among the most important mechanisms involved in the progression of atherosclerosis. It has also been suggested that a NO imbalance in coronary arteries may be involved in myocardial ischemia as a result of vasomotor dysfunction triggering plaque rupture and the thrombotic response. We used 5’ nuclease assays (TaqMan™ PCRs to study gene expression in coronary plaques collected by means of therapeutic directional coronary atherectomy from 15 patients with stable angina (SA and 15 with acute coronary syndromes (ACS without ST elevation. Total RNA was extracted from the 30 plaques and the cDNA was amplified in order to determine endothelial nitric oxide synthase (eNOS gene expression. Analysis of the results showed that the expression of eNOS was significantly higher (p<0.001 in the plaques from the ACS patients. Furthermore, isolated VSMCs from ACS and SA plaques confirmed the above pattern even after 25 plating passages. In situ RT-PCR was also carried out to co-localize the eNOS messengers and the VSMC phenotype.

  9. Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential

    Science.gov (United States)

    Boros, Gábor; Miko, Edit; Muramatsu, Hiromi; Weissman, Drew; Emri, Eszter; Rózsa, Dávid; Nagy, Georgina; Juhász, Attila; Juhász, István; van der Horst, Gijsbertus; Horkay, Irén; Remenyik, Éva; Karikó, Katalin; Emri, Gabriella

    2013-01-01

    UVB irradiation induces harmful photochemical reactions, including formation of cyclobutane pyrimidine dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2 days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine. PMID:24211294

  10. Brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Vivek, E-mail: vivek.gupta@mq.edu.au [Australian School of Advanced Medicine, Macquarie University (Australia); Chitranshi, Nitin; You, Yuyi [Australian School of Advanced Medicine, Macquarie University (Australia); Gupta, Veer [School of Medical Sciences, Edith Cowan University, Perth (Australia); Klistorner, Alexander; Graham, Stuart [Australian School of Advanced Medicine, Macquarie University (Australia); Save Sight Institute, Sydney University, Sydney (Australia)

    2014-11-21

    Highlights: • BDNF knockdown leads to activation of GSK3β in the neuronal cells. • BDNF knockdown can induce GSK3β activation beyond TrkB mediated effects. • BDNF impairment in vivo leads to age dependent activation of GSK3β in the retina. • Systemic treatment with TrkB agonist induces inhibition of retinal GSK3β. - Abstract: Glycogen synthase kinase 3β (GSK3β) is involved in several biochemical processes in neurons regulating cellular survival, gene expression, cell fate determination, metabolism and proliferation. GSK3β activity is inhibited through the phosphorylation of its Ser-9 residue. In this study we sought to investigate the role of BDNF/TrkB signalling in the modulation of GSK3β activity. BDNF/TrkB signalling regulates the GSK3β activity both in vivo in the retinal tissue as well as in the neuronal cells under culture conditions. We report here for the first time that BDNF can also regulate GSK3β activity independent of its effects through the TrkB receptor signalling. Knockdown of BDNF lead to a decline in GSK3β phosphorylation without having a detectable effect on the TrkB activity or its downstream effectors Akt and Erk1/2. Treatment with TrkB receptor agonist had a stimulating effect on the GSK3β phosphorylation, but the effect was significantly less pronounced in the cells in which BDNF was knocked down. The use of TrkB receptor antagonist similarly, manifested itself in the form of downregulation of GSK3β phosphorylation, but a combined TrkB inhibition and BDNF knockdown exhibited a much stronger negative effect. In vivo, we observed reduced levels of GSK3β phosphorylation in the retinal tissues of the BDNF{sup +/−} animals implicating critical role of BDNF in the regulation of the GSK3β activity. Concluding, BDNF/TrkB axis strongly regulates the GSK3β activity and BDNF also exhibits GSK3β regulatory effect independent of its actions through the TrkB receptor signalling.

  11. Bisabolyl-derived sesquiterpenes from tobacco 5-epi-aristolochene synthase-catalyzed cyclization of (2Z,6E)-farnesyl diphosphate.

    Science.gov (United States)

    Faraldos, Juan A; O'Maille, Paul E; Dellas, Nikki; Noel, Joseph P; Coates, Robert M

    2010-03-31

    We report the structures and stereochemistry of seven bisabolyl-derived sesquiterpenes arising from an unprecedented 1,6-cyclization (cisoid pathway) efficiently catalyzed by tobacco 5-epi-aristolochene synthase (TEAS). The use of (2Z,6E)-farnesyl diphosphate as an alternate substrate for recombinant TEAS resulted in a robust enzymatic cyclization to an array of products derived exclusively (>/=99.5%) from the cisoid pathway, whereas these same products account for ca. 2.5% of the total hydrocarbons obtained using (2E,6E)-farnesyl diphosphate. Chromatographic fractionations of extracts from preparative incubations with the 2Z,6E substrate afforded, in addition to the acyclic allylic alcohols (2Z,6E)-farnesol (6.7%) and nerolidol (3.6%), five cyclic sesquiterpene hydrocarbons and two cyclic sesquiterpene alcohols: (+)-2-epi-prezizaene (44%), (-)-alpha-cedrene (21.5%), (R)-(-)-beta-curcumene (15.5%), alpha-acoradiene (3.9%), 4-epi-alpha-acoradiene (1.3%), and equal amounts of alpha-bisabolol (1.8%) and epi-alpha-bisalolol (1.8%). The structures, stereochemistry, and enantiopurities were established by comprehensive spectroscopic analyses, optical rotations, chemical correlations with known sesquiterpenes, comparisons with literature data, and GC analyses. The major product, (+)-2-epi-prezizaene, is structurally related to the naturally occurring tricyclic alcohol, jinkohol (2-epi-prezizaan-7beta-ol). Cisoid cyclization pathways are proposed by which all five sesquiterpene hydrocarbons are derived from a common (7R)-beta-bisabolyl(+)/pyrophosphate(-) ion pair intermediate. The implications of the "cisoid" catalytic activity of TEAS are discussed.

  12. Neuroprotection of vestibular sensory cells from gentamicin ototoxicity obtained using nitric oxide synthase inhibitors, reactive oxygen species scavengers, brain-derived neurotrophic factors and calpain inhibitors.

    Science.gov (United States)

    Takumida, Masaya; Anniko, Matti; Shimizu, Akira; Watanabe, Hiroshi

    2003-01-01

    In order to devise a new treatment for inner ear disorders, the efficacy of a nitric oxide synthase inhibitor (L-N(G)-nitroarginine methylester [L-NAME]), a radical scavenger (D-methionine), a neurotrophin (brain-derived neurotrophic factor [BDNF]) and a calpain inhibitor (leupeptin) for protection from hair cell damage was investigated. The effects of these drugs on gentamicin-induced production of nitric oxide (NO) and reactive oxygen species (ROS) were studied by means of the fluorescence indicators 4,5-diaminofluorescein diacetate and dihydrotetramethylrosamine. The effect on gentamicin-induced vestibular hair cell damage was examined by using an in vitro LIVE/DEAD system. L-NAME inhibited the production of NO, D-methionine and BDNF restricted the production of ROS and leupeptin inhibited neither NO nor ROS. All the drugs used limited the vestibular hair cell damage caused by gentamicin. The combinations L-NAME + BDNF, L-NAME + leupeptin and D-methionine + BDNF had a significantly stronger preventive effect on hair cell damage. It is suggested that combined treatment with a radical inhibitor and either a neurotrophin or calpain inhibitor may help to treat inner ear disorders more effectively.

  13. Cystathionine-β-synthase-derived hydrogen sulfide is required for amygdalar long-term potentiation and cued fear memory in rats.

    Science.gov (United States)

    Chen, Hai-Bo; Wu, Wen-Ning; Wang, Wei; Gu, Xun-Hu; Yu, Bin; Wei, Bo; Yang, Yuan-Jian

    2017-04-01

    Hydrogen sulfide (H2S) is an endogenous gaseous molecule that functions as a neuromodulator in the brain. We previously reported that H2S regulated amygdalar synaptic plasticity and cued fear memory in rats. However, whether endogenous H2S is required for amygdalar long-term potentiation (LTP) induction and cued fear memory formation remains unclear. Here, we show that cystathionine-β-synthase (CBS), the predominant H2S-producing enzyme in the brain, was highly expressed in the amygdala of rats. Suppressing CBS activity by inhibitor prevented activity-triggered generation of H2S in the lateral amygdala (LA) region. Incubating brain slices with CBS inhibitor significantly prevented the induction of NMDA receptors (NMDARs)-dependent LTP in the thalamo-LA pathway, and intra-LA infusion of CBS inhibitor impaired cued fear memory in rats. Notably, treatment with H2S donor, but not CBS activator, significantly reversed the impairments of LTP and fear memory caused by CBS inhibition. Mechanismly, inhibition of CBS activity led to a reduction in NMDAR-mediated synaptic response in the thalamo-LA pathway, and treatment with H2S donor restored the function of NMDARs. Collectively, these results indicate that CBS-derived H2S is required for amygdalar synaptic plasticity and cued fear memory in rats, and the effects of endogenous H2S might involve the regulation of NMDAR function. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

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

  15. Gain and loss of an intron in a protein-coding gene in Archaea: the case of an archaeal RNA pseudouridine synthase gene

    Directory of Open Access Journals (Sweden)

    Yokobori Shin-ichi

    2009-08-01

    Full Text Available Abstract Background We previously found the first examples of splicing of archaeal pre-mRNAs for homologs of the eukaryotic CBF5 protein (also known as dyskerin in humans in Aeropyrum pernix, Sulfolobus solfataricus, S. tokodaii, and S. acidocaldarirus, and also showed that crenarchaeal species in orders Desulfurococcales and Sulfolobales, except for Hyperthermus butylicus, Pyrodictium occultum, Pyrolobus fumarii, and Ignicoccus islandicus, contain the (putative cbf5 intron. However, the exact timing of the intron insertion was not determined and verification of the putative secondary loss of the intron in some lineages was not performed. Results In the present study, we determined approximately two-thirds of the entire coding region of crenarchaeal Cbf5 sequences from 43 species. A phylogenetic analysis of our data and information from the available genome sequences suggested that the (putative cbf5 intron existed in the common ancestor of the orders Desulfurococcales and Sulfolobales and that probably at least two independent lineages in the order Desulfurococcales lost the (putative intron. Conclusion This finding is the first observation of a lineage-specific loss of a pre-mRNA intron in Archaea. As the insertion or deletion of introns in protein-coding genes in Archaea has not yet been seriously considered, our finding suggests the possible difficulty of accurately and completely predicting protein-coding genes in Archaea.

  16. Minicircle DNA-mediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Bandara, Nadeeka; Gurusinghe, Saliya; Chen, Haiying; Chen, Shuangfeng; Wang, Le-Xin; Lim, Shiang Y; Strappe, Padraig

    2016-04-01

    Non-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells is a pleiotropic diffusible gas with positive effects on maintaining vascular tone and promoting wound healing and angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic modification to express eNOS may promote NO production and subsequent cellular responses. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of angiogenesis-associated genes, CD31, VEGF-A, PDGFRα, FGF2, and FGFR2, were analysed by quantitative polymerase chain reaction. Minicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells (55.51 ± 3.3 %) and in rBMSC (18.65 ± 1.05 %) compared to P-GFP in HEK293T cells (43.4 ± 4.9 %) and rBMSC (15.21 ± 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 ± 3 %) compared to P-eNOS (9 ± 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and P-eNOS gene-modified rBMSCs formed longer (14.66 ± 0.55 mm and 13.58 ± 0.68 mm, respectively) and a greater number of tubules (56.33 ± 3.51 and 51 ± 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant

  17. The brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3, and induced nitric oxide synthase expressions after low-level laser therapy in an axonotmesis experimental model.

    Science.gov (United States)

    Gomes, Lessandra Esper Abdala; Dalmarco, Eduardo Monguilhott; André, Edison Sanfelice

    2012-11-01

    A robust body of evidence has shown that low-level laser therapy (LLLT) improves peripheral nerve regeneration. However, the biochemical background triggered in this process is not yet fully understood. The purpose of this study was to evaluate the mRNA expression of neurotrophic factors (brain-derived neurotrophic factor [BDNF], nerve growth factor [NGF], and neurotrophin-3, [NT-3]) and also an inflammatory marker (induced nitric oxide synthase [iNOS]) in an axonotmesis experimental model after low-level laser therapy. Thirty-six adult male Wistar rats (250-350 g) were subjected to right sciatic nerve crush injury, and 24 h later, the animals in the three different experimental groups (n=18) were irradiated on a daily basis with helium-neon laser (collimated HeNe laser, continuous emission, wavelength: 632.8 nm, power density: 0.5 mW/cm(2), irradiation time: 20 sec, energy density: 10 J/cm(2)) during 7, 14, and 21 consecutive days, respectively. The control group (n=18) underwent the same procedures, but with the equipment turned off. At the end of the experiments, animals were killed with an overdose of anesthesia to remove samples from the sciatic nerve lesion epicenter to determine the mRNA expression of BDNF, NGF, NT-3 and iNOS enzyme. Comparisons between groups showed that HeNe laser increased the mRNA expression of both BDNF and NGF factors after 14 days of LLLT, with peak expression at the 21st day. Increase in NT-3 mRNA expression was not observed. In addition, HeNe laser produced iNOS expression reduction, which played an important role in the inflammatory process. The reported data could have a relevant practical value because LLLT is a noninvasive procedure, and have revealed significant increase in neurotrophic factor expressions and inflammatory process reduction, opening the possibility of using LLLT as an important aid to nerve regeneration process.

  18. 2'-Deoxyuridine 5'-monophosphate substrate displacement in thymidylate synthase through 6-hydroxy-2H-naphtho[1,8-bc]furan-2-one derivatives.

    Science.gov (United States)

    Ferrari, Stefania; Calò, Samuele; Leone, Rosalida; Luciani, Rosaria; Costantino, Luca; Sammak, Susan; Di Pisa, Flavio; Pozzi, Cecilia; Mangani, Stefano; Costi, M Paola

    2013-11-27

    Thymidylate synthase (TS) is a target for antifolate-based chemotherapies of microbial and human diseases. Here, ligand-based, synthetic, and X-ray crystallography studies led to the discovery of 6-(3-cyanobenzoyloxy)-2-oxo-2H-naphto[1,8-bc]furan, a novel inhibitor with a Ki of 310 nM against Pneumocystis carinii TS. The X-ray ternary complex with Escherichia coli TS revealed, for the first time, displacement of the substrate toward the dimeric protein interface, thus providing new opportunities for further design of specific inhibitors of microbial pathogens.

  19. Genetic Construction of Truncated and Chimeric Metalloproteins Derived from the Alpha Subunit of Acetyl-CoA Synthase from Clostridium thermoaceticum

    Energy Technology Data Exchange (ETDEWEB)

    Huay-Keng Loke; Xiangshi Tan; Paul A. Lindahl

    2002-06-28

    In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha2beta2 tetrameric 310 kda bifunctional enzyme. ACS catalyzes the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA from CO (or CO2 in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains 7 metal-sulfur clusters of 4 different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kda beta subunit while the A-cluster, a Ni-X-Fe4S4 cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kda alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire alpha subunit was removed, as long as CO, rather than CO2 and a low-potential reductant, was used as a substrate. Truncating an {approx} 30 kda region from the N-terminus of the alpha subunit yielded a 49 kda protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO binding properties. Further truncation afforded a 23 kda protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe4S4]2+ clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 kda and 82 kda fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multi-centered metalloenzymes and to generate new complex metalloenzymes with interesting properties.

  20. Identification of 2-aminothiazole-4-carboxylate derivatives active against Mycobacterium tuberculosis H37Rv and the beta-ketoacyl-ACP synthase mtFabH.

    Directory of Open Access Journals (Sweden)

    Qosay Al-Balas

    Full Text Available BACKGROUND: Tuberculosis (TB is a disease which kills two million people every year and infects approximately over one-third of the world's population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration. METHODOLOGY/PRINCIPAL FINDINGS: Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM's novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H(37R(v and, dissociatively, against the beta-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H(37R(v with an MIC of 0.06 microg/ml (240 nM, but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido-5-(3-chlorophenylthiazole-4-carboxylate inhibited mtFabH with an IC(50 of 0.95+/-0.05 microg/ml (2.43+/-0.13 microM but was not active against the whole cell organism. CONCLUSIONS/SIGNIFICANCE: These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents.

  1. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    DEFF Research Database (Denmark)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2...

  2. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  3. Inducible nitric oxide synthase in renal transplantation

    NARCIS (Netherlands)

    Joles, JA; Vos, IH; Grone, HJ; Rabelink, TJ

    The importance of the endothelial isoform of nitric oxide synthase (eNOS) has been well established. Endothelium-derived nitric oxide has been shown to be essential for vascular homeostasis and modulation of eNOS has thus become a target in prevention of cardiovascular disease. The role of the

  4. Lipoic Acid Synthase (LASY)

    National Research Council Canada - National Science Library

    Indira Padmalayam; Sumera Hasham; Uday Saxena; Sivaram Pillarisetti

    2009-01-01

    Lipoic Acid Synthase (LASY) A Novel Role in Inflammation, Mitochondrial Function, and Insulin Resistance Indira Padmalayam 1 , Sumera Hasham 2 , Uday Saxena 1 and Sivaram Pillarisetti 1 1 Discovery Research, ReddyUS...

  5. Chitin synthase homologs in three ectomycorrhizal truffles.

    Science.gov (United States)

    Lanfranco, L; Garnero, L; Delpero, M; Bonfante, P

    1995-12-01

    Degenerate PCR primers were used to amplify a conserved gene portion coding chitin synthase from genomic DNA of six species of ectomycorrhizal truffles. DNA was extracted from both hypogeous fruitbodies and in vitro growing mycelium of Tuber borchii. A single fragment of about 600 bp was amplified for each species. The amplification products from Tuber magnatum, T. borchii and T. ferrugineum were cloned and sequenced, revealing a high degree of identity (91.5%) at the nucleotide level. On the basis of the deduced amino acid sequences these clones were assigned to class II chitin synthase. Southern blot experiments performed on genomic DNA showed that the amplification products derive from a single copy gene. Phylogenetic analysis of the nucleotide sequences of class II chitin synthase genes confirmed the current taxonomic position of the genus Tuber, and suggested a close relationship between T. magnatum and T. uncinatum.

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

    DEFF Research Database (Denmark)

    Yang, Ting; Gao, Liping; Hu, Hao

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first path-way-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...

  7. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-03-02

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

  8. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-01-01

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

  9. The elevation of intraocular pressure is associated with apoptosis and increased immunoreactivity for nitric oxide synthase in rat retina whereas the effectiveness of retina derived relaxing factor is unaffected.

    Science.gov (United States)

    Takır, Selçuk; Gürel-Gürevin, Ebru; Toprak, Ayça; Demirci-Tansel, Cihan; Uydeş-Doğan, B Sönmez

    2016-04-01

    Glaucoma is a progressive ocular disease that stands in the upper rank for the cause of blindness in worldwide. In the present study, we aimed to elucidate the possible disturbances occurred in the layers of retina due to an increase in intraocular pressure (IOP) and to verify the effectiveness of retina derived relaxing factor, i.e., RRF in this pathologic condition. The increase in IOP was induced by cauterization of the three of episcleral veins simultaneously in rats. After 8 weeks period, the retinas excised from the vein cauterized eyes were evaluated for the possible histopathological and ultrastructural alterations as well as for the relaxing effects on isolated bovine retinal and rat mesenteric arteries, in comparison with the retinas obtained from contralateral sham-operated eyes. In the retinas of IOP-elevated eyes, profound morphological deteriorations were determined in the ganglion and outer nuclear cell layers which were associated with an increased number of TUNEL positive cells in the ganglion and inner nuclear cell layers. Increased immunohistochemical stainings for three isoforms of nitric oxide synthase (NOS) were defined in almost all layers of the retinas of IOP-elevated eyes, in which eNOS was abundant particularly in the inner plexiform and ganglion cell layers. An irregular basal folding of retinal pigment epithelium (RPE) and an increased inter lamellar space of photoreceptor cell layer furtherly characterized the prominent degeneration of those layers in the retinas of IOP-elevated eyes. On the other hand, the relaxing effects of the retina obtained from IOP-elevated eyes were determined to be unchanged on the retinal and mesenteric arteries precontracted either with prostaglandin F2α (PGF2α, 30 μM) or potassium chloride (K(+), 100 mM), when compared with the relaxations of control retina obtained from contralateral sham-operated eyes. Overall, these findings suggested that the elevation of IOP induces prominent structural changes in

  10. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-05-10

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

  11. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  12. Monoterpene synthases from common sage (Salvia officinalis)

    Science.gov (United States)

    Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

    1999-01-01

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

  13. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Science.gov (United States)

    Alquézar, Berta; Rodríguez, Ana; de la Peña, Marcos; Peña, Leandro

    2017-01-01

    Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z)-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays. PMID:28883829

  14. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Berta Alquézar

    2017-08-01

    Full Text Available Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck genome sequence allowed us to characterize for the first time the terpene synthase (TPS family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays.

  15. Isolation of streptococcal hyaluronate synthase.

    OpenAIRE

    Prehm, P; Mausolf, A

    1986-01-01

    Hyaluronate synthase was isolated from protoblast membranes of streptococci by Triton X-114 extraction and cetylpyridinium chloride precipitation. It was identified as a 52,000-Mr protein, which bound to nascent hyaluronate and was affinity-labelled by periodate-oxidized UDP-glucuronic acid and UDP-N-acetylglucosamine. Antibodies directed against the 52,000-Mr protein inhibited hyaluronate synthesis. Mutants defective in hyaluronate synthase activity lacked the 52,000-Mr protein in membrane e...

  16. Folate binding site of flavin-dependent thymidylate synthase.

    Science.gov (United States)

    Koehn, Eric M; Perissinotti, Laura L; Moghram, Salah; Prabhakar, Arjun; Lesley, Scott A; Mathews, Irimpan I; Kohen, Amnon

    2012-09-25

    The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH(2)H(4)folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs.

  17. Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus.

    Science.gov (United States)

    Agger, Sean; Lopez-Gallego, Fernando; Schmidt-Dannert, Claudia

    2009-06-01

    Fungi are a rich source of bioactive secondary metabolites, and mushroom-forming fungi (Agaricomycetes) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared with the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpene-oxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5, functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae. Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as an alpha-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes delta-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homologue but instead was found to catalyse the highly specific synthesis of alpha-cuprenene. Coexpression of cop6 and the two monooxygenase genes next to it yields oxygenated alpha-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species.

  18. Mitochondrial ATP synthase is dispensable in blood-stage Plasmodium berghei rodent malaria but essential in the mosquito phase

    OpenAIRE

    Sturm, Angelika; Mollard, Vanessa; Cozijnsen, Anton; Goodman, Christopher D; McFadden, Geoffrey I.

    2015-01-01

    Mitochondrial ATP synthase is driven by chemiosmotic oxidation of pyruvate derived from glycolysis. Blood-stage malaria parasites eschew chemiosmosis, instead relying almost solely on glycolysis for their ATP generation, which begs the question of whether mitochondrial ATP synthase is necessary during the blood stage of the parasite life cycle. We knocked out the mitochondrial ATP synthase β subunit gene in the rodent malaria parasite, Plasmodium berghei, ablating the protein that converts AD...

  19. Control of malate synthase formation in Rhizopus nigricans.

    Science.gov (United States)

    Wegener, W S; Schell, J; Romano, A H

    1967-12-01

    The control of malate synthase formation in a fumaric acid-producing strain of Rhizopus nigricans has been found to be similar in most respects to that of isocitrate lyase, the companion enzyme of the glyoxylate bypass. A basal level is formed in a casein hydrolysate medium, which is repressed by glucose. Utilization of glucose during growth results in relief of glucose repression. Any factor which stimulates growth promotes relief of glucose repression by enhancing the incorporation of repressor metabolites derived from glucose into cell material. Thus, malate synthase formation was enhanced in glucose-containing media by the addition of zinc, or by an increase of the concentration of available nitrogen source in a synthetic medium. Both acetate and glycolate acted as apparent inducers of malate synthase, with glycolate the more effective of the two when added alone. Acetate induction was enhanced by Zn(++), however, whereas induction by glycolate was unaffected. This supports the concept that acetate stimulates formation of glyoxylate bypass enzymes by a derepression mechanism, whereas glycolate or a product derived from it acts directly as an inducer. Moreover, it is indicated that the malate synthases induced by acetate and glycolate are separate and distinct, as has been shown in Escherichia coli.

  20. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr

    2015-01-01

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

  1. Endothelial nitric oxide synthase in the microcirculation.

    Science.gov (United States)

    Shu, Xiaohong; Keller, T C Stevenson; Begandt, Daniela; Butcher, Joshua T; Biwer, Lauren; Keller, Alexander S; Columbus, Linda; Isakson, Brant E

    2015-12-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

  2. Catalysis by nitric oxide synthase.

    Science.gov (United States)

    Marletta, M A; Hurshman, A R; Rusche, K M

    1998-10-01

    The enzyme nitric oxide synthase catalyzes the oxidation of the amino acid L-arginine to L-citrulline and nitric oxide in an NADPH-dependent reaction. Nitric oxide plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and is a key component of the cytostatic/cytotoxic function of the immune system. Characterization of nitric oxide synthase substrates and cofactors has outlined the broad details of the overall reaction and suggested possibilities for chemical steps in the reaction; however, the molecular details of the reaction mechanism are still poorly understood. Recent evidence suggests a role for the reduced bound pterin in the first step of the reaction--the hydroxylation of L-arginine.

  3. Mitochondrial ATP synthase is dispensable in blood-stage Plasmodium berghei rodent malaria but essential in the mosquito phase.

    Science.gov (United States)

    Sturm, Angelika; Mollard, Vanessa; Cozijnsen, Anton; Goodman, Christopher D; McFadden, Geoffrey I

    2015-08-18

    Mitochondrial ATP synthase is driven by chemiosmotic oxidation of pyruvate derived from glycolysis. Blood-stage malaria parasites eschew chemiosmosis, instead relying almost solely on glycolysis for their ATP generation, which begs the question of whether mitochondrial ATP synthase is necessary during the blood stage of the parasite life cycle. We knocked out the mitochondrial ATP synthase β subunit gene in the rodent malaria parasite, Plasmodium berghei, ablating the protein that converts ADP to ATP. Disruption of the β subunit gene of the ATP synthase only marginally reduced asexual blood-stage parasite growth but completely blocked mouse-to-mouse transmission via Anopheles stephensi mosquitoes. Parasites lacking the β subunit gene of the ATP synthase generated viable gametes that fuse and form ookinetes but cannot progress beyond this stage. Ookinetes lacking the β subunit gene of the ATP synthase had normal motility but were not viable in the mosquito midgut and never made oocysts or sporozoites, thereby abrogating transmission to naive mice via mosquito bite. We crossed the self-infertile ATP synthase β subunit knockout parasites with a male-deficient, self-infertile strain of P. berghei, which restored fertility and production of oocysts and sporozoites, which demonstrates that mitochondrial ATP synthase is essential for ongoing viability through the female, mitochondrion-carrying line of sexual reproduction in P. berghei malaria. Perturbation of ATP synthase completely blocks transmission to the mosquito vector and could potentially be targeted for disease control.

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

    DEFF Research Database (Denmark)

    Yang, Ting; Gao, Liping; Hu, Hao

    2014-01-01

    (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...

  5. A functional cellulose synthase from ascidian epidermis

    OpenAIRE

    Matthysse, Ann G.; Deschet, Karine; Williams, Melanie; Marry, Mazz; White, Alan R.; Smith, William C.

    2004-01-01

    Among animals, urochordates (e.g., ascidians) are unique in their ability to biosynthesize cellulose. In ascidians cellulose is synthesized in the epidermis and incorporated into a protective coat know as the tunic. A putative cellulose synthase-like gene was first identified in the genome sequences of the ascidian Ciona intestinalis. We describe here a cellulose synthase gene from the ascidian Ciona savignyi that is expressed in the epidermis. The predicted C. savignyi cellulose synthase ami...

  6. Geranyllinalool synthases in solanaceae and other angiosperms constitute an ancient branch of diterpene synthases involved in the synthesis of defensive compounds

    NARCIS (Netherlands)

    Falara, V.; Alba, J.M.; Kant, M.R.; Schuurink, R.C.; Pichersky, E.

    2014-01-01

    Many angiosperm plants, including basal dicots, eudicots, and monocots, emit (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, which is derived from geranyllinalool, in response to biotic challenge. An Arabidopsis (Arabidopsis thaliana) geranyllinalool synthase (GLS) belonging to the e/f clade of the

  7. Design, synthesis and biological evaluation of N-alkyl or aryl substituted isoindigo derivatives as potential dual cyclin-dependent kinase 2 (CDK2)/glycogen synthase kinase 3β (GSK-3β) phosphorylation inhibitors.

    Science.gov (United States)

    Zhao, Ping; Li, Yanzhong; Gao, Guangwei; Wang, Shuai; Yan, Yun; Zhan, Xiaoping; Liu, Zenglu; Mao, Zhenmin; Chen, Shaoxiong; Wang, Liqun

    2014-10-30

    A series of N-alkyl or aryl substituted isoindigo derivatives have been synthesized and their anti-proliferative activity was evaluated by Sulforhodamine B (SRB) assay. Some of the target compounds exhibited significant antitumor activity, including compounds 6h and 6k (against K562 cells), 6i (against HeLa cells) and 6j (against A549 cells). N-(p-methoxy-phenyl)-isoindigo (6k) exhibited a high and selective anti-proliferative activity against K562 cells (IC50 7.8 μM) and induced the apoptosis of K562 cells in a dose-dependent manner. Compound 6k arrested the cell cycle at S phase in K562 cells by decreasing the expression of cyclin A and CDK2, which played critical roles in DNA replication and passage through G2 phase. Moreover, compound 6k down-regulated the expression of p-GSK-3β (Ser9), β-catenin and c-myc proteins, up-regulated the expression of GSK-3β, consequently, suppressed Wnt/β-catenin signaling pathway and induced the apoptosis of K562 cells. The binding mode of compound 6k with GSK-3β was simulated using molecular docking tools. All of these studies gave a better understanding to the molecular mechanisms of this class of agents and clues to develop dual CDK2/GSK-3β (Ser9) phosphorylation inhibitors applied in cancer chemotherapy. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  8. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

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

  9. Genetic structure and regulation of isoprene synthase in Poplar (Populus spp.).

    Science.gov (United States)

    Vickers, Claudia E; Possell, Malcolm; Nicholas Hewitt, C; Mullineaux, Philip M

    2010-07-01

    Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-D: -erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase; however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.

  10. Metabolic derangement of methionine and folate metabolism in mice deficient in methionine synthase reductase

    Science.gov (United States)

    Elmore, C. Lee; Wu, Xuchu; Leclerc, Daniel; Watson, Erica D.; Bottiglieri, Teodoro; Krupenko, Natalia I.; Krupenko, Sergey A.; Cross, James C.; Rozen, Rima; Gravel, Roy A.; Matthews, Rowena G.

    2007-01-01

    Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one-carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form methionine. In humans, methionine synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Complete ablation of methionine synthase activity in mice results in embryonic lethality. Other mouse models for hyperhomocyst(e)inemia have normal or reduced levels of methyltetrahydrofolate and are not embryonic lethal, although they have decreased ratios of AdoMet/AdoHcy and impaired methylation. We have constructed a mouse model with a gene trap insertion in the Mtrr gene specifying methionine synthase reductase, an enzyme essential for the activity of methionine synthase. This model is a hypomorph, with reduced methionine synthase reductase activity, thus avoiding the lethality associated with the absence of methionine synthase activity. Mtrrgt/gt mice have increased plasma homocyst(e)ine, decreased plasma methionine, and increased tissue methyltetrahydrofolate. Unexpectedly, Mtrrgt/gt mice do not show decreases in the AdoMet/AdoHcy ratio in most tissues. The different metabolite profiles in the various genetic mouse models for hyperhomocysteinemia may be useful in understanding biological effects of elevated homocyst(e)ine. PMID:17369066

  11. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...... with emotional lability, subjective mood fluctuations and cognitive function in healthy individuals. Thirty-seven healthy subjects were evaluated with neuropsychological tests and blood samples at baseline and 12-week follow-up. Total GSK-3β and serine-9-phosphorylated GSK-3β in peripheral blood mononuclear...... analysis revealed lower activity of GSK-3β in spring and summer compared with the fall season. No correlation was observed between GSK-3β activity and emotional lability, subjective mood fluctuations or cognitive function. The results suggest that intra- and interindividual variation in GSK-3β activity...

  12. Box H/ACA snoRNAs are preferred substrates for the trimethylguanosine synthase in the divergent unicellular eukaryote Trichomonas vaginalis

    Science.gov (United States)

    Simoes-Barbosa, Augusto; Chakrabarti, Kausik; Pearson, Michael; Benarroch, Delphine; Shuman, Stewart; Johnson, Patricia J.

    2012-01-01

    The 2,2,7-trimethylguanosine caps of eukaryal snRNAs and snoRNA are formed by the enzyme Tgs1, which catalyzes sequential guanine-N2 methylations of m7G caps. Atypically, in the divergent unicellular eukaryote Trichomonas vaginalis, spliceosomal snRNAs lack a guanosine cap and the recombinant T. vaginalis trimethylguanosine synthase (TvTgs) produces only m2,7G in vitro. Here, we show by direct metabolic labeling that endogenous T. vaginalis RNAs contain m7G, m2,7G, and m2,2,7G caps. Immunodepletion of TvTgs from cell extracts and TvTgs add-back experiments demonstrate that TvTgs produces m2,7G and m2,2,7G caps. Expression of TvTgs in yeast tgs1Δ cells leads to the formation of m2,7G and m2,2,7G caps and complementation of the lethality of a tgs1Δ mud2Δ strain. Whereas TvTgs is present in the nucleus and cytosol of T. vaginalis cells, TMG-containing RNAs are localized primarily in the nucleolus. Molecular cloning of anti-TMG affinity-purified T. vaginalis RNAs identified 16 box H/ACA snoRNAs, which are implicated in guiding RNA pseudouridylation. The ensemble of new T. vaginalis H/ACA snoRNAs allowed us to predict and partially validate an extensive map of pseudouridines in T. vaginalis rRNA. PMID:22847815

  13. NCBI nr-aa BLAST: CBRC-RMAC-20-0034 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-RMAC-20-0034 ref|ZP_01517190.1| Pseudouridine synthase, Rsu [Chloroflexus aggr...egans DSM 9485] gb|EAV09148.1| Pseudouridine synthase, Rsu [Chloroflexus aggregans DSM 9485] ZP_01517190.1 3e-11 40% ...

  14. Inhibitors of Fatty Acid Synthase for Prostate Cancer

    Science.gov (United States)

    2010-05-31

    ring of structure 3a; and coupling of various aldehydes and ,- unsaturated ethers to the 5 position of the quinine under acidic conditions to yield...share with orlistat a beta- lactone moiety as the distinguishing chemotype [70]. Beta-lactam derivatives of orlistat have also been described [71...Smith, J. W. Synthesis of novel beta‐ lactone  inhibitors of fatty acid synthase. J Med Chem, 2008,   51(17), 5285‐5296.  71.  Zhang, W., Richardson, R. D

  15. Genetics Home Reference: GM3 synthase deficiency

    Science.gov (United States)

    ... statistics provide? Why are some genetic conditions more common in particular ethnic groups? ... an enzyme called GM3 synthase, which carries out a chemical reaction that is the first step in the production ...

  16. Nitric oxide synthases: structure, function and inhibition

    National Research Council Canada - National Science Library

    Alderton, W K; Cooper, C E; Knowles, R G

    2001-01-01

    This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family...

  17. Identification of avian wax synthases.

    Science.gov (United States)

    Biester, Eva-Maria; Hellenbrand, Janine; Gruber, Jens; Hamberg, Mats; Frentzen, Margrit

    2012-02-04

    Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS) which have been identified in pro- and eukaryotic organisms. Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under in vivo and in vitro conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands. We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities.

  18. Identification of avian wax synthases

    Directory of Open Access Journals (Sweden)

    Biester Eva-Maria

    2012-02-01

    Full Text Available Abstract Background Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS which have been identified in pro- and eukaryotic organisms. Results Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under in vivo and in vitro conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands. Conclusions We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities.

  19. Terpene synthases from Cannabis sativa.

    Science.gov (United States)

    Booth, Judith K; Page, Jonathan E; Bohlmann, Jörg

    2017-01-01

    Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  20. Terpene synthases from Cannabis sativa.

    Directory of Open Access Journals (Sweden)

    Judith K Booth

    Full Text Available Cannabis (Cannabis sativa plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E-β-ocimene, (--limonene, (+-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  1. Endothelial nitric oxide synthase gene haplotypes and diabetic nephropathy among Asian Indians

    DEFF Research Database (Denmark)

    Ahluwalia, Tarun Veer Singh; Ahuja, Monica; Rai, Taranjit Singh

    2008-01-01

    Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease, including diabetic nephropathy. Endothelial-derived nitric oxide synthase (eNOS) gene polymorphisms affect eNOS activity and are associated with endothelial dysfunction. We evaluated the association...... of the constitutive endothelial nitric oxide synthase gene (eNOS) polymorphisms with type 2 diabetic nephropathy. We genotyped three polymorphisms of eNOS (Two SNPs: -786T > C, 894G > T and one 27-bp repeat polymorphism in Intron 4 (27VNTR)) in type 2 diabetic nephropathy patients (cases: n = 195) and type 2 diabetic...

  2. Isolation and function of spinach leaf β-ketoacyl-[acyl-carrier-protein] synthases

    OpenAIRE

    Shimakata, Takashi; Stumpf, Paul K.

    1982-01-01

    Crude spinach leaf extract readily forms the stearoyl derivative of acyl-carrier-protein (ACP) when acetyl-ACP and malonyl-ACP are incubated together. Palmitoyl-ACP is also elongated by malonyl-ACP to stearoyl-ACP. When β-ketoacyl-ACP synthase {3-oxoacyl-[ACP] synthase; acyl-[ACP]:malonyl-[ACP] C-acyltransferase (decarboxylating), EC 2.3.1.41} is purified with decanoyl-ACP as the assay substrate, palmitoyl-ACP elongation activity is lost. When palmitoyl-ACP is the assay substrate, another pro...

  3. Probing the mechanism of 1,4-conjugate elimination reactions catalyzed by terpene synthases.

    Science.gov (United States)

    Faraldos, Juan A; Gonzalez, Veronica; Li, Amang; Yu, Fanglei; Köksal, Mustafa; Christianson, David W; Allemann, Rudolf K

    2012-12-26

    The reaction mechanisms of (E)-β-farnesene synthase (EBFS) and isoprene synthase (ISPS), enzymes that catalyze a formal regiospecific 1,4-conjugate elimination of hydrogen diphosphate from (E,E)-farnesyl and dimethylallyl diphosphate (FDP and DMADP) to generate the semiochemicals (E)-β-farnesene and isoprene, respectively, were probed with substrate analogs and kinetic measurements. The results support stepwise reaction mechanisms through analogous enzyme-bound allylic cationic intermediates. For EBFS, we demonstrate that the elimination reaction can proceed via the enzyme-bound intermediate trans-nerolidyl diphosphate, while for ISPS the intermediacy of 2-methylbut-3-enyl 2-diphosphate can be inferred from the product outcome when deuterated DMADPs are used as substrates. Possible implications derived from the mechanistic details of the EBFS-catalyzed reaction for the evolution of sesquiterpene synthases are discussed.

  4. Occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants.

    Science.gov (United States)

    Ishida, Mariko; Kitao, Naoko; Mizuno, Kouichi; Tanikawa, Natsu; Kato, Misako

    2009-02-01

    Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-(14)C]adenine and [8-(14)C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue.

  5. 18-Hydroxydolabella-3,7-diene synthase - a diterpene synthase from Chitinophaga pinensis

    NARCIS (Netherlands)

    Dickschat, Jeroen S.; Rinkel, Jan; Rabe, Patrick; Kashkooli, Arman Beyraghdar; Bouwmeester, Harro J.

    2017-01-01

    The product obtained in vitro from a diterpene synthase encoded in the genome of the bacterium Chitinophaga pinensis, an enzyme previously reported to have germacrene A synthase activity during heterologous expression in Escherichia coli, was identified by extensive NMR-spectroscopic methods as

  6. Properties of phosphorylated thymidylate synthase.

    Science.gov (United States)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Terpene synthases are widely distributed in bacteria

    Science.gov (United States)

    Yamada, Yuuki; Kuzuyama, Tomohisa; Komatsu, Mamoru; Shin-ya, Kazuo; Omura, Satoshi; Cane, David E.; Ikeda, Haruo

    2015-01-01

    Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes. PMID:25535391

  8. Monoterpene synthase from Dracocephalum kotschyi and SPME-GC-MS analysis of its aroma profile

    Directory of Open Access Journals (Sweden)

    S. Saeidnia

    2014-04-01

    Full Text Available Dracocephalum kotschyi (Lamiaceae, as one of the remarkable aromatic plants, widely grows and also is cultivated in various temperate regions of Iran. There are diverse reports about the composition of the oil of this plant representing limonene derivatives as its major compounds. There is no report on cloning of mono- or sesquiterpene synthases from this plant. In the present study, the aroma profile of D. kotschyi has been extracted and analyzed via Headspace Solid-Phase Microextraction technique coupled with Gas Chromatography- Mass Spectroscopy. In order to determine the sequence of the active terpene synthase in this plant, first mRNA was prepared and cloning was performed by 3’ and 5’-RACEs-PCR method, then cDNA was sequenced and finally aligned with other recognized terpene synthases. The results showed that the plant leaves mainly comprised geranial (37.2%, limonene-10-al (28.5%, limonene (20.1% and 1,1-dimethoxy decane (14.5%. Sequencing the cDNA cloned from this plant revealed the presence of a monoterpene synthase absolutely similar to limonene synthase, responsible in formation of limonene, terpinolene, camphene and some other cyclic monoterpenes in its young leaves.

  9. Eugenol synthase genes in floral scent variation in Gymnadenia species.

    Science.gov (United States)

    Gupta, Alok K; Schauvinhold, Ines; Pichersky, Eran; Schiestl, Florian P

    2014-12-01

    Floral signaling, especially through floral scent, is often highly complex, and little is known about the molecular mechanisms and evolutionary causes of this complexity. In this study, we focused on the evolution of "floral scent genes" and the associated changes in their functions in three closely related orchid species of the genus Gymnadenia. We developed a benchmark repertoire of 2,571 expressed sequence tags (ESTs) in Gymnadenia odoratissima. For the functional characterization and evolutionary analysis, we focused on eugenol synthase, as eugenol is a widespread and important scent compound. We obtained complete coding complementary DNAs (cDNAs) of two copies of putative eugenol synthase genes in each of the three species. The proteins encoded by these cDNAs were characterized by expression and testing for activity in Escherichia coli. While G. odoratissima and Gymnadenia conopsea enzymes were found to catalyze the formation of eugenol only, the Gymnadenia densiflora proteins synthesize eugenol, as well as a smaller amount of isoeugenol. Finally, we showed that the eugenol and isoeugenol producing gene copies of G. densiflora are evolutionarily derived from the ancestral genes of the other species producing only eugenol. The evolutionary switch from production of one to two compounds evolved under relaxed purifying selection. In conclusion, our study shows the molecular bases of eugenol and isoeugenol production and suggests that an evolutionary transition in a single gene can lead to an increased complexity in floral scent emitted by plants.

  10. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-05-24

    May 24, 2010 ... NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained from the ... Key words: Periodontal diseases, nitric oxide synthases gene, DNA, PCR. INTRODUCTION ... various diseases' pathogenesis because of its dual role. *Corresponding author.

  11. Glutamate synthase: An archaeal horizontal gene transfer?

    Indian Academy of Sciences (India)

    (GOGAT) which is a key enzyme in ammonia assimilation in bacteria, algae and plants. It catalyzes the reductive transamidation of amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate (Temple et al 1998). Glutamate synthases differ according to their molecular weights, subunit compositions, ...

  12. Protective role of endothelial nitric oxide synthase

    NARCIS (Netherlands)

    Albrecht, Ester W J A; Stegeman, Coen A; Heeringa, Peter; Henning, Robert; van Goor, Harry

    Nitric oxide is a versatile molecule, with its actions ranging from haemodynamic regulation to anti-proliferative effects on vascular smooth muscle cells. Nitric oxide is produced by the nitric oxide synthases, endothelial NOS (eNOS), neural NOS (nNOS), and inducible NOS (iNOS). Constitutively

  13. Relationship between endothelial nitric oxide synthase gene ...

    African Journals Online (AJOL)

    Introduction: Endothelial nitric oxide synthase (eNOS), the enzyme in charge of nitric oxide production, plays a crucial role in vascular biology. However, the impact of single nucleotide polymorphisms (SNPs) affecting the gene encoding for eNOS (eNOS) on coronary artery diseases remains under debate and no data were ...

  14. Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

    National Research Council Canada - National Science Library

    Elisabeth Schmidtmann Ann-Christine Konig Anne Orwat Dario Leister Markus Hartl Iris Finkemeier

    2014-01-01

    Citrate synthase has a key role in the tricarboxylic (TCA) cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA...

  15. Producing alpha-olefins using polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Katz, Leonard; Steen, Eric J.; Keasling, Jay D.

    2018-01-02

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an .alpha.-olefin, such as 1-hexene or butadiene. The present invention also provides for a host cell comprising the PKS and when cultured produces the .alpha.-olefin.

  16. The tomato terpene synthase gene family

    NARCIS (Netherlands)

    Falara, V.; Akhtar, T.A.; Nguyen, T.T.H.; Spyropoulou, E.A.; Bleeker, P.M.; Schauvinhold, I.; Matsuba, Y.; Bonini, M.E.; Schilmiller, A.L.; Last, R.L.; Schuurink, R.C.; Pichersky, E.

    2011-01-01

    Compounds of the terpenoid class play many roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of Solanum lycopersicum (cultivated tomato) contains 40 terpene synthase (TPS) genes, including 28

  17. Expression of Deinococcus geothermalis trehalose synthase gene ...

    African Journals Online (AJOL)

    A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1692 bp reading-frame encoding 564 amino acids was amplified using polymerase chain reaction (PCR). The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl β-D-thiogalactopyranoside induction in ...

  18. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    Endothelial nitric oxide synthase (NOS3) is involved in key steps of immune response. Genetic factors predispose individuals to periodontal disease. This study's aim was to explore the association between NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained ...

  19. Terpene synthases of oregano (Origanum vulgare L.) and their roles in the pathway and regulation of terpene biosynthesis.

    Science.gov (United States)

    Crocoll, Christoph; Asbach, Julia; Novak, Johannes; Gershenzon, Jonathan; Degenhardt, Jörg

    2010-08-01

    The aroma, flavor and pharmaceutical value of cultivated oregano (Origanum vulgare L.) is a consequence of its essential oil which consists mostly of monoterpenes and sesquiterpenes. To investigate the biosynthetic pathway to oregano terpenes and its regulation, we identified and characterized seven terpene synthases, key enzymes of terpene biosynthesis, from two cultivars of O. vulgare. Heterologous expression of these enzymes showed that each forms multiple mono- or sesquiterpene products and together they are responsible for the direct production of almost all terpenes found in O. vulgare essential oil. The correlation of essential oil composition with relative and absolute terpene synthase transcript concentrations in different lines of O. vulgare demonstrated that monoterpene synthase activity is predominantly regulated on the level of transcription and that the phenolic monoterpene alcohol thymol is derived from gamma-terpinene, a product of a single monoterpene synthase. The combination of heterologously-expressed terpene synthases for in vitro assays resulted in blends of mono- and sesquiterpene products that strongly resemble those found in vivo, indicating that terpene synthase expression levels directly control the composition of the essential oil. These results will facilitate metabolic engineering and directed breeding of O. vulgare cultivars with higher quantity of essential oil and improved oil composition.

  20. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence

    Science.gov (United States)

    2014-01-01

    Background Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Methods Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Results Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Conclusions Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene. PMID:24716800

  1. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence.

    Science.gov (United States)

    Jagielska, Elżbieta; Płucienniczak, Andrzej; Dąbrowska, Magdalena; Dowierciał, Anna; Rode, Wojciech

    2014-04-09

    Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.

  2. Anti-obesogenic role of endothelial nitric oxide synthase

    Science.gov (United States)

    Sansbury, Brian E.; Hill, Bradford G.

    2015-01-01

    The prevalence of obesity has increased remarkably in the past four decades. Because obesity can promote the development of type 2 diabetes and cardiovascular disease, understanding the mechanisms that engender weight gain and discovering safe anti-obesity therapies are of critical importance. In particular, the gaseous signaling molecule, nitric oxide (NO), appears to be a central factor regulating adiposity and systemic metabolism. Obese and diabetic states are characterized by a deficit in bioavailable NO, with such decreases commonly attributed to downregulation of endothelial NO synthase (eNOS), loss of eNOS activity, or quenching of NO by its reaction with oxygen radicals. Gain-of-function studies, in which vascular-derived NO has been increased pharmacologically or genetically, reveal remarkable actions of NO on body composition and systemic metabolism. This review addresses the metabolic actions of eNOS and the potential therapeutic utility of harnessing its anti-obesogenic effects. PMID:25189393

  3. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...... endothelium is consistent with a role for NO in the control of blood flow in human skeletal muscle....

  4. Asymmetric dimethylarginine, oxidative stress, and vascular nitric oxide synthase in essential hypertension

    DEFF Research Database (Denmark)

    Wang, Dan; Strandgaard, Svend; Iversen, Jens

    2009-01-01

    We reported impaired endothelium-derived relaxation factor/nitric oxide (EDRF/NO) responses and constitutive nitric oxide synthase (cNOS) activity in subcutaneous vessels dissected from patients with essential hypertension (n = 9) compared with normal controls (n = 10). We now test the hypothesis...... and hypertensive subjects, the individual values for plasma levels of ADMA and HODE were both significantly (P oxidative stress in a group of hypertensive...

  5. Fatty Acid Synthase Activity as a Target for c-Met Driven Prostate Cancer

    Science.gov (United States)

    2013-07-01

    cancer potentially due to increased fecal fat excretion. In addition, several families of plant-derived flavonoid compounds including...Apoptosis by Flavonoids Is Associated with Their Ability to Inhibit Fatty Acid Synthase Activity. J. Biol. Chem., 2005. 280(7): p. 5636-5645. 156... flavonoids , represent a source of relatively nontoxic, orally available and affordable compounds that are known to affect a number of different

  6. Inducible nitric oxide synthase (iNOS) in tumor biology: the two sides of the same coin

    NARCIS (Netherlands)

    Lechner, Matthias; Lirk, Philipp; Rieder, Josef

    2005-01-01

    Inducible nitric oxide synthase (iNOS) is one of three key enzymes generating nitric oxide (NO) from the amino acid l-arginine. iNOS-derived NO plays an important role in numerous physiological (e.g. blood pressure regulation, wound repair and host defence mechanisms) and pathophysiological

  7. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.; (MSU)

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  8. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  9. Defective ceramide synthases in mice cause reduced amplitudes in electroretinograms and altered sphingolipid composition in retina and cornea.

    Science.gov (United States)

    Brüggen, Bianca; Kremser, Christiane; Bickert, Andreas; Ebel, Philipp; Vom Dorp, Katharina; Schultz, Konrad; Dörmann, Peter; Willecke, Klaus; Dedek, Karin

    2016-07-01

    Complex sphingolipids are strongly expressed in neuronal tissue and contain ceramides in their backbone. Ceramides are synthesized by six ceramide synthases (CerS1-6). Although it is known that each tissue has a unique profile of ceramide synthase expression and ceramide synthases are implicated in several neurodegenerative disorders, the expression of ceramide synthase isoforms has not been investigated in the retina. Here we demonstrate CerS1, CerS2 and CerS4 expression in mouse retina and cornea, with CerS4 ubiquitously expressed in all retinal neurons and Müller cells. To test whether ceramide synthase deficiency affects retinal function, we compared electroretinograms and retina morphology between wild-type and CerS1-, CerS2- and CerS4-deficient mice. Electroretinograms were strongly reduced in amplitude in ceramide synthase-deficient mice, suggesting that signalling in the outer retina is affected. However, the number of photoreceptors and cone outer segment length were unaltered and no changes in retinal layer thickness or synaptic structures were found. Mass spectrometric analyses of ceramides, hexosyl-ceramides and sphingomyelins showed that C20 to C24 acyl-containing species were decreased whereas C16-containing species were increased in the retina of ceramide synthase-deficient mice. Similar but smaller changes were also found in the cornea. Thus, we hypothesize that the replacement of very long-chain fatty acyl residues by shorter C16 residues may affect the electrical properties of retina and cornea, and alter receptor-mediated signal transduction, vesicle-mediated synaptic transmission or corneal light transmission. Future studies need to identify the molecular targets of ceramides or derived sphingolipids in light signal transduction and transmission in the eye. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  10. Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

    Science.gov (United States)

    Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

    2015-03-01

    The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in

  11. Trinuclear Metal Clusters in Catalysis by Terpenoid Synthases

    OpenAIRE

    Aaron, Julie A.; Christianson, David. W.

    2010-01-01

    Terpenoid synthases are ubiquitous enzymes that catalyze the formation of structurally and stereochemically diverse isoprenoid natural products. Many isoprenoid coupling enzymes and terpenoid cyclases from bacteria, fungi, protists, plants, and animals share the class I terpenoid synthase fold. Despite generally low amino acid sequence identity among these examples, class I terpenoid synthases contain conserved metal binding motifs that coordinate to a trinuclear metal cluster. This cluster n...

  12. Differential modulation of nitric oxide synthases in aging: therapeutic opportunities

    Directory of Open Access Journals (Sweden)

    Stêfany Bruno De Assis Cau

    2012-06-01

    Full Text Available Vascular aging is the term that describes the structural and functional disturbances of the vasculature with advancing aging. The molecular mechanisms of aging-associated endothelial dysfunction are complex, but reduced nitric oxide (NO bioavailability and altered vascular expression and activity of NO synthase (NOS enzymes have been implicated as major players. Impaired vascular relaxation in aging has been attributed to reduced endothelial NOS (eNOS-derived NO, while increased inducible NOS (iNOS expression seems to account for nitrosative stress and disrupted vascular homeostasis. Although eNOS is considered the main source of NO in the vascular endothelium, neuronal NOS (nNOS also contributes to endothelial cells-derived NO, a mechanism that is reduced in aging. Pharmacological modulation of NO generation and expression/activity of NOS isoforms may represent a therapeutic alternative to prevent the progression of cardiovascular diseases. Accordingly, this review will focus on drugs that modulate NO bioavailability, such as nitrite anions and NO-releasing non-steroidal anti-inflammatory drugs, hormones (dehydroepiandrosterone and estrogen, statins, resveratrol and folic acid, since they may be useful to treat/to prevent aging-associated vascular dysfunction. The impact of these therapies on life quality in elderly and longevity will be discussed.

  13. Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes.

    Science.gov (United States)

    Tilbrook, Kimberley; Poirier, Yves; Gebbie, Leigh; Schenk, Peer M; McQualter, Richard B; Brumbley, Stevens M

    2014-10-01

    Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome-targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the β-oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  14. The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

    Directory of Open Access Journals (Sweden)

    Alexander Krah

    Full Text Available The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

  15. The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex

    NARCIS (Netherlands)

    Vain, T.; Crowell, E.F.; Timpano, H.; Biot, E.; Desprez, T.; Mansoori Zangir, N.; Trindade, L.M.; Pagant, S.; Robert, S.; Hofte, H.; Gonneau, M.; Vernhettes, S.

    2014-01-01

    Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis

  16. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... Full Length Research Paper. Sequence analysis of cereal sucrose synthase genes and isolation of sorghum sucrose synthase gene fragment. T. Sivasudha1* and P. A. Kumar2. 1Department of Environmental Biotechnology, Bharathidasan University, Tiruchy-620 024, India. 2NRC on Plant Biotechnology, ...

  17. ATP synthases from archaea: the beauty of a molecular motor.

    Science.gov (United States)

    Grüber, Gerhard; Manimekalai, Malathy Sony Subramanian; Mayer, Florian; Müller, Volker

    2014-06-01

    Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    Directory of Open Access Journals (Sweden)

    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  19. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage.

    Science.gov (United States)

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Komur, Baran; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  20. Partition separation and characterization of the polyhydroxyalkanoates synthase produced from recombinant Escherichia coli using an aqueous two-phase system.

    Science.gov (United States)

    Lan, John Chi-Wei; Yeh, Chun-Yi; Wang, Chih-Chi; Yang, Yu-Hsuan; Wu, Ho-Shing

    2013-10-01

    Polyhydroxyalkanoates (PHAs) are renewable and biodegradable polyesters which can be synthesized either by numerous of microorganisms in vivo or synthase in vitro. The synthesis of PHAs in vitro requires an efficient separation for high yield of purified enzyme. The recombinant Escherichia coli harboring phaC gene derived from Ralstonia eutropha H16 was cultivated in the chemically defined medium for overexpression of synthase in the present work. The purification and characteristics of PHA synthase from clarified feedstock by using aqueous two-phase systems (ATPS) was investigated. The optimized concentration of ATPS for partitioning PHA synthase contained polyethylene glycol 6000 (30%, w/w) and potassium phosphate (8%, w/w) with 3.25 volume ratio in the absence of NaCl at pH 8.7 and 4°C. The results showed that the partition coefficient of enzyme activity and protein content are 6.07 and 0.22, respectively. The specific activity, selectivity, purification fold and recovery of phaC(Re) achieved 1.76 U mg⁻¹, 29.05, 16.23 and 95.32%, respectively. Several metal ions demonstrated a significant effect on activity of purified enzyme. The purified enzyme displayed maximum relative activity as operating condition at pH value of 7.5 and 37°C. As compared to conventional purification processes, ATPS can be a promising technique applied for rapid recovery of PHA synthase and preparation of large quantity of PHA synthase on synthesis of P(3HB) in vitro. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  1. Kinetic characterization and allosteric inhibition of the Yersinia pestis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase).

    Science.gov (United States)

    Haymond, Amanda; Johny, Chinchu; Dowdy, Tyrone; Schweibenz, Brandon; Villarroel, Karen; Young, Richard; Mantooth, Clark J; Patel, Trishal; Bases, Jessica; San Jose, Geraldine; Jackson, Emily R; Dowd, Cynthia S; Couch, Robin D

    2014-01-01

    The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development.

  2. Kinetic characterization and allosteric inhibition of the Yersinia pestis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase.

    Directory of Open Access Journals (Sweden)

    Amanda Haymond

    Full Text Available The methylerythritol phosphate (MEP pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP to methylerythritol phosphate (MEP, catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9 then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase. It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space for rational drug development.

  3. Refractory epilepsy and mitochondrial dysfunction due to GM3 synthase deficiency.

    Science.gov (United States)

    Fragaki, Konstantina; Ait-El-Mkadem, Samira; Chaussenot, Annabelle; Gire, Catherine; Mengual, Raymond; Bonesso, Laurent; Bénéteau, Marie; Ricci, Jean-Ehrland; Desquiret-Dumas, Valérie; Procaccio, Vincent; Rötig, Agnès; Paquis-Flucklinger, Véronique

    2013-05-01

    We report two children, born from consanguineous parents, who presented with early-onset refractory epilepsy associated with psychomotor delay, failure to thrive, blindness and deafness. Polarographic and spectrophotometric analyses in fibroblasts and liver revealed a respiratory chain (RC) dysfunction. Surprisingly, we identified a homozygous nonsense mutation in the GM3 synthase gene by using exome sequencing. GM3 synthase catalyzes the formation of GM3 ganglioside from lactosylceramide, which is the first step in the synthesis of complex ganglioside species. Mass spectrometry analysis revealed that the complete absence of GM3 ganglioside and its biosynthetic derivatives was associated with an upregulation of the alternative globoside pathway in fibroblasts. The accumulation of Gb3 and Gb4 globosides likely has a role in RC dysfunction and in the decrease of mitochondrial membrane potential leading to apoptosis, which we observed in fibroblasts. We show for the first time that GM3 synthase deficiency, responsible for early-onset epilepsy syndrome, leads to a secondary RC dysfunction. Our study highlights the role of secondary mitochondrial disorders that can interfere with the diagnosis and the evolution of other metabolic diseases.

  4. Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize.

    Directory of Open Access Journals (Sweden)

    Hao Wu

    Full Text Available The opaque-2 (o2 mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2 called "Quality Protein Maize" (QPM have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms.

  5. Identification and molecular cloning of a heparosan synthase from Pasteurella multocida type D.

    Science.gov (United States)

    DeAngelis, Paul L; White, Carissa L

    2002-03-01

    Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of approximately 500-3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of beta4GlcUA-alpha4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.

  6. Crystallization and preliminary crystallographic analysis of mannosyl-3-phosphoglycerate synthase from Rubrobacter xylanophilus

    Energy Technology Data Exchange (ETDEWEB)

    Sá-Moura, Bebiana [IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto (Portugal); Albuquerque, Luciana; Empadinhas, Nuno [Centro de Neurociências e Biologia Celular, Departamento de Zoologia, Universidade de Coimbra, Coimbra (Portugal); Costa, Milton S. da [Departamento de Bioquímica, Universidade de Coimbra, Coimbra (Portugal); Pereira, Pedro José Barbosa; Macedo-Ribeiro, Sandra, E-mail: sribeiro@ibmc.up.pt [IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto (Portugal)

    2008-08-01

    The enzyme mannosyl-3-phosphoglycerate synthase from R. xylanophilus has been expressed, purified and crystallized. The crystals belong to the hexagonal space group P6{sub 5}22 and diffract to 2.2 Å resolution. Rubrobacter xylanophilus is the only Gram-positive bacterium known to synthesize the compatible solute mannosylglycerate (MG), which is commonly found in hyperthermophilic archaea and some thermophilic bacteria. Unlike the salt-dependent pattern of accumulation observed in (hyper)thermophiles, in R. xylanophilus MG accumulates constitutively. The synthesis of MG in R. xylanophilus was tracked from GDP-mannose and 3-phosphoglycerate, but the genome sequence of the organism failed to reveal any of the genes known to be involved in this pathway. The native enzyme was purified and its N-terminal sequence was used to identify the corresponding gene (mpgS) in the genome of R. xylanophilus. The gene encodes a highly divergent mannosyl-3-phosphoglycerate synthase (MpgS) without relevant sequence homology to known mannosylphosphoglycerate synthases. In order to understand the specificity and enzymatic mechanism of this novel enzyme, it was expressed in Escherichia coli, purified and crystallized. The crystals thus obtained belonged to the hexagonal space group P6{sub 5}22 and contained two protein molecules per asymmetric unit. The structure was solved by SIRAS using a mercury derivative.

  7. A mechanism of paraquat toxicity involving nitric oxide synthase

    Science.gov (United States)

    Day, Brian J.; Patel, Manisha; Calavetta, Lisa; Chang, Ling-Yi; Stamler, Jonathan S.

    1999-01-01

    Paraquat (PQ) is a well described pneumotoxicant that produces toxicity by redox cycling with cellular diaphorases, thereby elevating intracellular levels of superoxide (O2⨪). NO synthase (NOS) has been shown to participate in PQ-induced lung injury. Current theory holds that NO reacts with O2⨪ generated by PQ to produce the toxin peroxynitrite. We asked whether NOS might alternatively function as a PQ diaphorase and reexamined the question of whether NO/O2⨪ reactions were toxic or protective. Here, we show that: (i) neuronal NOS has PQ diaphorase activity that inversely correlates with NO formation; (ii) PQ-induced endothelial cell toxicity is attenuated by inhibitors of NOS that prevent NADPH oxidation, but is not attenuated by those that do not; (iii) PQ inhibits endothelium-derived, but not NO-induced, relaxations of aortic rings; and (iv) PQ-induced cytotoxicity is potentiated in cytokine-activated macrophages in a manner that correlates with its ability to block NO formation. These data indicate that NOS is a PQ diaphorase and that toxicity of such redox-active compounds involves a loss of NO-related activity. PMID:10535996

  8. Elucidating nitric oxide synthase domain interactions by molecular dynamics.

    Science.gov (United States)

    Hollingsworth, Scott A; Holden, Jeffrey K; Li, Huiying; Poulos, Thomas L

    2016-02-01

    Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing L-Arg to NO and L-citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH-derived electrons are transferred to the heme-containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN-to-heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme-FMN-calmodulin NOS complex based on the recent biophysical studies using a 105-ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis. © 2015 The Protein Society.

  9. Phylogenomic and functional domain analysis of polyketide synthases in Fusarium

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Daren W.; Butchko, Robert A.; Baker, Scott E.; Proctor, Robert H.

    2012-02-01

    Fusarium species are ubiquitous in nature, cause a range of plant diseases, and produce a variety of chemicals often referred to as secondary metabolites. Although some fungal secondary metabolites affect plant growth or protect plants from other fungi and bacteria, their presence in grain based food and feed is more often associated with a variety of diseases in plants and in animals. Many of these structurally diverse metabolites are derived from a family of related enzymes called polyketide synthases (PKSs). A search of genomic sequence of Fusarium verticillioides, F. graminearum, F. oxysporum and Nectria haematococca (anamorph F. solani) identified a total of 58 PKS genes. To gain insight into how this gene family evolved and to guide future studies, we conducted a phylogenomic and functional domain analysis. The resulting genealogy suggested that Fusarium PKSs represent 34 different groups responsible for synthesis of different core metabolites. The analyses indicate that variation in the Fusarium PKS gene family is due to gene duplication and loss events as well as enzyme gain-of-function due to the acquisition of new domains or of loss-of-function due to nucleotide mutations. Transcriptional analysis indicate that the 16 F. verticillioides PKS genes are expressed under a range of conditions, further evidence that they are functional genes that confer the ability to produce secondary metabolites.

  10. Structure-function studies on nitric oxide synthases.

    Science.gov (United States)

    Li, Huiying; Poulos, Thomas L

    2005-01-01

    Nitric oxide synthase (NOS) catalyzes the oxidation of one l-arginine guanidinium N atom to nitric oxide (NO). NOS consists of a heme domain linked to a flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD) reductase that shuttles electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to the heme. This review summarizes various aspects of NOS structure and function derived from crystal structures coupled with a wealth of biochemical and biophysical data. This includes the binding of diatomic ligands, especially the product, NO, whose binding to the heme iron blocks enzyme activity. An unusual feature of NOS catalysis is the strict requirement for the essential cofactor, tetrahydrobiopterin (H4B). It now is generally agreed that H4B serves as an electron donor to the heme-oxy complex. The reason NOS may have recruited H4B as an electron transfer cofactor is to provide rapid coupled proton/electron transfer required for O2 activation. NOS is a highly regulated enzyme which is controlled by calmodulin (CaM) at the level of electron transfer within the FMN/FAD reductase and between the reductase and heme domains. Recent crystal structures provide a basis for developing models on the structural underpinnings of NOS regulation. In addition to the complex and fascinating functional and regulatory features of NOS, NOS is an important therapeutic target. Crystal structures have revealed the structural basis of isoform-selective inhibition by a group of dipeptide inhibitors which opens the way for structure-based inhibitor design.

  11. Oxide Synthase Expression by p38 MAP Kinase

    Directory of Open Access Journals (Sweden)

    Tuija Turpeinen

    2011-01-01

    Full Text Available The role of dual specificity phosphatase 1 (DUSP1 in inducible nitric oxide synthase (iNOS expression in A549 human pulmonary epithelial cells, J774 mouse macrophages and primary mouse bone marrow-derived macrophages (BMMs was investigated. iNOS expression was induced by a cytokine mixture (TNF, IFNγ and IL-1β in A549 cells and by LPS in J774 cells, and it was inhibited by p38 MAPK inhibitors SB202190 and BIRB 796. Stimulation with cytokine mixture or LPS enhanced also DUSP1 expression. Down-regulation of DUSP1 by siRNA increased p38 MAPK phosphorylation and iNOS expression in A549 and J774 cells. In addition, LPS-induced iNOS expression was enhanced in BMMs from DUSP1(−/− mice as compared to that in BMMs from wild-type mice. The results indicate that DUSP1 suppresses iNOS expression by limiting p38 MAPK activity in human and mouse cells. Compounds that enhance DUSP1 expression or modulate its function may be beneficial in diseases complicated with increased iNOS-mediated NO production.

  12. Identification of cystathionine γ-synthase and threonine synthase from Cicer arietinum and Lens culinaris.

    Science.gov (United States)

    Morneau, Dominique J K; Jaworski, Allison F; Aitken, Susan M

    2013-04-01

    In plants, cystathionine γ-synthase (CGS) and threonine synthase (TS) compete for the branch-point metabolite O-phospho-L-homoserine. These enzymes are potential targets for metabolic engineering studies, aiming to alter the flux through the competing methionine and threonine biosynthetic pathways, with the goal of increasing methionine production. Although CGS and TS have been characterized in the model organisms Escherichia coli and Arabidopsis thaliana, little information is available on these enzymes in other, particularly plant, species. The functional CGS and TS coding sequences from the grain legumes Cicer arietinum (chickpea) and Lens culinaris (lentil) identified in this study share approximately 80% amino acid sequence identity with the corresponding sequences from Glycine max. At least 7 active-site residues of grain legume CGS and TS are conserved in the model bacterial enzymes, including the catalytic base. Putative processing sites that remove the targeting sequence and result in functional TS were identified in the target species.

  13. [Thymidylate synthase-catalyzed reaction mechanism].

    Science.gov (United States)

    Rode, Wojciech; Jarmuńa, Adam

    2015-01-01

    Thymidylate synthase ThyA (EC 2.1.1.45;-encoded by the Tyms gene), having been for 60 years a molecular target in chemotherapy, catalyses the dUMP pyrimidine ring C(5) methylation reaction, encompassing a transfer of one-carbon group (the methylene one, thus at the formaldehyde oxidation level) from 6R-N5,10-methylenetetrahydrofolate, coupled with a reduction of this group to the methyl one, with concomitant generation of 7,8-dihydrofolate and thymidylate. New facts are presented, concerning (i) molecular mechanism of the catalyzed reaction, including the substrate selectivity mechanism, (ii) mechanism of inhibition by a particular inhibitor, N4-hydroxy-dCMP, (iii) structural properties of the enzyme, (iv) cellular localization, (v) potential posttranslational modifications of the enzyme protein and their influence on the catalytic properties and (vi) non-catalytic activities of the enzyme.

  14. Catalytic site interactions in yeast OMP synthase

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Barr, Eric W.; Jensen, Kaj Frank

    2014-01-01

    The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry...... and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here...... shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding....

  15. CLYBL is a polymorphic human enzyme with malate synthase and β-methylmalate synthase activity

    Science.gov (United States)

    Strittmatter, Laura; Li, Yang; Nakatsuka, Nathan J.; Calvo, Sarah E.; Grabarek, Zenon; Mootha, Vamsi K.

    2014-01-01

    CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/β-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to β-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/β-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown. PMID:24334609

  16. Biosynthetic potential of sesquiterpene synthases: product profiles of Egyptian Henbane premnaspirodiene synthase and related mutants.

    Science.gov (United States)

    Koo, Hyun Jo; Vickery, Christopher R; Xu, Yi; Louie, Gordon V; O'Maille, Paul E; Bowman, Marianne; Nartey, Charisse M; Burkart, Michael D; Noel, Joseph P

    2016-07-01

    The plant terpene synthase (TPS) family is responsible for the biosynthesis of a variety of terpenoid natural products possessing diverse biological functions. TPSs catalyze the ionization and, most commonly, rearrangement and cyclization of prenyl diphosphate substrates, forming linear and cyclic hydrocarbons. Moreover, a single TPS often produces several minor products in addition to a dominant product. We characterized the catalytic profiles of Hyoscyamus muticus premnaspirodiene synthase (HPS) and compared it with the profile of a closely related TPS, Nicotiana tabacum 5-epi-aristolochene synthase (TEAS). The profiles of two previously studied HPS and TEAS mutants, each containing nine interconverting mutations, dubbed HPS-M9 and TEAS-M9, were also characterized. All four TPSs were compared under varying temperature and pH conditions. In addition, we solved the X-ray crystal structures of TEAS and a TEAS quadruple mutant complexed with substrate and products to gain insight into the enzymatic features modulating product formation. These informative structures, along with product profiles, provide new insight into plant TPS catalytic promiscuity.

  17. Quaternary structure of human fatty acid synthase by electron cryomicroscopy

    Science.gov (United States)

    Brink, Jacob; Ludtke, Steven J.; Yang, Chao-Yuh; Gu, Zei-Wei; Wakil, Salih J.; Chiu, Wah

    2002-01-01

    We present the first three-dimensional reconstruction of human fatty acid synthase obtained by electron cryomicroscopy and single-particle image processing. The structure shows that the synthase is composed of two monomers, arranged in an antiparallel orientation, which is consistent with biochemical data. The monomers are connected to each other at their middle by a bridge of density, a site proposed to be the combination of the interdomain regions of the two monomers. Each monomer subunit appears to be subdivided into three structural domains. With this reconstruction of the synthase, we propose a location for the enzyme's two fatty acid synthesis sites. PMID:11756679

  18. Class II recombinant phosphoribosyl diphosphate synthase from spinach

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows...... an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K(ATP) and K(Rib-5-P) values of 170 and 110 micrometer, respectively, and a V(max) value of 13.1 micromol (min x...

  19. Crystallization and Preliminary X-ray Analysis of Allene Oxide Synthase, Cytochrome P450 CYP74A2, from Parthenium argentatum

    Science.gov (United States)

    Oxylipins are oxygenated derivatives of fatty acids and pivotal signaling molecules in plants and animals. Allene oxide synthase (AOS) is a key cytochrome P450 CYP74 enzyme involved in the biosynthesis of plant oxylipin jasmonates to convert 13(S)-hydroperoxide to allene oxide. Guayule (Parthenium a...

  20. Chalcone synthase genes from milk thistle (Silybum marianum ...

    Indian Academy of Sciences (India)

    Leyva et al. 1995), UV treatments and blue light (Hartmann et al. 1998; Wade et al. 2001; Zhou et al. 2007), elicitor treatments such as salicylic acid and. Keywords. chalcone synthase; real-time PCR; silymarin; anthocyanin; Silybum marianum.

  1. Molecular devices for the regulation of chloroplast ATP synthase

    NARCIS (Netherlands)

    Hisabori, T.; Konno, H.; Ichimura, H.; Strotmann, H.; Bald, D.

    2002-01-01

    In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential

  2. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J; Qvortrup, K

    1991-01-01

    Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase...... in the human duodenum, jejunum, ileum and colon by immunohistochemistry. PGH synthase immunoreactivity appeared to be similar in all segments of the intestine. Most smooth muscle cells seemed to contain PGH synthase; however, the reaction in the lamina muscularis mucosae was much stronger than...... in the longitudinal and circular muscle layers. Endothelial cells in capillaries and larger vessels showed a positive reaction. In addition, unidentified cells in subserosa, at the level of Auerbach's plexus and in the submucosa were stained. We concluded that the smooth muscle cells of the human gut has a rather...

  3. Functional and evolutionary relationships between terpene synthases from Australian Myrtaceae.

    Science.gov (United States)

    Keszei, Andras; Brubaker, Curt L; Carter, Richard; Köllner, Tobias; Degenhardt, Jörg; Foley, William J

    2010-06-01

    Myrtaceae is one of the chemically most variable and most significant essential oil yielding plant families. Despite an abundance of chemical information, very little work has focussed on the biochemistry of terpene production in these plants. We describe 70 unique partial terpene synthase transcripts and eight full-length cDNA clones from 21 myrtaceous species, and compare phylogenetic relationships and leaf oil composition to reveal clades defined by common function. We provide further support for the correlation between function and phylogenetic relationships by the first functional characterisation of terpene synthases from Myrtaceae: a 1,8-cineole synthase from Eucalyptus sideroxylon and a caryophyllene synthase from Eucalyptusdives. Copyright 2010 Elsevier Ltd. All rights reserved.

  4. A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Hassan, Sherif S; Azim, Sofiya

    2017-11-20

    For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Plant terpenoid synthases: Molecular biology and phylogenetic analysis

    OpenAIRE

    Bohlmann, Jörg; Meyer-Gauen, Gilbert; Croteau, Rodney

    1998-01-01

    This review focuses on the monoterpene, sesquiterpene, and diterpene synthases of plant origin that use the corresponding C10, C15, and C20 prenyl diphosphates as substrates to generate the enormous diversity of carbon skeletons characteristic of the terpenoid family of natural products. A description of the enzymology and mechanism of terpenoid cyclization is followed by a discussion of molecular cloning and heterologous expression of terpenoid synthases. Sequence relatedness and phylogeneti...

  6. Regulation of CDP-diacylglycerol synthase activity in Saccharomyces cerevisiae.

    OpenAIRE

    Homann, M J; Henry, S A; Carman, G M

    1985-01-01

    The addition of ethanolamine or choline to inositol-containing growth medium resulted in a reduction of CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) activity in Saccharomyces cerevisiae. The reduction of activity did not occur in the absence of inositol. CDP-diacylglycerol synthase activity was not regulated in a S. cerevisiae mutant strain (opi1; an inositol biosynthesis regulatory mutant) by the addition of phospholipid precursors to the growth medium.

  7. Concerted versus stepwise mechanism in thymidylate synthase.

    Science.gov (United States)

    Islam, Zahidul; Strutzenberg, Timothy S; Gurevic, Ilya; Kohen, Amnon

    2014-07-16

    Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design.

  8. Nitric Oxide Synthases in Heart Failure

    Science.gov (United States)

    Carnicer, Ricardo; Crabtree, Mark J.; Sivakumaran, Vidhya

    2013-01-01

    Abstract Significance: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca2+ homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology. Recent Advances: Major strides in understanding the role of normal and abnormal constitutive NOS in the heart have revealed molecular targets by which NO modulates myocyte function and morphology, the role and nature of post-translational modifications of NOS, and factors controlling nitroso-redox balance. Localized and differential signaling from NOS1 (neuronal) versus NOS3 (endothelial) isoforms are being identified, as are methods to restore NOS function in heart disease. Critical Issues: Abnormal NOS signaling plays a key role in many cardiac disorders, while targeted modulation may potentially reverse this pathogenic source of oxidative stress. Future Directions: Improvements in the clinical translation of potent modulators of NOS function/dysfunction may ultimately provide a powerful new treatment for many hearts diseases that are fueled by nitroso-redox imbalance. Antioxid. Redox Signal. 18, 1078–1099. PMID:22871241

  9. Tapentadol and nitric oxide synthase systems.

    Science.gov (United States)

    Bujalska-Zadrożny, Magdalena; Wolińska, Renata; Gąsińska, Emilia; Nagraba, Łukasz

    2015-04-01

    Tapentadol, a new analgesic drug with a dual mechanism of action (μ-opioid receptor agonism and norepinephrine reuptake inhibition), is indicated for the treatment of moderate to severe acute and chronic pain. In this paper, the possible additional involvement of the nitric oxide synthase (NOS) system in the antinociceptive activity of tapentadol was investigated using an unspecific inhibitor of NOS, L-NOArg, a relatively specific inhibitor of neuronal NOS, 7-NI, a relatively selective inhibitor of inducible NOS, L-NIL, and a potent inhibitor of endothelial NOS, L-NIO. Tapentadol (1-10 mg/kg, intraperitoneal) increased the threshold for mechanical (Randall-Selitto test) and thermal (tail-flick test) nociceptive stimuli in a dose-dependent manner. All four NOS inhibitors, administered intraperitoneally in the dose range 0.1-10 mg/kg, potentiated the analgesic action of tapentadol at a low dose of 2 mg/kg in both models of pain. We conclude that NOS systems participate in tapentadol analgesia.

  10. Electric field driven torque in ATP synthase.

    Directory of Open Access Journals (Sweden)

    John H Miller

    Full Text Available FO-ATP synthase (FO is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1 overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring.

  11. Human Isoprenoid Synthase Enzymes as Therapeutic Targets

    Science.gov (United States)

    Park, Jaeok; Matralis, Alexios; Berghuis, Albert; Tsantrizos, Youla

    2014-07-01

    The complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids in the human body, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently, pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

  12. Structures of human constitutive nitric oxide synthases.

    Science.gov (United States)

    Li, Huiying; Jamal, Joumana; Plaza, Carla; Pineda, Stephanie Hai; Chreifi, Georges; Jing, Qing; Cinelli, Maris A; Silverman, Richard B; Poulos, Thomas L

    2014-10-01

    Mammals produce three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The overproduction of NO by nNOS is associated with a number of neurodegenerative disorders; therefore, a desirable therapeutic goal is the design of drugs that target nNOS but not the other isoforms. Crystallography, coupled with computational approaches and medicinal chemistry, has played a critical role in developing highly selective nNOS inhibitors that exhibit exceptional neuroprotective properties. For historic reasons, crystallography has focused on rat nNOS and bovine eNOS because these were available in high quality; thus, their structures have been used in structure-activity-relationship studies. Although these constitutive NOSs share more than 90% sequence identity across mammalian species for each NOS isoform, inhibitor-binding studies revealed that subtle differences near the heme active site in the same NOS isoform across species still impact enzyme-inhibitor interactions. Therefore, structures of the human constitutive NOSs are indispensible. Here, the first structure of human neuronal NOS at 2.03 Å resolution is reported and a different crystal form of human endothelial NOS is reported at 1.73 Å resolution.

  13. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan′s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  14. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  15. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J; Qvortrup, Klaus

    1991-01-01

    Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase in th...... large capacity for PGH synthesis and the present results may provide a basis for a better understanding of both normal physiological functions as well as intestinal disease states involving disorders of prostaglandin synthesis.......Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase...... in the human duodenum, jejunum, ileum and colon by immunohistochemistry. PGH synthase immunoreactivity appeared to be similar in all segments of the intestine. Most smooth muscle cells seemed to contain PGH synthase; however, the reaction in the lamina muscularis mucosae was much stronger than...

  16. Enzymatic Properties and Mutational Studies of Chalcone Synthase from Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Mahiran Basri

    2012-08-01

    Full Text Available PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products. These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant’s active site.

  17. Enzymatic Properties and Mutational Studies of Chalcone Synthase from Physcomitrella patens

    Science.gov (United States)

    Rahman, Raja Noor Zaliha Raja Abdul; Zakaria, Iffah Izzati; Salleh, Abu Bakar; Basri, Mahiran

    2012-01-01

    PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant’s active site. PMID:22949824

  18. Inducible nitric oxide synthase haplotype associated with migraine and aura.

    Science.gov (United States)

    de O S Mansur, Thiago; Gonçalves, Flavia M; Martins-Oliveira, Alisson; Speciali, Jose G; Dach, Fabiola; Lacchini, Riccardo; Tanus-Santos, Jose E

    2012-05-01

    Migraine is a complex neurological disorder with a clear neurogenic inflammatory component apparently including enhanced nitric oxide (NO) formation. Excessive NO amounts possibly contributing to migraine are derived from increased expression and activity of inducible NO synthase (iNOS). We tested the hypothesis that two functional, clinically relevant iNOS genetic polymorphisms (C(-1026)A-rs2779249 and G2087A-rs2297518) are associated with migraine with or without aura. We studied 142 healthy women without migraine (control group) and 200 women with migraine divided into two groups: 148 with migraine without aura (MWA) and 52 with aura (MA). Genotypes were determined by real-time polymerase chain reaction using the Taqman(®) allele discrimination assays. The PHASE 2.1 software was used to estimate the haplotypes. The A allele for the G2087A polymorphism was more commonly found in the MA group than in the MWA group (28 vs. 18%; P 0.05). The haplotype combining both A alleles for the two polymorphisms was more commonly found in the MA group than in the control group or in the MWA group (19 vs. 10 or 8%; P = 0.0245 or 0.0027, respectively). Our findings indicate that the G2087A and the C(-1026)A polymorphism in the iNOS gene affect the susceptibility to migraine with aura when their effects are combined within haplotypes, whereas the G2087A affects the susceptibility to aura in migraine patients. These finding may have therapeutic implications when examining the effects of selective iNOS inhibitors.

  19. Nitric oxide synthases: regulation and function

    Science.gov (United States)

    Förstermann, Ulrich; Sessa, William C.

    2012-01-01

    Nitric oxide (NO), the smallest signalling molecule known, is produced by three isoforms of NO synthase (NOS; EC 1.14.13.39). They all utilize l-arginine and molecular oxygen as substrates and require the cofactors reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-)5,6,7,8-tetrahydrobiopterin (BH4). All NOS bind calmodulin and contain haem. Neuronal NOS (nNOS, NOS I) is constitutively expressed in central and peripheral neurons and some other cell types. Its functions include synaptic plasticity in the central nervous system (CNS), central regulation of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral nitrergic nerves. Nitrergic nerves are of particular importance in the relaxation of corpus cavernosum and penile erection. Phosphodiesterase 5 inhibitors (sildenafil, vardenafil, and tadalafil) require at least a residual nNOS activity for their action. Inducible NOS (NOS II) can be expressed in many cell types in response to lipopolysaccharide, cytokines, or other agents. Inducible NOS generates large amounts of NO that have cytostatic effects on parasitic target cells. Inducible NOS contributes to the pathophysiology of inflammatory diseases and septic shock. Endothelial NOS (eNOS, NOS III) is mostly expressed in endothelial cells. It keeps blood vessels dilated, controls blood pressure, and has numerous other vasoprotective and anti-atherosclerotic effects. Many cardiovascular risk factors lead to oxidative stress, eNOS uncoupling, and endothelial dysfunction in the vasculature. Pharmacologically, vascular oxidative stress can be reduced and eNOS functionality restored with renin- and angiotensin-converting enzyme-inhibitors, with angiotensin receptor blockers, and with statins. PMID:21890489

  20. Characterisation of the tryptophan synthase alpha subunit in maize

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    Gierl Alfons

    2008-04-01

    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA and β (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex.

  1. PKMiner: a database for exploring type II polyketide synthases

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    Kim Jinki

    2012-08-01

    Full Text Available Abstract Background Bacterial aromatic polyketides are a pharmacologically important group of natural products synthesized by type II polyketide synthases (type II PKSs in actinobacteria. Isolation of novel aromatic polyketides from microbial sources is currently impeded because of the lack of knowledge about prolific taxa for polyketide synthesis and the difficulties in finding and optimizing target microorganisms. Comprehensive analysis of type II PKSs and the prediction of possible polyketide chemotypes in various actinobacterial genomes will thus enable the discovery or synthesis of novel polyketides in the most plausible microorganisms. Description We performed a comprehensive computational analysis of type II PKSs and their gene clusters in actinobacterial genomes. By identifying type II PKS subclasses from the sequence analysis of 280 known type II PKSs, we developed highly accurate domain classifiers for these subclasses and derived prediction rules for aromatic polyketide chemotypes generated by different combinations of type II PKS domains. Using 319 available actinobacterial genomes, we predicted 231 type II PKSs from 40 PKS gene clusters in 25 actinobacterial genomes, and polyketide chemotypes corresponding to 22 novel PKS gene clusters in 16 genomes. These results showed that the microorganisms capable of producing aromatic polyketides are specifically distributed within a certain suborder of Actinomycetales such as Catenulisporineae, Frankineae, Micrococcineae, Micromonosporineae, Pseudonocardineae, Streptomycineae, and Streptosporangineae. Conclusions We could identify the novel candidates of type II PKS gene clusters and their polyketide chemotypes in actinobacterial genomes by comprehensive analysis of type II PKSs and prediction of aromatic polyketides. The genome analysis results indicated that the specific suborders in actinomycetes could be used as prolific taxa for polyketide synthesis. The chemotype-prediction rules with

  2. PKMiner: a database for exploring type II polyketide synthases.

    Science.gov (United States)

    Kim, Jinki; Yi, Gwan-Su

    2012-08-08

    Bacterial aromatic polyketides are a pharmacologically important group of natural products synthesized by type II polyketide synthases (type II PKSs) in actinobacteria. Isolation of novel aromatic polyketides from microbial sources is currently impeded because of the lack of knowledge about prolific taxa for polyketide synthesis and the difficulties in finding and optimizing target microorganisms. Comprehensive analysis of type II PKSs and the prediction of possible polyketide chemotypes in various actinobacterial genomes will thus enable the discovery or synthesis of novel polyketides in the most plausible microorganisms. We performed a comprehensive computational analysis of type II PKSs and their gene clusters in actinobacterial genomes. By identifying type II PKS subclasses from the sequence analysis of 280 known type II PKSs, we developed highly accurate domain classifiers for these subclasses and derived prediction rules for aromatic polyketide chemotypes generated by different combinations of type II PKS domains. Using 319 available actinobacterial genomes, we predicted 231 type II PKSs from 40 PKS gene clusters in 25 actinobacterial genomes, and polyketide chemotypes corresponding to 22 novel PKS gene clusters in 16 genomes. These results showed that the microorganisms capable of producing aromatic polyketides are specifically distributed within a certain suborder of Actinomycetales such as Catenulisporineae, Frankineae, Micrococcineae, Micromonosporineae, Pseudonocardineae, Streptomycineae, and Streptosporangineae. We could identify the novel candidates of type II PKS gene clusters and their polyketide chemotypes in actinobacterial genomes by comprehensive analysis of type II PKSs and prediction of aromatic polyketides. The genome analysis results indicated that the specific suborders in actinomycetes could be used as prolific taxa for polyketide synthesis. The chemotype-prediction rules with the suggested type II PKS modules derived using this resource

  3. Synthesis and evaluation of C-5 modified 2'-deoxyuridine monophosphates as inhibitors of M. tuberculosis thymidylate synthase.

    Science.gov (United States)

    Alexandrova, Liudmila A; Chekhov, Vladimir O; Shmalenyuk, Eduard R; Kochetkov, Sergey N; El-Asrar, Rania Abu; Herdewijn, Piet

    2015-11-15

    A series of 5'-monophosphates of 5-substituted 2'-deoxyuridine analogs, which recently demonstrated in vitro substantial suppression of two strains of Mycobacterium tuberculosis growth (virulent laboratory H37Rv and multiple resistant MS-115), has been synthesized and evaluated as potential inhibitors of M. tuberculosis thymidylate synthases: classical (ThyA) and flavin dependent thymidylate synthase (ThyX). A systematic SAR study and docking revealed 5-undecyloxymethyl-2'-deoxyuridine 5'-monophosphate 3b, displaying an IC50 value against ThyX of 8.32 μM. All derivatives lack activity against the ThyA. It can be assumed that the mechanism of action of 3b may be partially associated with the inhibition of the ThyX. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Substrate channeling between the human dihydrofolate reductase and thymidylate synthase.

    Science.gov (United States)

    Wang, Nuo; McCammon, J Andrew

    2016-01-01

    In vivo, as an advanced catalytic strategy, transient non-covalently bound multi-enzyme complexes can be formed to facilitate the relay of substrates, i. e. substrate channeling, between sequential enzymatic reactions and to enhance the throughput of multi-step enzymatic pathways. The human thymidylate synthase and dihydrofolate reductase catalyze two consecutive reactions in the folate metabolism pathway, and experiments have shown that they are very likely to bind in the same multi-enzyme complex in vivo. While reports on the protozoa thymidylate synthase-dihydrofolate reductase bifunctional enzyme give substantial evidences of substrate channeling along a surface "electrostatic highway," attention has not been paid to whether the human thymidylate synthase and dihydrofolate reductase, if they are in contact with each other in the multi-enzyme complex, are capable of substrate channeling employing surface electrostatics. This work utilizes protein-protein docking, electrostatics calculations, and Brownian dynamics to explore the existence and mechanism of the substrate channeling between the human thymidylate synthase and dihydrofolate reductase. The results show that the bound human thymidylate synthase and dihydrofolate reductase are capable of substrate channeling and the formation of the surface "electrostatic highway." The substrate channeling efficiency between the two can be reasonably high and comparable to that of the protozoa. © 2015 The Protein Society.

  5. A Comparative Analysis of Acyl-Homoserine Lactone Synthase Assays.

    Science.gov (United States)

    Shin, Daniel; Frane, Nicole D; Brecht, Ryan M; Keeler, Jesse; Nagarajan, Rajesh

    2015-12-01

    Quorum sensing is cell-to-cell communication that allows bacteria to coordinate attacks on their hosts by inducing virulent gene expression, biofilm production, and other cellular functions, including antibiotic resistance. AHL synthase enzymes synthesize N-acyl-l-homoserine lactones, commonly referred to as autoinducers, to facilitate quorum sensing in Gram-negative bacteria. Studying the synthases, however, has proven to be a difficult road. Two assays, including a radiolabeled assay and a colorimetric (DCPIP) assay are well-documented in literature to study AHL synthases. In this paper, we describe additional methods that include an HPLC-based, C-S bond cleavage and coupled assays to investigate this class of enzymes. In addition, we compare and contrast each assay for both acyl-CoA- and acyl-ACP-utilizing synthases. The expanded toolkit described in this study should facilitate mechanistic studies on quorum sensing signal synthases and expedite discovery of antivirulent compounds. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Heterologous expression of an active chitin synthase from Rhizopus oryzae.

    Science.gov (United States)

    Salgado-Lugo, Holjes; Sánchez-Arreguín, Alejandro; Ruiz-Herrera, José

    2016-12-01

    Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly. Copyright © 2016. Published by Elsevier Inc.

  7. Para-aminobenzoic acid (PABA synthase enhances thermotolerance of mushroom Agaricus bisporus.

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    Zhonglei Lu

    Full Text Available Most mushrooms are thermo-sensitive to temperatures over 23°C, which greatly restricts their agricultural cultivation. Understanding mushroom's innate heat-tolerance mechanisms may facilitate genetic improvements of their thermotolerance. Agaricus bisporus strain 02 is a relatively thermotolerant mushroom strain, while strain 8213 is quite thermo-sensitive. Here, we compared their responses at proteomic level to heat treatment at 33°C. We identified 73 proteins that are differentially expressed between 02 and 8213 or induced upon heat stress in strain 02 itself, 48 of which with a known identity. Among them, 4 proteins are constitutively more highly expressed in 02 than 8213; and they can be further upregulated in response to heat stress in 02, but not in 8213. One protein is encoded by the para-aminobenzoic acid (PABA synthase gene Pabs, which has been shown to scavenge the reactive oxygen species in vitro. Pabs mRNA and its chemical product PABA show similar heat stress induction pattern as PABA synthase protein and are more abundant in 02, indicating transcriptional level upregulation of Pabs upon heat stress. A specific inhibitor of PABA synthesis impaired thermotolerance of 02, while exogenous PABA or transgenic overexpression of 02 derived PABA synthase enhanced thermotolerance of 8213. Furthermore, compared to 8213, 02 accumulated less H2O2 but more defense-related proteins (e.g., HSPs and Chitinase under heat stress. Together, these results demonstrate a role of PABA in enhancing mushroom thermotolerance by removing H2O2 and elevating defense-related proteins.

  8. Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase.

    Science.gov (United States)

    Stull, Frederick W; Bernard, Steffen M; Sapra, Aparna; Smith, Janet L; Zuiderweg, Erik R P; Palfey, Bruce A

    2016-06-14

    Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2'-deoxythymidine 5'-monophosphate (dTMP) from 2'-deoxyuridine 5'-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans and a very different mechanism. Here we report nuclear magnetic resonance evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogues also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.

  9. Flavin-Dependent Thymidylate Synthase as a New Antibiotic Target

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    Michael Choi

    2016-05-01

    Full Text Available In humans de novo synthesis of 2′-deoxythymidine-5′-monophosphate (dTMP, an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate synthase (TSase and dihydrofolate reductase (DHFR. The enzyme flavin-dependent thymidylate synthase (FDTS represents an alternative enzymatic pathway to synthesize dTMP, which is not present in human cells. A number of pathogenic bacteria, however, depend on this enzyme in lieu of or in conjunction with the analogous human pathway. Thus, inhibitors of this enzyme may serve as antibiotics. Here, we review the similarities and differences of FDTS vs. TSase including aspects of their structure and chemical mechanism. In addition, we review current progress in the search for inhibitors of flavin dependent thymidylate synthase as potential novel therapeutics.

  10. Flavin-Dependent Thymidylate Synthase as a New Antibiotic Target.

    Science.gov (United States)

    Choi, Michael; Karunaratne, Kalani; Kohen, Amnon

    2016-05-20

    In humans de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP), an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate synthase (TSase) and dihydrofolate reductase (DHFR). The enzyme flavin-dependent thymidylate synthase (FDTS) represents an alternative enzymatic pathway to synthesize dTMP, which is not present in human cells. A number of pathogenic bacteria, however, depend on this enzyme in lieu of or in conjunction with the analogous human pathway. Thus, inhibitors of this enzyme may serve as antibiotics. Here, we review the similarities and differences of FDTS vs. TSase including aspects of their structure and chemical mechanism. In addition, we review current progress in the search for inhibitors of flavin dependent thymidylate synthase as potential novel therapeutics.

  11. Plant terpenoid synthases: Molecular biology and phylogenetic analysis

    Science.gov (United States)

    Bohlmann, Jörg; Meyer-Gauen, Gilbert; Croteau, Rodney

    1998-01-01

    This review focuses on the monoterpene, sesquiterpene, and diterpene synthases of plant origin that use the corresponding C10, C15, and C20 prenyl diphosphates as substrates to generate the enormous diversity of carbon skeletons characteristic of the terpenoid family of natural products. A description of the enzymology and mechanism of terpenoid cyclization is followed by a discussion of molecular cloning and heterologous expression of terpenoid synthases. Sequence relatedness and phylogenetic reconstruction, based on 33 members of the Tps gene family, are delineated, and comparison of important structural features of these enzymes is provided. The review concludes with an overview of the organization and regulation of terpenoid metabolism, and of the biotechnological applications of terpenoid synthase genes. PMID:9539701

  12. Acetolactate synthase inhibiting herbicides bind to the regulatory site.

    Science.gov (United States)

    Subramanian, M V; Loney-Gallant, V; Dias, J M; Mireles, L C

    1991-05-01

    Acetolactate synthase from spontaneous mutants of tobacco (Nicotiana tabacum; KS-43 and SK-53) and cotton (Gossypium hirsutum; PS-3, PSH-91, and DO-2) selected in tissue culture for resistance to a triazolopyrimidine sulfonanilide showed varying degrees of insensitivity to feedback inhibitor(s) valine and/or leucine. A similar feature was evident in the enzyme isolated from chlorsulfuron-resistant weed biotypes, Kochia scoparia and Stellaria media. Dual inhibition analyses of triazolopyrimidine sulfonanilide, thifensulfuron, and imazethapyr versus feedback inhibitor leucine revealed that the three herbicides were competitive with the amino acid for binding to acetolactate synthase from wild-type cotton cultures. Acetolactate synthase inhibiting herbicides may bind to the regulatory site on the enzyme.

  13. Chlorine gas exposure causes systemic endothelial dysfunction by inhibiting endothelial nitric oxide synthase-dependent signaling.

    Science.gov (United States)

    Honavar, Jaideep; Samal, Andrey A; Bradley, Kelley M; Brandon, Angela; Balanay, Joann; Squadrito, Giuseppe L; MohanKumar, Krishnan; Maheshwari, Akhil; Postlethwait, Edward M; Matalon, Sadis; Patel, Rakesh P

    2011-08-01

    Chlorine gas (Cl(2)) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl(2) exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl(2) promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl(2) for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl(2) dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl(2) (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (∼ 60%) in aorta isolated from Cl(2)-exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl(2) exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl(2)-exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl(2) exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.

  14. Type III polyketide synthase is involved in the biosynthesis of protocatechuic acid in Aspergillus niger.

    Science.gov (United States)

    Lv, Yangyong; Xiao, Jing; Pan, Li

    2014-11-01

    Genomic studies have shown that not only plants but also filamentous fungi contain type III polyketide synthases. To study the function of type III polyketide synthase (AnPKSIII) in Aspergillus niger, a deletion strain (delAnPKSIII) and an overexpression strain (oeAnPKSIII) were constructed in A. niger MA169.4, a derivative of the wild-type (WT) A. niger ATCC 9029 that produces large quantities of gluconic acid. Alterations in the metabolites were analyzed by HPLC when the extract of the overexpression strain was compared with extracts of the WT and deletion strains. Protocatechuic acid (PCA; 3,4-dihydroxybenzoic acid, 3.2 mg/l) was isolated and identified as the main product of AnPKSIII when inductively expressed in A. niger MA169.4. The molecular weight of PCA was 154.1 (m/z 153.1 [M-H](-)), was detected by ESI-MS in the negative ionization mode, and (1)H and (13)C NMR data confirmed its structure.

  15. Polyketide synthases of bacterial symbionts in sponges--evolution-based applications in natural products research.

    Science.gov (United States)

    Hochmuth, Thomas; Piel, Jörn

    2009-01-01

    Marine sponges are an unusually rich source of bioactive natural products with clinical potential. They also often harbor rich communities of symbiotic bacteria that have often been suspected as the true producers of sponge-derived compounds. To date, these bacteria can in most cases not be cultivated, but culture-independent methods, such as isolating and analyzing biosynthetic gene clusters using metagenomic strategies, have recently provided first insights into their chemical potential. This review summarizes recent work of our laboratory on the study of polyketide synthases (PKSs). These studies revealed two evolutionarily distinct, unusual PKS types that are commonly found in sponge metagenomes and were shown to be of bacterial origin. One, the sup PKS, dominates sponge metagenomic DNA libraries, occurs widespread in bacteriosponges and is to date exclusively known from such animals. Data suggest that it is a type of synthase that generates methyl-branched fatty acids, which are commonly present in sponges. The other PKS type, termed trans-acyltransferase (AT) PKS, is responsible for the biosynthesis of complex, bioactive polyketides, such as the onnamides, and also occurs in free-living bacteria. The diversity of PKS genes present in a single sponge metagenome can be enormous. However, the phylogenetic approaches outlined in this review can provide valuable insights into the PKS function and structures of polyketides and can assist in the targeted isolation of gene clusters.

  16. Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum

    Energy Technology Data Exchange (ETDEWEB)

    Pasquo, Alessandra [ENEA Casaccia Research Centre, Dipartimento BIOTEC, Sezione Genetica e Genomica Vegetale, PO Box 2400, I-00100 Rome (Italy); Bonamore, Alessandra; Franceschini, Stefano; Macone, Alberto; Boffi, Alberto; Ilari, Andrea, E-mail: andrea.ilari@uniroma1.it [Istituto di Biologia e Patologia Molecolari, CNR (IBPM) and Department Of Biochemical Sciences, University of Roma ‘La Sapienza’, Piazza Aldo Moro 5, 00179 Roma (Italy); ENEA Casaccia Research Centre, Dipartimento BIOTEC, Sezione Genetica e Genomica Vegetale, PO Box 2400, I-00100 Rome (Italy)

    2008-04-01

    The cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from T. flavum, a protein which catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids, are reported. Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 Å resolution using a synchrotron-radiation source. The crystals belong to the trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 86.31, c = 118.36 Å. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 Å resolution. The model is under construction.

  17. Early growth and development impairments in patients with ganglioside GM3 synthase deficiency.

    Science.gov (United States)

    Wang, H; Wang, A; Wang, D; Bright, A; Sency, V; Zhou, A; Xin, B

    2016-05-01

    Ganglioside GM3 synthase is a key enzyme involved in the biosynthesis of gangliosides. GM3 synthase deficiency (GSD) causes a complete absence of GM3 and all downstream biosynthetic derivatives. The individuals affected by this disorder manifest severe irritability, intractable seizures and profound intellectual disability. However, we have found that most newborns seem symptom-free for a period of time after birth. In order to further understand the onset of the disease, we investigated the early growth and development of patients with this condition through this study. We compared 37 affected individuals with their normal siblings and revealed that all children with GSD had relatively normal intrauterine growth and development, as their weight, length and head circumference were similar to their normal siblings at birth. However, the disease progresses quickly after birth and causes significant constitutional impairments of growth and development by 6 months of age. Neither breastfeeding nor gastrostomy tube placement made significant difference on growth and development as all groups of patients showed the similar pattern. We conclude that GSD causes significant postnatal growth and developmental impairments and the amount of gangliosides in breast milk and general nutritional intervention do not seem to alter these outcomes. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Neuronal Nitric Oxide Synthase Induction in the Antitumorigenic and Neurotoxic Effects of 2-Methoxyestradiol

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    Magdalena Gorska

    2014-08-01

    Full Text Available Objective: 2-Methoxyestradiol, one of the natural 17β-estradiol derivatives, is a novel, potent anticancer agent currently being evaluated in advanced phases of clinical trials. The main goal of the study was to investigate the anticancer activity of 2-methoxy-estradiol towards osteosarcoma cells and its possible neurodegenerative effects. We used an experimental model of neurotoxicity and anticancer activity of the physiological agent, 2-methoxyestradiol. Thus, we used highly metastatic osteosarcoma 143B and mouse immortalized hippocampal HT22 cell lines. The cells were treated with pharmacological (1 μM, 10 μM concentrations of 2-methoxyestradiol. Experimental: Neuronal nitric oxide synthase and 3-nitrotyrosine protein levels were determined by western blotting. Cell viability and induction of cell death were measured by MTT and PI/Annexin V staining and a DNA fragmentation ELISA kit, respectively. Intracellular levels of nitric oxide were determined by flow cytometry. Results: Here we demonstrated that the signaling pathways of neurodegenerative diseases and cancer may overlap. We presented evidence that 2-methoxyestradiol, in contrast to 17β-estradiol, specifically affects neuronal nitric oxide synthase and augments 3-nitrotyrosine level leading to osteosarcoma and immortalized hippocampal cell death. Conclusions: We report the dual facets of 2-methoxyestradiol, that causes cancer cell death, but on the other hand may play a key role as a neurotoxin.

  19. Rapid Discovery and Functional Characterization of Terpene Synthases from Four Endophytic Xylariaceae.

    Science.gov (United States)

    Wu, Weihua; Tran, William; Taatjes, Craig A; Alonso-Gutierrez, Jorge; Lee, Taek Soon; Gladden, John M

    2016-01-01

    Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of volatile organic compounds (VOCs) with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs) derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS. Out of the 26 TPS's profiled, 12 TPS's were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity.

  20. Rapid Discovery and Functional Characterization of Terpene Synthases from Four Endophytic Xylariaceae.

    Directory of Open Access Journals (Sweden)

    Weihua Wu

    Full Text Available Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of volatile organic compounds (VOCs with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME and GC-MS. Out of the 26 TPS's profiled, 12 TPS's were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity.

  1. Hemoglobin derivatives

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003371.htm Hemoglobin derivatives To use the sharing features on this page, please enable JavaScript. Hemoglobin derivatives are altered forms of hemoglobin . Hemoglobin is ...

  2. The Polymorphisms in Methylenetetrahydrofolate Reductase, Methionine Synthase, Methionine Synthase Reductase, and the Risk of Colorectal Cancer

    Science.gov (United States)

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results. PMID:22719222

  3. Stability of alkyl-dihydroxyacetonephosphate synthase in human control and peroxisomal disorder fibroblasts

    NARCIS (Netherlands)

    Biermann, J.; Gootjes, J.; Wanders, R. J.; van den Bosch, H.

    1999-01-01

    Alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) is a peroxisomal enzyme that plays a key role in ether phospholipid biosynthesis. To determine the turnover of alkyl-DHAP synthase in several peroxisomal disorders, pulse-chase experiments were performed. In control fibroblasts, mature

  4. A Bicarbonate Cofactor Modulates 1,4-Dihydroxy-2-naphthoyl-Coenzyme A Synthase in Menaquinone Biosynthesis of Escherichia coli*

    Science.gov (United States)

    Jiang, Ming; Chen, Minjiao; Guo, Zu-Feng; Guo, Zhihong

    2010-01-01

    1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase-fold protein catalyzing an intramolecular Claisen condensation in the menaquinone biosynthetic pathway. We have characterized this enzyme from Escherichia coli and found that it is activated by bicarbonate in a concentration-dependent manner. The bicarbonate binding site has been identified in the crystal structure of a virtually identical ortholog (96.8% sequence identity) from Salmonella typhimurium through comparison with a bicarbonate-insensitive orthologue. Kinetic properties of the enzyme and its site-directed mutants of the bicarbonate binding site indicate that the exogenous bicarbonate anion is essential to the enzyme activity. With this essential catalytic role, the simple bicarbonate anion is an enzyme cofactor, which is usually a small organic molecule derived from vitamins, a metal ion, or a metal-containing polyatomic anionic complex. This finding leads to classification of the DHNA-CoA synthases into two evolutionarily conserved subfamilies: type I enzymes that are bicarbonate-dependent and contain a conserved glycine at the bicarbonate binding site; and type II enzymes that are bicarbonate-independent and contain a conserved aspartate at the position similar to the enzyme-bound bicarbonate. In addition, the unique location of the enzyme-bound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the α-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases. PMID:20643650

  5. Flavin-Dependent Thymidylate Synthase ThyX Activity: Implications for the Folate Cycle in Bacteria▿ †

    Science.gov (United States)

    Leduc, Damien; Escartin, Frédéric; Nijhout, H. Frederik; Reed, Michael C.; Liebl, Ursula; Skouloubris, Stéphane; Myllykallio, Hannu

    2007-01-01

    Although flavin-dependent ThyX proteins show thymidylate synthase activity in vitro and functionally complement thyA defects in heterologous systems, direct proof of their cellular functions is missing. Using insertional mutagenesis of Rhodobacter capsulatus thyX, we constructed the first defined thyX inactivation mutant. Phenotypic analyses of the obtained mutant strain confirmed that R. capsulatus ThyX is required for de novo thymidylate synthesis. Full complementation of the R. capsulatus thyX::spec strain to thymidine prototrophy required not only the canonical thymidylate synthase ThyA but also the dihydrofolate reductase FolA. Strikingly, we also found that addition of exogenous methylenetetrahydrofolate transiently inhibited the growth of the different Rhodobacter strains used in this work. To rationalize these experimental results, we used a mathematical model of bacterial folate metabolism. This model suggests that a very low dihydrofolate reductase activity is enough to rescue significant thymidylate synthesis in the presence of ThyX proteins and is in agreement with the notion that intracellular accumulation of folates results in growth inhibition. In addition, our observations suggest that the presence of flavin-dependent thymidylate synthase X provides growth benefits under conditions in which the level of reduced folate derivatives is compromised. PMID:17890305

  6. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    DEFF Research Database (Denmark)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with ...

  7. Cloning and expression analysis of an anthocyanidin synthase gene ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 93; Issue 2. Cloning and expression analysis of an anthocyanidin synthase gene homologue from Brassica carinata. Mingli Yan Suping Ding Lili Liu Xiaoming Yin Jiabin Shu. Research Note Volume 93 Issue 2 August 2014 pp 513-516 ...

  8. Expression of Inducible Nitric Oxide Synthase in the Epithelial ...

    African Journals Online (AJOL)

    Conclusion: iNOS was over expressed in OKCs when compared with DC and RC suggesting that iNOS may contribute to the aggressive behavior of OKC. This is yet another evidence to support that OKC is the neoplasm. Keywords: Dentigerous cyst, Immunohistochemistry, Inducible nitric oxide synthase, Odontogenic ...

  9. Predicting the catalytic sites of isopenicillin N synthase (IPNS ...

    African Journals Online (AJOL)

    Predicting the catalytic sites of isopenicillin N synthase (IPNS) related non-haem iron-dependent oxygenases and oxidases (NHIDOX) through a structural superimposition ... With the advancement of protein structural analysis software, it is possible to predict the catalytic sites of protein that shared a structural resemblance.

  10. Functional isopenicillin N synthase in an animal genome

    NARCIS (Netherlands)

    Roelofs, D.; Timmermans, M.J.T.N.; Hensbergen, P.; van Leeuwen, H.; Koopman, J.; Faddeeva, A.; Suring, W.; de Boer, T.E.; Mariën, J.; Boer, R.; Bovenberg, R.; van Straalen, N.M.

    2013-01-01

    Horizontal transfer of genes is widespread among prokaryotes, but is less common between microorganisms and animals. Here, we present evidence for the presence of a gene encoding functional isopenicillin N synthase, an enzyme in the β-lactam antibiotics biosynthesis pathway, in the genome of the

  11. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    DEFF Research Database (Denmark)

    Edman, U; Edman, J C; Lundgren, B

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar...

  12. Role of Endothelial Nitric Oxide Synthase Gene Polymorphisms ...

    African Journals Online (AJOL)

    Background: Previous studies indicated an association between endothelial nitric oxide synthase (eNOS) activity and maintenance of pregnancy, but it is rather controversial whether polymorphisms of the gene encoding for eNOS are associated with recurrent spontaneous abortions (RSAs). Aim: The aim was to investigate ...

  13. A functional isopenicillin N synthase in an animal genome

    NARCIS (Netherlands)

    Roelofs, D.; Timmermans, M.J.T.N.; Hensbergen, P.J.; van Leeuwen, H.; Koopman, J.; Faddeeva-Vakhrusheva, A.; Suring, W.J.; de Boer, T.E.; Mariën, A.G.H.; Boer, R.; Bovenberg, R.; van Straalen, N.M.

    Horizontal transfer of genes is widespread among prokaryotes, but is less common between microorganisms and animals. Here, we present evidence for the presence of a gene encoding functional isopenicillin N synthase, an enzyme in the β-lactam antibiotics biosynthesis pathway, in the genome of the

  14. Endothelial nitric oxide synthase polymorphism G298T in ...

    Indian Academy of Sciences (India)

    Supplementary data: Endothelial nitric oxide synthase polymorphism G298T in association with oxidative DNA damage in coronary atherosclerosis. Rajesh G. Kumar, Mrudula K. Spurthi, Kishore G. Kumar, Sanjib K. Sahu and Surekha H. Rani. J. Genet. 91, 349–352. Table 1. The demographic and clinical data of the CHD ...

  15. Incidence of UMP synthase deficiency in South African Holstein cattle

    African Journals Online (AJOL)

    Deficiency of uridine monophosphate synthase (DUMPS) is an inherited recessive metabolic defect identified in Holstein cattle. Since heterorygous carriers transmit the defective gene 50% of the time, one fourth of the offspring from matings between two carriers are expected to be homozygous for DUMPS. This is a lethal ...

  16. Analysis of genetic variation of inducible nitric oxide synthase and ...

    African Journals Online (AJOL)

    user

    2011-02-21

    Feb 21, 2011 ... The genetic diversity of 100 Malaysian native chickens was investigated using polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) for two candidate genes: inducible nitric oxide synthase (INOS) and natural resistance-associated macrophage protein 1 (NRAMP1). The two genes.

  17. Inhibition of Inducible Nitric Oxide Synthase, Cycleooxygenase-2 ...

    African Journals Online (AJOL)

    HP

    Purpose: To explore the antioxidant properties of the methanol extract of Pericarpium Zanthoxyli and its effect on inducible nitric oxide synthase (iNOS), cycleooxygenase-2 (COX-2) and lipopolysaccharides (LPS)-induced cell damage in macrophage cells. Methods: Anti-oxidant activities were tested by measuring free ...

  18. Endothelial nitric oxide synthase gene Glu298Asp polymorphism ...

    African Journals Online (AJOL)

    Administrator

    2011-09-12

    Sep 12, 2011 ... eNOS haplotypes associated with gestational hypertension or preeclampsia. Pharmacogenomics, 9(10):. 1467-73. Serrano NC, Casas JP, Diaz LA, Paez C, Mesa CM, Cifuentes R,. Monterrosa A, Bautista A, Hawe E, Hingorani AD, Vallance P, Lopez-. Jaramillo P (2004). Endothelial NO synthase genotype ...

  19. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are

  20. Molecular cloning and expression profiling of a chalcone synthase ...

    Indian Academy of Sciences (India)

    Lamiophlomis rotata is a renowned Chinese medicinal plant. Chalcone synthase (CHS) is important in flavonoid and isoflavonoid biosynthesis, catalysing the formation of naringenin chalcone in plants. A full-length cDNA encoding the CHS gene was cloned from L. rotata based on the highly conserved CHS gene ...

  1. Biosynthesis of polyketides by trans-AT polyketide synthases.

    Science.gov (United States)

    Piel, Jörn

    2010-07-01

    This review discusses the biosynthesis of natural products that are generated by trans-AT polyketide synthases, a family of catalytically versatile enzymes that have recently been recognized as one of the major group of proteins involved in the production of bioactive polyketides. 436 references are cited.

  2. Inhibition of Inducible Nitric Oxide Synthase, Cycleooxygenase-2 ...

    African Journals Online (AJOL)

    Inhibition of Inducible Nitric Oxide Synthase, Cycleooxygenase-2 and Lipid Peroxidation by Methanol Extract of Pericarpium Zanthoxyli. ... Production of iNOS induced by LPS was significantly (p < 0.05) inhibited by the extract, suggesting that the extract inhibits nitric oxide (NO) production by suppressing iNOS expression.

  3. Nucleotide variation at the methionine synthase locus in an ...

    African Journals Online (AJOL)

    Nucleotide variation at the methionine synthase (MetE) locus within and among populations of an endangered forest tree Fokienia hodginsii in Vietnam was investigated in the present study. A total of 12 populations were sampled across Vietnam. The length of the sequenced locus varied from 1567 to 1559 bp. A total of 42 ...

  4. Aldosterone synthase C-344T, angiotensin II type 1 receptor ...

    Indian Academy of Sciences (India)

    Analysis of genetic and biochemical data revealed that in this population the CT and TT genotypes of aldosterone synthase C-344T polymorphism, frequency of alcohol consumption and aldosterone levels were significantly high among the total as well as male hypertensives, while the AC and CC genotypes of angiotensin ...

  5. Templating effects in aristolochene synthase catalysis: elimination versus cyclisation.

    Science.gov (United States)

    Faraldos, Juan A; González, Verónica; Senske, Michael; Allemann, Rudolf K

    2011-10-21

    Analysis of the products generated by mutants of aristolochene synthase from P. roqueforti (PR-AS) revealed the prominent structural role played by the aliphatic residue Leu 108 in maintaining the productive conformation of farnesyl diphosphate to ensure C1-C10 (σ-bond) ring-closure and hence (+)-aristolochene production.

  6. The role of aristolochene synthase in diphosphate activation.

    Science.gov (United States)

    Faraldos, Juan A; Gonzalez, Veronica; Allemann, Rudolf K

    2012-03-28

    Analysis of the role of amino acids involved in diphosphate binding in the Michaelis complex of aristolochene synthase from P. roqueforti (PR-AS) reveals mechanistic details about leaving group (PPi) activation and the nature of the active site acid. This journal is © The Royal Society of Chemistry 2012

  7. Functional Characterization of Sesquiterpene Synthase from Polygonum minus

    Directory of Open Access Journals (Sweden)

    Su-Fang Ee

    2014-01-01

    Full Text Available Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS has a complete open reading frame (ORF of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of β-sesquiphellandrene.

  8. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... comparative analysis of grass genomes and as a source of beneficial genes for agriculture. Recent studies have shown that ... sequencing of sucrose synthase gene fragment from sor- ghum using primers designed at their ... Sequencing was carried out by Sanger dideoxy DNA sequencing method. Results.

  9. Characterising the cellulose synthase complexes of cell walls

    NARCIS (Netherlands)

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the

  10. Contribution of granule bound starch synthase in kernel modification

    African Journals Online (AJOL)

    ACSS

    The role of gbssI and gbssII genes, encoding granule bound starch synthase enzyme I and II, respectively, in quality protein maize (QPM) were studied at different days after pollination. (DAP). Total RNA was used for first strand cDNA synthesis using the ImpromIISriptTM reverse transcriptase. No detectable levels of gbssI ...

  11. Isolation of developing secondary xylem specific cellulose synthase ...

    Indian Academy of Sciences (India)

    The present study aimed at identifying developing secondary xylem specific cellulose synthase genes from Eucalyptus tereticornis, a species predominantly used in paper and pulp industries in the tropics. The differential expression analysis of the three EtCesA genes using qRT-PCR revealed 49 to 87 fold relative ...

  12. Isolation of developing secondary xylem specific cellulose synthase ...

    Indian Academy of Sciences (India)

    ondary cell wall deposition. The present study aimed at identifying developing secondary xylem specific cellulose synthase genes from Eucalyptus tereticornis, a species predominantly used in paper and pulp industries in the tropics. The differen- tial expression analysis of the three EtCesA genes using qRT-PCR revealed ...

  13. Endothelial nitric oxide synthase polymorphism G298T in ...

    Indian Academy of Sciences (India)

    http://www.ias.ac.in/article/fulltext/jgen/091/03/0349-0352. Keywords. coronary artery disease; endothelial nitric oxide synthase; myocardial infarction; reactive oxygen species. Author Affiliations. Rajesh G. Kumar1 Mrudula K. Spurthi1 Kishore G. Kumar1 Sanjib K. Sahu2 Surekha H. Rani1. Department of Genetics, Osmania ...

  14. Detailed characterization of the substrate specificity of mouse wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  15. A new salicylate synthase AmS is identified for siderophores biosynthesis in Amycolatopsis methanolica 239(T).

    Science.gov (United States)

    Xie, Feng; Dai, Shengwang; Shen, Jinzhao; Ren, Biao; Huang, Pei; Wang, Qiushui; Liu, Xueting; Zhang, Buchang; Dai, Huanqin; Zhang, Lixin

    2015-07-01

    Siderophores are important for the growth of bacteria or the applications in treatment of iron overload-associated diseases due to the iron-chelating property. Salicylate synthase played a key role in the biosynthesis of some NRPS-derived siderophores by the providing of an iron coordination moiety as the initial building block. A new salicylate synthase, namely AmS, was identified in the biosynthesis pathway of siderophore amychelin in Amycolatopsis methanolica 239(T), since it shunt chorismate, an integrant precursor, from primary to secondary metabolite flow. The amino acid sequence alignment and phylogenetic analysis showed that AmS grouped into a new cluster. In vitro assays of AmS revealed its wide temperature tolerance ranged from 0 to 40 °C and narrow pH tolerant ranged from 7.0 to 9.0. AmS was resistant to organic solvents and non-ionic detergents. Moreover, AmS converted chorismate to salicylate with K m of 129.05 μM, k cat of 2.20 min(-1) at optimal conditions, indicating its low substrate specificity and comparable velocity to reported counterparts (Irp9 and MbtI). These properties of AmS may improve the iron-seizing ability of A. methanolica to compete with its neighbors growing in natural environments. Most importantly, serine and cysteine residues were found to be important for the catalytic activity of AmS. This study presented AmS as a new cluster of salicylate synthase and the reaction mechanism and potential applications of salicylate synthase were highlighted as well.

  16. Significance of nitric oxide synthases: Lessons from triple nitric oxide synthases null mice

    Directory of Open Access Journals (Sweden)

    Masato Tsutsui

    2015-01-01

    Full Text Available Nitric oxide (NO is synthesized by three distinct NO synthases (neuronal, inducible, and endothelial NOSs, all of which are expressed in almost all tissues and organs in humans. The regulatory roles of NOSs in vivo have been investigated in pharmacological studies with non-selective NOS inhibitors. However, the specificity of the inhibitors continues to be an issue of debate, and the authentic significance of NOSs is still poorly understood. To address this issue, we generated mice in which all three NOS genes are completely disrupted. The triple NOSs null mice exhibited cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced EDHF responses, with a shorter survival. The triple NOSs null mice also displayed metabolic abnormalities, including metabolic syndrome and high-fat diet-induced severe dyslipidemia. Furthermore, the triple NOSs null mice showed renal abnormalities (nephrogenic diabetes insipidus and pathological renal remodeling, lung abnormalities (accelerated pulmonary fibrosis, and bone abnormalities (increased bone mineral density and bone turnover. These results provide evidence that NOSs play pivotal roles in the pathogenesis of a wide variety of disorders. This review summarizes the latest knowledge on the significance of NOSs in vivo, based on lessons learned from experiments with our triple mutant model.

  17. Antioxidant Functions of Nitric Oxide Synthase in a Methicillin Sensitive Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Manisha Vaish

    2013-01-01

    Full Text Available Nitric oxide and its derivative peroxynitrites are generated by host defense system to control bacterial infection. However certain Gram positive bacteria including Staphylococcus aureus possess a gene encoding nitric oxide synthase (SaNOS in their chromosome. In this study it was determined that under normal growth conditions, expression of SaNOS was highest during early exponential phase of the bacterial growth. In oxidative stress studies, deletion of SaNOS led to increased susceptibility of the mutant cells compared to wild-type S. aureus. While inhibition of SaNOS activity by the addition of L-NAME increased sensitivity of the wild-type S. aureus to oxidative stress, the addition of a nitric oxide donor, sodium nitroprusside, restored oxidative stress tolerance of the SaNOS mutant. The SaNOS mutant also showed reduced survival after phagocytosis by PMN cells with respect to wild-type S. aureus.

  18. Transgene silencing of sucrose synthase in alfalfa stem vascular tissue by a truncated phosphoenolpyruvate carboxylase: sucrose synthase construct

    Science.gov (United States)

    An important role of sucrose synthase (SUS, EC 2.4.1.13) in plants is to provide UDP-glucose needed for cellulose synthesis in cell walls. We examined if over-expressing SUS in alfalfa (Medicago sativa L.) would increase cellulose content of stem cell walls. Alfalfa plants were transformed with two ...

  19. Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Kragelund, Birthe B; von Wettstein-Knowles, Penny

    2007-01-01

    activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high...

  20. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Nicholas C Carpita

    2009-04-20

    Five specific objectives of this project are to develop strategies to identify the genes that encode the catalytic components of "mixed-linkage" (1→3),(1→4)-beta-D-glucans in grasses, to determine the protein components of the synthase complex, and determine the biochemical mechanism of synthesis. We have used proteomic approaches to define intrinsic and extrinsic polypeptides of Golgi membranes that are associated with polysaccharide synthesis and trafficking. We were successful in producing recombinant catalytic domains of cellulose synthase genes and discovered that they dimerize upon concentration, indicating that two CesA proteins form the catalytic unit. We characterized a brittle stalk2 mutant as a defect in a COBRA-like protein that results in compromised lignin-cellulose interactions that decrease tissue flexibility. We used virus-induced gene silencing of barley cell wall polysaccharide synthesis by BSMV in an attempt to silence specific members of the cellulose synthase-like gene family. However, we unexpectedly found that regardless of the specificity of the target gene, whole gene interaction networks were silenced. We discovered the cause to be an antisense transcript of the cellulose synthase gene initiated small interfering RNAs that spread silencing to related genes.

  1. Artichoke, Cynarin and Cyanidin Downregulate the Expression of Inducible Nitric Oxide Synthase in Human Coronary Smooth Muscle Cells

    OpenAIRE

    Ning Xia; Andrea Pautz; Ursula Wollscheid; Gisela Reifenberg; Ulrich Förstermann; Huige Li

    2014-01-01

    Artichoke (Cynara scolymus L.) is one of the world’s oldest medicinal plants with multiple health benefits. We have previously shown that artichoke leaf extracts and artichoke flavonoids upregulate the gene expression of endothelial-type nitric oxide synthase (eNOS) in human endothelial cells. Whereas NO produced by the eNOS is a vasoprotective molecule, NO derived from the inducible iNOS plays a pro-inflammatory role in the vasculature. The present study was aimed to investigate the effects ...

  2. Expressing an (E)-β-farnesene synthase in the chloroplast of tobacco affects the preference of green peach aphid and its parasitoid.

    Science.gov (United States)

    Wang, Gen-Ping; Yu, Xiu-Dao; Fan, Jia; Wang, Cheng-She; Xia, Lan-Qin

    2015-09-01

    (E)-β-Farnesene (EβF) synthase catalyses the production of EβF, which for many aphids is the main or only component of the alarm pheromone causing the repellence of aphids and also functions as a kairomone for aphids' natural enemies. Many plants possess EβF synthase genes and can release EβF to repel aphids. In order to effectively recruit the plant-derived EβF synthase genes for aphid control, by using chloroplast transit peptide (CTP) of the small subunit of Rubisco (rbcS) from wheat (Triticum aestivum L.), we targeted AaβFS1, an EβF synthase gene from sweet wormwood (Artemisia annua L.), to the chloroplast of tobacco to generate CTP + AaβFS1 transgenic lines. The CTP + AaβFS1 transgenic tobacco plants could emit EβF at a level up to 19.25 ng/day per g fresh tissues, 4-12 fold higher than the AaβFS1 transgenic lines without chloroplast targeting. Furthermore, aphid/parasitoid behavioral bioassays demonstrated that the CTP + AaβFS1 transgenic tobacco showed enhanced repellence to green peach aphid (Myzus persicae) and attracted response of its parasitoid Diaeretiella rapae, thus affecting aphid infestation at two trophic levels. These data suggest that the chloroplast is an ideal subcellular compartment for metabolic engineering of plant-derived EβF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control. © 2014 Institute of Botany, Chinese Academy of Sciences.

  3. Evolutionary and mechanistic insights from the reconstruction of α-humulene synthases from a modern (+)-germacrene A synthase.

    Science.gov (United States)

    Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J; Faraldos, Juan A; Allemann, Rudolf K

    2014-10-15

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases.

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

    Science.gov (United States)

    Engprasert, Surang; Taura, Futoshi; Kawamukai, Makoto; Shoyama, Yukihiro

    2004-11-18

    Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25DeltacrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  5. Heterologous Gene Expression of N-Terminally Truncated Variants of LipPks1 Suggests a Functionally Critical Structural Motif in the N-terminus of Modular Polyketide Synthase

    DEFF Research Database (Denmark)

    Yuzawa, Satoshi; Bailey, Constance B.; Fujii, Tatsu A.

    2017-01-01

    Streptomyces-derived, Well-characterized modular, polyketide synthase (PKS). Using this enzyme as a model, we experimentally investigated the effects of alternative TSSs using a heterologous host, Streptomyces venezuelae. One of the TSSs employed boosted the protein level by 59-fold and the product yield by 23...

  6. octene derivatives

    Indian Academy of Sciences (India)

    Ultrasonication; trimethylsilyloxy-derivatives; bicyclo[2.2.2]octene; Diels–Alder reaction; crystal structure; C–H. . . O and π...π interactions. 1. ... of silyl- derivatives were studied.8 The structural studies indicated a self-assembly ..... Technology (DST), New Delhi, India for the financial assistance. References. 1. Zhao F, Zhang ...

  7. Financial Derivatives

    DEFF Research Database (Denmark)

    Wigan, Duncan

    2013-01-01

    Contemporary derivatives mark the development of capital and constitute a novel form of ownership. By reconfiguring the temporal, spatial and legal character of ownership derivatives present a substantive challenge to the tax collecting state. While fiscal systems are nationally bounded and inheren...

  8. Targeting Bacterial Nitric Oxide Synthase with Aminoquinoline-Based Inhibitors.

    Science.gov (United States)

    Holden, Jeffrey K; Lewis, Matthew C; Cinelli, Maris A; Abdullatif, Ziad; Pensa, Anthony V; Silverman, Richard B; Poulos, Thomas L

    2016-10-04

    Nitric oxide is produced in Gram-positive pathogens Bacillus anthracis and Staphylococcus aureus by the bacterial isoform of nitric oxide synthase (NOS). Inhibition of bacterial nitric oxide synthase (bNOS) has been identified as a promising antibacterial strategy for targeting methicillin-resistant S. aureus [Holden, J. K., et al. (2015) Chem. Biol. 22, 785-779]. One class of NOS inhibitors that demonstrates antimicrobial efficacy utilizes an aminoquinoline scaffold. Here we report on a variety of aminoquinolines that target the bacterial NOS active site, in part, by binding to a hydrophobic patch that is unique to bNOS. Through mutagenesis and crystallographic studies, our findings demonstrate that aminoquinolines are an excellent scaffold for further aiding in the development of bNOS specific inhibitors.

  9. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    expression analysis and proteomics have pointed to abnormalities in mitochondrial oxidative phosphorylation and cellular stress in muscle of type 2 diabetic subjects, and recent work suggests that impaired mitochondrial activity is another early defect in the pathogenesis of type 2 diabetes. This review......Insulin resistance in skeletal muscle is a major hallmark of type 2 diabetes and an early detectable abnormality in the development of this disease. The cellular mechanisms of insulin resistance include impaired insulin-mediated muscle glycogen synthesis and increased intramyocellular lipid content......, whereas impaired insulin activation of muscle glycogen synthase represents a consistent, molecular defect found in both type 2 diabetic and high-risk individuals. Despite several studies of the insulin signaling pathway believed to mediate dephosphorylation and hence activation of glycogen synthase...

  10. Sucrose Synthase Expression during Cold Acclimation in Wheat 1

    Science.gov (United States)

    Crespi, Martin D.; Zabaleta, Eduardo J.; Pontis, Horacio G.; Salerno, Graciela L.

    1991-01-01

    When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4°C) above 0°C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23°C to 4°C. The level of SS diminished when plants were moved back to 23°C. Northern blots of poly(A)+ RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress. ImagesFigure 1Figure 2Figure 3 PMID:16668270

  11. Dihydrodipicolinate synthase in opaque and floury maize mutants

    NARCIS (Netherlands)

    Varisi, V.A.; Medici, L.O.; Meer, van der I.M.; Lea, P.J.; Azevedo, J.L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine

  12. Use of linalool synthase in genetic engineering of scent production

    Science.gov (United States)

    Pichersky, Eran

    1998-01-01

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

  13. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal...... and sex showed no such changes. Our data support the hypothesis that NO is a pathogenic factor in MS, and that NOS IR is strongly expressed in brain regions appearing normal by MRI...

  14. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yang, Chang-Qing; Wu, Xiu-Ming; Ruan, Ju-Xin; Hu, Wen-Li; Mao, Yin-Bo; Chen, Xiao-Ya; Wang, Ling-Jian

    2013-12-01

    Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Piriformospora indica requires kaurene synthase activity for successful plant colonization.

    Science.gov (United States)

    Li, Liang; Chen, Xi; Ma, Chaoyang; Wu, Hongqing; Qi, Shuting

    2016-05-01

    Ent-kaurene (KS) synthases and ent-kaurene-like (KSL) synthases are involved in the biosynthesis of phytoalexins and/or gibberellins which play a role in plant immunity and development. The relationship between expression of five synthase genes (HvKSL1, HvKS2, HvKS4, HvKS5, HvKSL4) and plant colonization by the endophytic fungus Piriformospora indica was assessed in barley (Hordeum vulgare). The KS gene family is differently up-regulated at 1, 3 and 7 day after P. indica inoculation. By comparison, the HvKSL4 gene expression pattern is more significantly affected by UV irradiation and P. indica colonization. The characterizations of two silencing lines (HvKSL1-RNAi, HvKSL4-RNAi) also were analyzed. HvKSL1-RNAi and HvKSL4-RNAi lines in the first generation lead to less dark green leaves and slower plant development. Further, reduced spikelet fertility in progenies of RNAi plants heterozygous for HvKSL1 were observed, but not for HvKSL4. T2 generation of HvKSL1-RNAi line showed semi-dwarf phenotype while the wild type phenotype could be restored by applying GA3. Silencing of HvKSL4 and HvKSL1 resulted in reduced colonization by P. indica especially in the HvKSL1-RNAi line. These results probably suggest the presence of two ent-KS synthase in barley, one (HvKSL1) that participates in the biosynthesis of GAs and another (HvKSL4) that is involved in the biosynthesis of phytoalexins. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  16. Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

    Science.gov (United States)

    Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M

    2016-02-23

    Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases.

  17. Characterization of tryptophan synthase alpha subunit mutants of Arabidopsis thaliana.

    Science.gov (United States)

    Radwanski, E R; Barczak, A J; Last, R L

    1996-12-13

    Three mutations in the Arabidopsis thaliana gene encoding the alpha subunit of tryptophan synthase were isolated by selection for resistance to 5-methylanthranilate or 5-fluoroindole, toxic analogs of tryptophan pathway intermediates. Plants homozygous for trp3-1 and trp3-2 are light-conditional tryptophan auxotrophs, while trp3-100 is a more leaky mutant. Genetic complementation crosses demonstrated that the three mutations are allelic to each other, and define a new complementation group. All three mutants have decreased steady-state levels of tryptophan synthase alpha protein, and the trp3-100 polypeptide exhibits altered electrophoretic mobility. All three mutations were shown to be in the TSA1 (tryptophan synthase alpha subunit) structural gene by several criteria. Firstly, the trp3-1 mutation is linked to TSA1 on the bottom of chromosome 3. Secondly, the trp3-1 mutation was complemented when transformed with the wild-type TSA1 gene. Finally, DNA sequence analysis of the TSA1 gene revealed a single transition mutation in each trp3 mutant.

  18. Substrate range of benzylsuccinate synthase from Azoarcus sp. strain T.

    Science.gov (United States)

    Beller, H R; Spormann, A M

    1999-09-01

    Benzylsuccinate synthase, which catalyzes the anaerobic addition of the methyl carbon of toluene to fumarate, has recently been reported in several denitrifying and sulfate-reducing, toluene-degrading bacteria. In substrate range studies with partially purified benzylsuccinate synthase from denitrifying Azoarcus sp. strain T, benzylsuccinate analogs were observed as a result of fumarate addition to the following toluene surrogates: xylenes, monofluorotoluenes, benzaldehyde, and 1-methyl-1-cyclohexene (but not 4-methyl-l-cyclohexene or methylcyclohexane). Benzylsuccinate was also observed as a result of toluene addition to maleate, but no products were observed from assays with toluene and either crotonate or trans-glutaconate. Toluene-maleate addition, like toluene-fumarate addition, resulted in highly stereospecific formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid]. The previously reported finding that the methyl H atom abstracted from toluene is retained in the succinyl moiety of benzylsuccinate was found to apply to several toluene surrogates. The implications of these observations for the mechanism of benzylsuccinate synthase will be discussed.

  19. Multi-substrate terpene synthases: their occurrence and physiological significance

    Directory of Open Access Journals (Sweden)

    Leila Pazouki

    2016-07-01

    Full Text Available Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15, and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5, mono- (C10 and diterpenes (C20. Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles.

  20. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance.

    Science.gov (United States)

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles.

  1. A Single Point Mutation in the Gene Encoding Gb3/CD77 Synthase Causes a Rare Inherited Polyagglutination Syndrome*

    Science.gov (United States)

    Suchanowska, Anna; Kaczmarek, Radoslaw; Duk, Maria; Lukasiewicz, Jolanta; Smolarek, Dorota; Majorczyk, Edyta; Jaskiewicz, Ewa; Laskowska, Anna; Wasniowska, Kazimiera; Grodecka, Magdalena; Lisowska, Elwira; Czerwinski, Marcin

    2012-01-01

    Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NORint, and NOR2, in which Gal(α1–4), GalNAc(β1–3)Gal(α1–4), and Gal(α1–4)GalNAc(β1–3)Gal(α1–4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1–4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1–4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1–4)Gal and Gal(α1–4)GalNAc moieties. PMID:22965229

  2. A single point mutation in the gene encoding Gb3/CD77 synthase causes a rare inherited polyagglutination syndrome.

    Science.gov (United States)

    Suchanowska, Anna; Kaczmarek, Radoslaw; Duk, Maria; Lukasiewicz, Jolanta; Smolarek, Dorota; Majorczyk, Edyta; Jaskiewicz, Ewa; Laskowska, Anna; Wasniowska, Kazimiera; Grodecka, Magdalena; Lisowska, Elwira; Czerwinski, Marcin

    2012-11-02

    Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR(int), and NOR2, in which Gal(α1-4), GalNAc(β1-3)Gal(α1-4), and Gal(α1-4)GalNAc(β1-3)Gal(α1-4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1-4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1-4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1-4)Gal and Gal(α1-4)GalNAc moieties.

  3. Structural Analysis of Thymidylate Synthase from Kaposi's Sarcoma-Associated Herpesvirus with the Anticancer Drug Raltitrexed.

    Directory of Open Access Journals (Sweden)

    Yong Mi Choi

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is a highly infectious human herpesvirus that causes Kaposi's sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2'-deoxyuridine-5'-monophosphate (dUMP to thymidine-5'-monophosphate (dTMP using 5,10-methylenetetrahydrofolate (mTHF as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo, complexes with dUMP (binary, and complexes with both dUMP and raltitrexed (ternary were determined at 1.7 Å, 2.0 Å, and 2.4 Å, respectively. While the ternary complex structures of human thymidylate synthase and E. coli thymidylate synthase had a closed conformation, the ternary complex structure of KSHV thymidylate synthase was observed in an open conformation, similar to that of rat thymidylate synthase. The complex structures of KSHV thymidylate synthase did not have a covalent bond between the sulfhydryl group of Cys219 and C6 atom of dUMP, unlike the human thymidylate synthase. The catalytic Cys residue demonstrated a dual conformation in the apo structure, and its sulfhydryl group was oriented toward the C6 atom of dUMP with no covalent bond upon ligand binding in the complex structures. These structural data provide the potential use of antifolates such as raltitrexed as a viral induced anticancer drug and structural basis to design drugs for targeting the thymidylate synthase of KSHV.

  4. Structural Analysis of Thymidylate Synthase from Kaposi's Sarcoma-Associated Herpesvirus with the Anticancer Drug Raltitrexed.

    Science.gov (United States)

    Choi, Yong Mi; Yeo, Hyun Ku; Park, Young Woo; Lee, Jae Young

    2016-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a highly infectious human herpesvirus that causes Kaposi's sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2'-deoxyuridine-5'-monophosphate (dUMP) to thymidine-5'-monophosphate (dTMP) using 5,10-methylenetetrahydrofolate (mTHF) as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo), complexes with dUMP (binary), and complexes with both dUMP and raltitrexed (ternary) were determined at 1.7 Å, 2.0 Å, and 2.4 Å, respectively. While the ternary complex structures of human thymidylate synthase and E. coli thymidylate synthase had a closed conformation, the ternary complex structure of KSHV thymidylate synthase was observed in an open conformation, similar to that of rat thymidylate synthase. The complex structures of KSHV thymidylate synthase did not have a covalent bond between the sulfhydryl group of Cys219 and C6 atom of dUMP, unlike the human thymidylate synthase. The catalytic Cys residue demonstrated a dual conformation in the apo structure, and its sulfhydryl group was oriented toward the C6 atom of dUMP with no covalent bond upon ligand binding in the complex structures. These structural data provide the potential use of antifolates such as raltitrexed as a viral induced anticancer drug and structural basis to design drugs for targeting the thymidylate synthase of KSHV.

  5. Suites of terpene synthases explain differential terpenoid production in ginger and turmeric tissues.

    Directory of Open Access Journals (Sweden)

    Hyun Jo Koo

    Full Text Available The essential oils of ginger (Zingiber officinale and turmeric (Curcuma longa contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+-germacrene D synthase and (S-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (--caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+-α-turmerone and (+-β-turmerone, are produced from (--α-zingiberene and (--β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase.

  6. Suites of Terpene Synthases Explain Differential Terpenoid Production in Ginger and Turmeric Tissues

    Science.gov (United States)

    Koo, Hyun Jo; Gang, David R.

    2012-01-01

    The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (−)-caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+)-α-turmerone and (+)-β-turmerone, are produced from (−)-α-zingiberene and (−)-β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase. PMID:23272109

  7. β-Cyanoalanine Synthase Is a Mitochondrial Cysteine Synthase-Like Protein in Spinach and Arabidopsis1

    Science.gov (United States)

    Hatzfeld, Yves; Maruyama, Akiko; Schmidt, Ahlert; Noji, Masaaki; Ishizawa, Kimiharu; Saito, Kazuki

    2000-01-01

    β-Cyano-alanine synthase (CAS; EC 4.4.1.9) plays an important role in cyanide metabolism in plants. Although the enzymatic activity of β-cyano-Ala synthase has been detected in a variety of plants, no cDNA or gene has been identified so far. We hypothesized that the mitochondrial cysteine synthase (CS; EC 4.2.99.8) isoform, Bsas3, could actually be identical to CAS in spinach (Spinacia oleracea) and Arabidopsis. An Arabidopsis expressed sequence tag database was searched for putative Bsas3 homologs and four new CS-like isoforms, ARAth;Bsas1;1, ARAth;Bsas3;1, ARAth;Bsas4;1, and ARAth;Bsas4;2, were identified in the process. ARAth;Bsas3;1 protein was homologous to the mitochondrial SPIol;Bsas3;1 isoform from spinach, whereas ARAth;Bsas4;1 and ARAth;Bsas4;2 proteins defined a new class within the CS-like proteins family. In contrast to spinach SPIol;Bsas1;1 and SPIol;Bsas2;1 recombinant proteins, spinach SPIol;Bsas3;1 and Arabidopsis ARAth;Bsas3;1 recombinant proteins exhibited preferred substrate specificities for the CAS reaction rather than for the CS reaction, which identified these Bsas3 isoforms as CAS. Immunoblot studies supported this conclusion. This is the first report of the identification of CAS synthase-encoding cDNAs in a living organism. A new nomenclature for CS-like proteins in plants is also proposed. PMID:10889265

  8. Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium.

    Science.gov (United States)

    Jin, Zhehao; Kim, Jin-Hee; Park, Sang Un; Kim, Soo-Un

    2016-12-01

    Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.

  9. Global Derivatives

    DEFF Research Database (Denmark)

    Andersen, Torben Juul

    approaches to dealing in the global business environment." - Sharon Brown-Hruska, Commissioner, Commodity Futures Trading Commission, USA. "This comprehensive survey of modern risk management using derivative securities is a fine demonstration of the practical relevance of modern derivatives theory to risk......""In Global Derivatives: A Strategic Risk Management Perspective", Torben Juul Andersen has succeeded to gather in one book a complete and thorough summary and an easy-to-read explanation of all types of derivative instruments and their background, and their use in modern management of risk......." - Steen Parsholt, Chairman and CEO, Aon Nordic Region. "Andersen has done a wonderful job of developing a comprehensive text that deals with risk management in global markets. I would recommend this book to any student or businessman who has a need to better understand the risks and risk management...

  10. CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Lei, Lei; Singh, Abhishek; Bashline, Logan; Li, Shundai; Yingling, Yaroslava G.; Gu, Ying

    2015-10-06

    Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress.

  11. Nitric-oxide synthase trafficking inducer is a pleiotropic regulator of endothelial cell function and signaling.

    Science.gov (United States)

    Chakraborty, Shreeta; Ain, Rupasri

    2017-04-21

    Endothelial nitric-oxide synthase (eNOS) and its bioactive product, nitric oxide (NO), mediate many endothelial cell functions, including angiogenesis and vascular permeability. For example, vascular endothelial growth factor (VEGF)-mediated angiogenesis is inhibited upon reduction of NO bioactivity both in vitro and in vivo Moreover, genetic disruption or pharmacological inhibition of eNOS attenuates angiogenesis during tissue repair, resulting in delayed wound closure. These observations emphasize that eNOS-derived NO can promote angiogenesis. Intriguingly, eNOS activity is regulated by nitric-oxide synthase trafficking inducer (NOSTRIN), which sequesters eNOS, thereby attenuating NO production. This has prompted significant interest in NOSTRIN's function in endothelial cells. We show here that NOSTRIN affects the functional transcriptome of endothelial cells by down-regulating several genes important for invasion and angiogenesis. Interestingly, the effects of NOSTRIN on endothelial gene expression were independent of eNOS activity. NOSTRIN also affected the expression of secreted cytokines involved in inflammatory responses, and ectopic NOSTRIN overexpression functionally restricted endothelial cell proliferation, invasion, adhesion, and VEGF-induced capillary tube formation. Furthermore, NOSTRIN interacted directly with TNF receptor-associated factor 6 (TRAF6), leading to the suppression of NFκB activity and inhibition of AKT activation via phosphorylation. Interestingly, TNF-α-induced NFκB pathway activation was reversed by NOSTRIN. We found that the SH3 domain of NOSTRIN is involved in the NOSTRIN-TRAF6 interaction and is required for NOSTRIN-induced down-regulation of endothelial cell proteins. These results have broad biological implications, as aberrant NOSTRIN expression leading to deactivation of the NFκB pathway, in turn triggering an anti-angiogenic cascade, might inhibit tumorigenesis and cancer progression. © 2017 by The American Society for

  12. Sphingomyelin synthase 2 deficiency inhibits the induction of murine colitis-associated colon cancer.

    Science.gov (United States)

    Ohnishi, Toshio; Hashizume, Chieko; Taniguchi, Makoto; Furumoto, Hidehiro; Han, Jia; Gao, Rongfen; Kinami, Shinichi; Kosaka, Takeo; Okazaki, Toshiro

    2017-09-01

    Sphingomyelin synthase 2 (SMS2) is the synthetic enzyme of sphingomyelin (SM), which regulates membrane fluidity and microdomain structure. SMS2 plays a role in LPS-induced lung injury and inflammation; however, its role in inflammation-mediated tumorigenesis is unclear. We investigated the effect of SMS2 deficiency on dextran sodium sulfate (DSS)-induced murine colitis and found inhibition of DSS-induced inflammation in SMS2-deficient (SMS2-/-) mice. DSS treatment induced a significant increase in ceramide levels, with a decrease of SM levels in SMS2-/- colon tissue, and demonstrated attenuation of the elevation of both inflammation-related gene expression and proinflammatory cytokines and chemokines, leukocyte infiltration, and MAPK and signal transducer and activator of transcription 3 activation. After undergoing transplantation of wild-type bone marrow, SMS2-/- mice also exhibited inhibition of DSS-induced inflammation in the colon, which suggested that SMS2 deficiency in bone marrow-derived immune cells was not involved in the inhibition of colitis. Finally, in an azoxymethane/DSS-induced cancer model, SMS2 deficiency significantly decreased tumor incidence in the colon. Our results demonstrate that SMS2 deficiency inhibits DSS-induced colitis and subsequent colitis-associated colon cancer via inhibition of colon epithelial cell-mediated inflammation; therefore, inhibition of SMS2 may be a potential therapeutic target for human colitis and colorectal cancer.-Ohnishi, T., Hashizume, C., Taniguchi, M., Furumoto, H., Han, J., Gao, R., Kinami, S., Kosaka, T., Okazaki, T. Sphingomyelin synthase 2 deficiency inhibits the induction of murine colitis-associated colon cancer. © FASEB.

  13. Triazolopyrimidines as a New Herbicidal Lead for Combating Weed Resistance Associated with Acetohydroxyacid Synthase Mutation.

    Science.gov (United States)

    Liu, Yu-Chao; Qu, Ren-Yu; Chen, Qiong; Yang, Jing-Fang; Cong-Wei, Niu; Zhen, Xi; Yang, Guang-Fu

    2016-06-22

    Acetohydroxyacid synthase (AHAS; also known as acetolactate synthase; EC 2.2.1.6, formerly EC 4.1.3.18) is the first common enzyme in the biosynthetic pathway leading to the branched-chain amino acids in plants and a wide range of microorganisms. Weed resistance to AHAS-inhibiting herbicides, increasing at an exponential rate, is becoming a global problem and leading to an urgent demand of developing novel compounds against both resistant and wild AHAS. In the present work, a series of novel 2-aroxyl-1,2,4-triazolopyrimidine derivatives (a total of 55) were designed and synthesized with the aim to discover an antiresistant lead compound. Fortunately, the screening results indicated that many of the newly synthesized compounds showed a better, even excellent, inhibition effect against both the wild-type Arabidopsis thaliana AHAS and P197L mutants. Among them, compounds 5-3 to 5-17, compounds 5-19 to 5-26, compounds 5-28 to 5-45, and compound 5-48 have the lower values of resistance factor (RF) and display a potential power to overcome resistance associated with the P197L mutation in the enzyme levels. Further greenhouse in vivo assay showed that compounds 5-15 and 5-20 displayed "moderate" to "good" herbicidal activity against both the wild type-and the resistant (P197L mutation) Descurainia sophia, even at a rate as low as 0.9375 (g of ai/ha). The above results indicated that these two compounds could be used as new leads for the future development of antiresistance herbicides.

  14. AcEST: BP913978 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 34 5.0 tr|A3XJS4|A3XJS4_9FLAO Pseudouridine synthase OS=Leeuwenhoekiell... 33 6.5...udouridine synthase OS=Leeuwenhoekiella blandensis MED217 GN=MED217_04192 PE=3 SV=1 Length = 265 Score = 33.

  15. AcEST: BP915836 [AcEST

    Lifescience Database Archive (English)

    Full Text Available udouridine synthase OS=Leeuwenhoekiell... 33 5.8 tr|A8L3M4|A8L3M4_FRASN Acyltransferase 3 OS=Frankia sp. (st...9FLAO Pseudouridine synthase OS=Leeuwenhoekiella blandensis MED217 GN=MED217_04192 PE=3 SV=1 Length = 265 Sc

  16. Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling.

    Science.gov (United States)

    Dave, Bhuvanesh; Granados-Principal, Sergio; Zhu, Rui; Benz, Stephen; Rabizadeh, Shahrooz; Soon-Shiong, Patrick; Yu, Ke-Da; Shao, Zhimin; Li, Xiaoxian; Gilcrease, Michael; Lai, Zhao; Chen, Yidong; Huang, Tim H-M; Shen, Haifa; Liu, Xuewu; Ferrari, Mauro; Zhan, Ming; Wong, Stephen T C; Kumaraswami, Muthiah; Mittal, Vivek; Chen, Xi; Gross, Steven S; Chang, Jenny C

    2014-06-17

    We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.

  17. Identification and Heterologous Expression of the Topopyrone Nonaketide Synthase Gene from Phoma sp.

    Science.gov (United States)

    Kashiwa, Nobuyuki; Ebizuka, Yutaka; Fujii, Isao

    2016-01-01

    Non-reducing iterative type I polyketide synthase genes, pnk1 and pnk2, were cloned from the fungus Phoma sp. BAUA2861, which produces the topoisomerase I inhibitors, topopyrones A to D. Heterologous expression of these polyketide synthase genes under the α-amylase promoter in Aspergillus oryzae was carried out to identify their functions. The pnk2 transformant produced topopyrones C, D, and haematommone. Therefore, the pnk2 gene was found to encode for the topopyrone nonaketide synthase.

  18. Derivative chameleons

    Energy Technology Data Exchange (ETDEWEB)

    Noller, Johannes, E-mail: johannes.noller08@imperial.ac.uk [Theoretical Physics, Blackett Laboratory, Imperial College London, Prince Consort Road, London, SW7 2BZ (United Kingdom)

    2012-07-01

    We consider generalized chameleon models where the conformal coupling between matter and gravitational geometries is not only a function of the chameleon field φ, but also of its derivatives via higher order co-ordinate invariants (such as ∂{sub μ}φ∂{sup μ}φ,□φ,...). Specifically we consider the first such non-trivial conformal factor A(φ,∂{sub μ}φ∂{sup μ}φ). The associated phenomenology is investigated and we show that such theories have a new generic mass-altering mechanism, potentially assisting the generation of a sufficiently large chameleon mass in dense environments. The most general effective potential is derived for such derivative chameleon setups and explicit examples are given. Interestingly this points us to the existence of a purely derivative chameleon protected by a shift symmetry for φ → φ+c. We also discuss potential ghost-like instabilities associated with mass-lifting mechanisms and find another, mass-lowering and instability-free, branch of solutions. This suggests that, barring fine-tuning, stable derivative models are in fact typically anti-chameleons that suppress the field's mass in dense environments. Furthermore we investigate modifications to the thin-shell regime and prove a no-go theorem for chameleon effects in non-conformal geometries of the disformal type.

  19. Hydrophilic, Potent, and Selective 7-Substituted 2-Aminoquinolines as Improved Human Neuronal Nitric Oxide Synthase Inhibitors.

    Science.gov (United States)

    Pensa, Anthony V; Cinelli, Maris A; Li, Huiying; Chreifi, Georges; Mukherjee, Paramita; Roman, Linda J; Martásek, Pavel; Poulos, Thomas L; Silverman, Richard B

    2017-08-24

    Neuronal nitric oxide synthase (nNOS) is a target for development of antineurodegenerative agents. Most nNOS inhibitors mimic l-arginine and have poor bioavailability. 2-Aminoquinolines showed promise as bioavailable nNOS inhibitors but suffered from low human nNOS inhibition, low selectivity versus human eNOS, and significant binding to other CNS targets. We aimed to improve human nNOS potency and selectivity and reduce off-target binding by (a) truncating the original scaffold or (b) introducing a hydrophilic group to interrupt the lipophilic, promiscuous pharmacophore and promote interaction with human nNOS-specific His342. We synthesized both truncated and polar 2-aminoquinoline derivatives and assayed them against recombinant NOS enzymes. Although aniline and pyridine derivatives interact with His342, benzonitriles conferred the best rat and human nNOS inhibition. Both introduction of a hydrophobic substituent next to the cyano group and aminoquinoline methylation considerably improved isoform selectivity. Most importantly, these modifications preserved Caco-2 permeability and reduced off-target CNS binding.

  20. Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Hagen, A; Poust, S; De Rond, T; Fortman, JL; Katz, L; Petzold, CJ; Keasling, JD

    2015-10-26

    Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design–build–test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS’ first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to “debug” PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.

  1. Electricity derivatives

    CERN Document Server

    Aïd, René

    2015-01-01

    Offering a concise but complete survey of the common features of the microstructure of electricity markets, this book describes the state of the art in the different proposed electricity price models for pricing derivatives and in the numerical methods used to price and hedge the most prominent derivatives in electricity markets, namely power plants and swings. The mathematical content of the book has intentionally been made light in order to concentrate on the main subject matter, avoiding fastidious computations. Wherever possible, the models are illustrated by diagrams. The book should allow prospective researchers in the field of electricity derivatives to focus on the actual difficulties associated with the subject. It should also offer a brief but exhaustive overview of the latest techniques used by financial engineers in energy utilities and energy trading desks.

  2. Citrate synthase purified from Tetrahymena mitochondria is identical with Tetrahymena 14-nm filament protein.

    Science.gov (United States)

    Kojima, H; Chiba, J; Watanabe, Y; Numata, O

    1995-07-01

    A 14-nm filament protein (designated as 49K protein) was purified from a ciliated protozoan, Tetrahymena, using the polymerization and depolymerization procedure. Previous studies in our laboratory showed that its primary structure shared a high sequence identity with citrate synthases known so far and that the 49K protein possessed citrate synthase activity. To ascertain whether or not Tetrahymena's mitochondrial citrate synthase is identical to the 49K protein, citrate synthase was purified from Tetrahymena mitochondria using ammonium sulfate fractionation, Butyl-Toyopearl and SP-Toyopearl column chromatographies, based on monitoring of the enzymatic activity. The molecular weight of the purified citrate synthase was estimated to be 49 kDa, as was that of the 49K protein and the enzyme cross-reacted with an anti-49K protein antiserum. The purified citrate synthase showed much the same optimum pH, optimum KCl concentration, effects of substrate concentrations (acetyl-CoA and oxaloacetate), and inhibitory effect by ATP as those of purified 49K protein. Furthermore, an anti-49K protein monoclonal antibody strongly suppressed the enzymatic activity of the purified citrate synthase. Thus, we suggest that mitochondrial citrate synthase and the 49K protein are identical and that the 49K protein has dual functions in the cytoskeleton in cytoplasm and as a TCA cycle enzyme, citrate synthase, in mitochondria.

  3. Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha[OPEN

    Science.gov (United States)

    Zhuang, Xun; Jiang, Zuodong; Jia, Qidong; Babbitt, Patricia C.

    2016-01-01

    Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases. PMID:27650333

  4. Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Ha, S B; Smith, A P; Howden, R; Dietrich, W M; Bugg, S; O'Connell, M J; Goldsbrough, P B; Cobbett, C S

    1999-06-01

    Phytochelatins (PCs), a family of heavy metal-inducible peptides important in the detoxification of heavy metals, have been identified in plants and some microorganisms, including Schizosaccharomyces pombe, but not in animals. PCs are synthesized enzymatically from glutathione (GSH) by PC synthase in the presence of heavy metal ions. In Arabidopsis, the CAD1 gene, identified by using Cd-sensitive, PC-deficient cad1 mutants, has been proposed to encode PC synthase. Using a positional cloning strategy, we have isolated the CAD1 gene. Database searches identified a homologous gene in S. pombe, and a mutant with a targeted deletion of this gene was also Cd sensitive and PC deficient. Extracts of Escherichia coli cells expressing a CAD1 cDNA or the S. pombe gene catalyzing GSH-dependent, heavy metal-activated synthesis of PCs in vitro demonstrated that both genes encode PC synthase activity. Both enzymes were activated by a range of metal ions. In contrast, reverse transcription-polymerase chain reaction experiments showed that expression of the CAD1 mRNA is not influenced by the presence of Cd. A comparison of the two predicted amino acid sequences revealed a highly conserved N-terminal region, which is presumed to be the catalytic domain, and a variable C-terminal region containing multiple Cys residues, which is proposed to be involved in activation of the enzyme by metal ions. Interestingly, a similar gene was identified in the nematode, Caenorhabditis elegans, suggesting that PCs may also be expressed in some animal species.

  5. A transcribed polyketide synthase gene from Xanthoria elegans.

    Science.gov (United States)

    Brunauer, Georg; Muggia, Lucia; Stocker-Wörgötter, Elfie; Grube, Martin

    2009-01-01

    We characterize the transcript of a polyketide synthase gene (PKS) from the cultured mycobiont of Xanthoria elegans (XePKS1) using SMART-rapid amplification of cDNA ends (RACE) cDNA synthesis. Sequence analysis of the cloned cDNA reveals an open reading frame of 2144 amino acid residues. It contains features of a non-reducing fungal type I PKS with an N-terminal starter unit: acyl carrier protein (ACP) transacetylase domain, ketosynthase, acyltransferase, two acyl carrier protein domains, and a thioesterase domain. XePKS1 was the only paralogue detected in the cDNA and the genomic DNA of the cultured X. elegans mycobiont by using a degenerate PCR approach targeted at the conserved regions of non-reducing type I PKS genes. The hypothetical protein is phylogenetically related to genes that are basal to a clade of dihydroxynaphthalene synthases (non-reducing clade II) and anthraquinone type synthases of non-lichenized fungi (non-reducing clade I). According to hplc and tlc analyses, the cultured mycobiont exclusively produced anthraquinones and its precursors. Therefore, we discuss whether the characterized paralogue is involved in anthraquinone production, which raises the possibility of a paraphyletic origin of lichen anthraquinone biosynthesis. The cDNA of XePKS1 was the first full-length coding sequence of a lichen PKS to be published. This proves SMART RACE to be a suitable tool for obtaining full-length coding sequences of genes from environmental samples and organisms, which are hardly amenable to standard molecular approaches or genomic sequencing.

  6. Role of nitric oxide synthase inhibition in the acute hypertensive response to intracerebroventricular cadmium

    Science.gov (United States)

    Demontis, Maria Piera; Varoni, Maria Vittoria; Volpe, Anna Rita; Emanueli, Costanza; Madeddu, Paolo

    1998-01-01

    In the rat, intracerebroventricular (i.c.v.) injection of cadmium, a pollutant with long biological half-life, causes a sustained increase in blood pressure at doses that are ineffective by peripheral route. Since cadmium inhibits calcium-calmodulin constitutive nitric oxide (NO) synthase in cytosolic preparations of rat brain, this mechanism may be responsible for the acute pressor action of this heavy metal.To test this possibility, we evaluated the effect of i.c.v. injection of 88 nmol cadmium in normotensive unanaesthetized Wistar rats, which were i.c.v. pre-treated with: (1) saline (control), (2) L-arginine (L-Arg), to increase the availability of substrate for NO biosynthesis, (3) D-arginine (D-Arg), (4) 3-[4-morpholinyl]-sydnonimine-hydrochloride (SIN-1), an NO donor, or (5) CaCl2, a cofactor of brain calcium-calmodulin-dependent cNOSI. In additional experiments, the levels of L-citrulline (the stable equimolar product derived from enzymatic cleavage of L-Arg by NO synthase) were determined in the brain of vehicle- or cadmium-treated rats.The pressor response to cadmium reached its nadir at 5 min (43±4 mmHg) and lasted over 20 min in controls. L-Citrulline/protein content was reduced from 35 up to 50% in the cerebral cortex, pons, hippocampus, striatus, hypothalamus (P<0.01) of cadmium-treated rats compared with controls. Central injection of NG nitro-L-arginine-methylester (L-NAME) also reduced the levels of L-citrulline in the brain.Both the magnitude and duration of the response were attenuated by 1.21 and 2.42 μmol SIN-1 (32±3 and 15±4 mmHg, P<0.05), or 1 μmol CaCl2 (6±4 mmHg, P<0.05). Selectivity of action exerted by SIN-1 was confirmed by the use of another NO donor, S-nitroso-N-acetyl-penicillamine (SNAP). Both L-Arg and D-Arg caused a mild but significant attenuation in the main phase of the pressor response evoked by cadmium. However, only L-Arg reduced the magnitude of the delayed, pressor response. Despite their similarity in

  7. Localization of nitric oxide synthase in the adult rat brain.

    Science.gov (United States)

    Rodrigo, J; Springall, D R; Uttenthal, O; Bentura, M L; Abadia-Molina, F; Riveros-Moreno, V; Martínez-Murillo, R; Polak, J M; Moncada, S

    1994-07-29

    The distribution of the immunoreactivity to nitric oxide synthase has been examined from rostral to caudal areas of the rat central nervous system using light microscopy. Endogenous nitric oxide synthase was located using a specific polyclonal antiserum, produced against affinity purified nitric oxide synthase from whole rat brain, following the avidin-biotin peroxidase procedure. Immunoreactive cell bodies and processes showed a widespread distribution in the brain. In the telencephalon, immunoreactive structures were distributed in all areas of the cerebral cortex, the ventral endopiriform nucleus and claustrum, the main and accessory olfactory bulb, the anterior and posterior olfactory nuclei, the precommisural hippocampus, the taenia tecta, the nucleus accumbens, the stria terminalis, the caudate putamen, the olfactory tubercle and islands of Calleja, septum, globus pallidus and substantia innominata, hippocampus and amygdala. In the diencephalon, the immunoreactivity was largely found in both the hypothalamus and thalamus. In the hypothalamus, immunoreactive cell bodies were characteristically located in the perivascular-neurosecretory systems and mamillary bodies. In addition, immunoreactive nerve fibres were detected in the median eminence of the infundibular stem. The mesencephalon showed nitric oxide synthase immunoreactivity in the ventral tegmental area, the interpeduncular nucleus, the rostral linear nucleus of the raphe and the dorsal raphe nucleus. Immunoreactive structures were also found in the nuclei of the central grey, the peripeduncular nucleus and substantia nigra pars lateralis, the geniculate nucleus and in the superior and inferior colliculi. The pons displayed immunoreactive structures principally in the pedunculopontine and laterodorsal tegmental nuclei, the ventral tegmental nucleus, the reticulotegmental pontine nucleus, the parabrachial nucleus and locus coeruleus. In the medulla oblongata, immunoreactive neurons and processes were

  8. [Localization of nitric oxide synthase in the chicken vestibular system].

    Science.gov (United States)

    Nie, Guohui; Wang, Jibao

    2002-08-01

    To locate nitric oxide synthase (NOS) in the chicken vestibular system. The frozen section were processed for NADPH-d histochemistry in a solution containing NADPH and nitroblue tetnazolium (NBT) to demonstrate NOS positive reactivity. NOS positive staining, black-blue in color, was seen at the nerve ending, nerve fibers of the utricul and saculla and ampiculium. Ganglion cells had different activity. The shape of the cells seems to be round or oral. Collectively, data indicate the presence of active NOS in these tissue and suggest modulation of vestibular neurotransmission by nitric oxide.

  9. Inhibition of (+)-aristolochene synthase with iminium salts resembling eudesmane cation.

    Science.gov (United States)

    Faraldos, Juan A; Allemann, Rudolf K

    2011-03-04

    Trigonal iminium halides of (4aS,7S)-1,4a-dimethyl- and (4aS,7S)-4a-methyl-7-(prop-1-en-2-yl)-2,3,4,4a,5,6,7,8-octahydroquinolinium ions, aimed to mimic transition states associated with the aristolochene synthase-catalyzed cyclization of (-)-germacrene A to eudesmane cation, were evaluated under standard kinetic steady-state conditions. In the presence of inorganic diphosphate, these analogues were shown to competitively inhibit the enzyme, suggesting a stabilizing role for the diphosphate leaving group in this apparently endothermic transformation.

  10. Complex derivatives

    Science.gov (United States)

    Battiston, Stefano; Caldarelli, Guido; Georg, Co-Pierre; May, Robert; Stiglitz, Joseph

    2013-03-01

    The intrinsic complexity of the financial derivatives market has emerged as both an incentive to engage in it, and a key source of its inherent instability. Regulators now faced with the challenge of taming this beast may find inspiration in the budding science of complex systems.

  11. Eugenol Production in Achenes and Receptacles of Strawberry Fruits Is Catalyzed by Synthases Exhibiting Distinct Kinetics1[W][OPEN

    Science.gov (United States)

    Aragüez, Irene; Osorio, Sonia; Hoffmann, Thomas; Rambla, José Luis; Medina-Escobar, Nieves; Granell, Antonio; Botella, Miguel Ángel; Schwab, Wilfried; Valpuesta, Victoriano

    2013-01-01

    Eugenol is a volatile that serves as an attractant for pollinators of flowers, acts as a defense compound in various plant tissues, and contributes to the aroma of fruits. Its production in a cultivated species such as strawberry (Fragaria × ananassa), therefore, is important for the viability and quality of the fruit. We have identified and functionally characterized three strawberry complementary DNAs (cDNAs) that encode proteins with high identity to eugenol synthases from several plant species. Based on a sequence comparison with the wild relative Fragaria vesca, two of these cDNAs, FaEGS1a and FaEGS1b, most likely correspond to transcripts derived from allelic gene variants, whereas the third cDNA, FaEGS2, corresponds to a different gene. Using coniferyl acetate as a substrate, FaEGS1a and FaEGS1b catalyze the in vitro formation of eugenol, while FaEGS2 catalyzes the formation of eugenol and also of isoeugenol with a lower catalytic efficiency. The expression of these genes is markedly higher in the fruit than in other tissues of the plant, with FaEGS1a and FaEGS1b mostly expressed in the green achenes, whereas FaEGS2 expression is almost restricted to the red receptacles. These expression patterns correlate with the eugenol content, which is highest in the achene at the green stage and in the receptacle at the red stage. The transient expression of the corresponding cDNAs in strawberry fruit and the subsequent volatile analyses confirm FaEGSs as genuine eugenol synthases in planta. These results provide new insights into the diversity of phenylpropene synthases in plants. PMID:23983228

  12. A Tandem Array of ent-Kaurene Synthases in Maize with Roles in Gibberellin and More Specialized Metabolism.

    Science.gov (United States)

    Fu, Jingye; Ren, Fei; Lu, Xuan; Mao, Hongjie; Xu, Meimei; Degenhardt, Jörg; Peters, Reuben J; Wang, Qiang

    2016-02-01

    While most commonly associated with its role in gibberellin phytohormone biosynthesis, ent-kaurene also serves as an intermediate in more specialized diterpenoid metabolism, as exemplified by the more than 800 known derived natural products. Among these are the maize kauralexins. However, no ent-kaurene synthases (KSs) have been identified from maize. The maize gibberellin-deficient dwarf-5 (d5) mutant has been associated with a loss of KS activity. The relevant genetic lesion has been previously mapped, and was found here to correlate with the location of the KS-like gene ZmKSL3. Intriguingly, this forms part of a tandem array with two other terpene synthases (TPSs). Although one of these, ZmTPS1, has been previously reported to encode a sesquiterpene synthase, and both ZmTPS1 and that encoded by the third gene, ZmKSL5, have lost the N-terminal γ-domain prototypically associated with KS(L)s, all three genes fall within the KS(L) or TPS-e subfamily. Here it is reported that all three genes encode enzymes that are targeted to the plastid in planta, where diterpenoid biosynthesis is initiated, and which all readily catalyze the production of ent-kaurene. Consistent with the closer phylogenetic relationship of ZmKSL3 with previously identified KSs from cereals, only transcription of this gene is affected in d5 plants. On the other hand, the expression of all three of these genes is inducible, suggesting a role in more specialized metabolism, such as that of the kauralexins. Thus, these results clarify not only gibberellin phytohormone, but also diterpenoid phytoalexin biosynthesis in this important cereal crop plant. © 2016 American Society of Plant Biologists. All Rights Reserved.

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

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

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

  14. Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.

    Science.gov (United States)

    Wilson, Wayne A; Pradhan, Prajakta; Madhan, Nayasha; Gist, Galen C; Brittingham, Andrew

    2017-07-01

    Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily.

    Science.gov (United States)

    Martin, Diane M; Fäldt, Jenny; Bohlmann, Jörg

    2004-08-01

    Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (-)-limonene synthase, (-)-alpha/beta-pinene synthase, and (-)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-alpha-farnesene synthase, and E-alpha-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.

  16. In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

    Directory of Open Access Journals (Sweden)

    Jose Antonio Cuesta-Seijo

    2016-01-01

    Full Text Available Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs. While the overall starch synthase (SS reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

  17. Subcellular targeting domains of sphingomyelin synthase 1 and 2.

    Science.gov (United States)

    Yeang, Calvin; Ding, Tingbo; Chirico, William J; Jiang, Xian-Cheng

    2011-12-14

    Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. Furthermore, its product SM has been implicated in atherogenic processes such as retention of lipoproteins in the blood vessel intima. There are two mammalian sphingomyelin synthases: SMS1 and SMS2. SMS1 is found exclusively in the Golgi at steady state, whereas SMS2 exists in the Golgi and plasma membrane. Conventional motifs responsible for protein targeting to the plasma membrane or Golgi are either not present in, or unique to, SMS1 and SMS2. In this study, we examined how SMS1 and SMS2 achieve their respective subcellular localization patterns. Brefeldin A treatment prevented SMS1 and SMS2 from exiting the ER, demonstrating that they transit through the classical secretory pathway. We created truncations and chimeras of SMS1 and SMS2 to define their targeting signals. We found that SMS1 contains a C-terminal Golgi targeting signal and that SMS2 contains a C-terminal plasma membrane targeting signal.

  18. Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Houghton, P.J.; Germain, G.S.; Hazelton, B.J.; Pennington, J.W.; Houghton, J.A. (Saint Jude Children' s Research Hospital, Memphis, TN (USA))

    1989-02-01

    GC{sub 3}/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methane sulfonate, and three clones deficient in thymidylate synthase activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS{sup {minus}}c{sub 1} and TS{sup {minus}}c{sub 3}), whereas one clone (TS{sup {minus}}c{sub 2}) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2{prime}-deoxyuridine 5{prime}-monophosphate-binding capacity and with incorporation of 2{prime}-deoxy(6-{sup 3}H)uridine into DNA. In the presence of optimal thymidine, growth rates were only 5-18% that of the parental clone (GC{sub 3}/c1), which grew equally well in thymidine-deficient or -replete medium. Analysis of poly(A){sup +} RNA showed normal levels of a 1.6-kilobase transcript in TS{sup {minus}}c{sub 1} and TS{sup minus}c{sub 2} but decreased levels in TS{sup {minus}}c{sub 3}. Clone TS{sup minus}c{sub 3} was 32-, 750-, and >100,000-fold more resistant than the parental clone to 5-fluorouracil, 5-fluoro-2{prime}-deoxyuridine, and methotrexate, respectively. When inoculated into athymic nude mice, each TS{sup {minus}} clone produced tumors, demonstrating continued ability to proliferate in vivo.

  19. Thymidylate synthase enhancer region: Novel allele in Indians.

    Science.gov (United States)

    Dhawan, Dipali; Padh, Harish

    2017-02-01

    Thymidylate synthase (TS) is the major target for fluoropyrimidine drugs like 5-Fluorouracil (5-FU). There are polymorphic tandem repeats in the TYMS gene enhancer region (TSER). The number of tandem repeats varies in different populations. The aim of this study was to determine the frequencies of the TSER tandem repeats (rs34743033) and compare the observed frequencies with those of other populations. This study genotyped 350 healthy individuals by Polymerase Chain Reaction (PCR). A novel allele *1 (only a single repeat) was observed in four individuals, the individuals were heterozygous (TSER*1/*2) for TYMS. Another variant rs2853542 affecting the expression of Thymidylate synthase was also analysed. The observed genotype frequencies were compared with frequencies observed in other populations for understanding differences between various population groups. There was a statistically significant difference between Indians and Chinese, Kenyans, Ghanians, African-Americans, Americans of European Ancestry, British, Hungarians, Turkish, Australians and Brazilians. This study identified a novel single repeat in the TYMS gene which might have an impact on the expression of this gene, which needs to be confirmed by functional studies.

  20. CTP limitation increases expression of CTP synthase in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jørgensen, C.M.; Hammer, Karin; Martinussen, Jan

    2003-01-01

    CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which beta-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold...... syntheses are inhibited. Expression of pyrG responds only to the cellular level of CTP, since expression of pyrG has no correlation to alterations in UTP, GTP, and ATP pool sizes. In the untranslated pyrG leader sequence a potential terminator structure can be identified, and this structure is required...... on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing...

  1. Ack kinase regulates CTP synthase filaments during Drosophila oogenesis.

    Science.gov (United States)

    Strochlic, Todd I; Stavrides, Kevin P; Thomas, Sam V; Nicolas, Emmanuelle; O'Reilly, Alana M; Peterson, Jeffrey R

    2014-11-01

    The enzyme CTP synthase (CTPS) dynamically assembles into macromolecular filaments in bacteria, yeast, Drosophila, and mammalian cells, but the role of this morphological reorganization in regulating CTPS activity is controversial. During Drosophila oogenesis, CTPS filaments are transiently apparent in ovarian germline cells during a period of intense genomic endoreplication and stockpiling of ribosomal RNA. Here, we demonstrate that CTPS filaments are catalytically active and that their assembly is regulated by the non-receptor tyrosine kinase DAck, the Drosophila homologue of mammalian Ack1 (activated cdc42-associated kinase 1), which we find also localizes to CTPS filaments. Egg chambers from flies deficient in DAck or lacking DAck catalytic activity exhibit disrupted CTPS filament architecture and morphological defects that correlate with reduced fertility. Furthermore, ovaries from these flies exhibit reduced levels of total RNA, suggesting that DAck may regulate CTP synthase activity. These findings highlight an unexpected function for DAck and provide insight into a novel pathway for the developmental control of an essential metabolic pathway governing nucleotide biosynthesis. © 2014 The Authors.

  2. Molecular and functional evolution of the fungal diterpene synthase genes.

    Science.gov (United States)

    Fischer, Marc J C; Rustenhloz, Camille; Leh-Louis, Véronique; Perrière, Guy

    2015-10-19

    Terpenes represent one of the largest and most diversified families of natural compounds and are used in numerous industrial applications. Terpene synthase (TPS) genes originated in bacteria as diterpene synthase (di-TPS) genes. They are also found in plant and fungal genomes. The recent availability of a large number of fungal genomes represents an opportunity to investigate how genes involved in diterpene synthesis were acquired by fungi, and to assess the consequences of this process on the fungal metabolism. In order to investigate the origin of fungal di-TPS, we implemented a search for potential fungal di-TPS genes and identified their presence in several unrelated Ascomycota and Basidiomycota species. The fungal di-TPS phylogenetic tree is function-related but is not associated with the phylogeny based on housekeeping genes. The lack of agreement between fungal and di-TPS-based phylogenies suggests the presence of Horizontal Gene Transfer (HGTs) events. Further evidence for HGT was provided by conservation of synteny of di-TPS and neighbouring genes in distantly related fungi. The results obtained here suggest that fungal di-TPSs originated from an ancient HGT event of a single di-TPS gene from a plant to a fungus in Ascomycota. In fungi, these di-TPSs allowed for the formation of clusters consisting in di-TPS, GGPPS and P450 genes to create functional clusters that were transferred between fungal species, producing diterpenes acting as hormones or toxins, thus affecting fungal development and pathogenicity.

  3. An uncultivated crenarchaeota contains functional bacteriochlorophyll a synthase.

    Science.gov (United States)

    Meng, Jun; Wang, Fengping; Wang, Feng; Zheng, Yanping; Peng, Xiaotong; Zhou, Huaiyang; Xiao, Xiang

    2009-01-01

    A fosmid clone 37F10 containing an archaeal 16S rRNA gene was screened out from a metagenomic library of Pearl River sediment, southern China. Sequence analysis of the 35 kb inserted fragment of 37F10 found that it contains a single 16S rRNA gene belonging to Miscellaneous Crenarchaeotal Group (MCG) and 36 open reading frames (ORFs). One ORF (orf11) encodes putative bacteriochlorophyll a synthase (bchG) gene. Bacteriochlorophyll a synthase gene has never been reported in a member of the domain Archaea, in accordance with the fact that no (bacterio)-chlorophyll has ever been detected in any cultivated archaea. The putative archaeal bchG (named as ar-bchG) was cloned and heterologously expressed in Escherichia coli. The protein was found to be capable of synthesizing bacteriochlorophyll a by esterification of bacteriochlorophyllide a with phytyl diphosphate or geranylgeranyl diphosphate. Furthermore, phylogenetic analysis clearly indicates that the ar-bchG diverges before the bacterial bchGs. Our results for the first time demonstrate that a key and functional enzyme for bacteriochlorophyll a biosynthesis does exist in Archaea.

  4. Tomato linalool synthase is induced in trichomes by jasmonic acid

    Science.gov (United States)

    van Schie, Chris C. N.; Haring, Michel A.

    2007-01-01

    Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato. PMID:17440821

  5. Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production.

    Science.gov (United States)

    Miller, R T; Martásek, P; Roman, L J; Nishimura, J S; Masters, B S

    1997-12-09

    Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CaM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of L-arginine to L-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O2.-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O2.- during catalytic turnover in the presence of L-arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O2.- production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O2.- production by nNOS and the CYS-331 mutant is CaM-dependent and that O2.- production can be modulated by substrates and inhibitors of nNOS. O2.- was also produced by the reductase domain of nNOS; however, it did not display the same CaM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O2.-, but that the reductase domain is both necessary and sufficient for O2.- production.

  6. Review Article: The Role of Nitric Oxide Synthase in Post-Operative ...

    African Journals Online (AJOL)

    Nitric Oxide (NO) is produced by nitric oxide synthase (NOS) isoenzymes. Inducible nitric oxide synthase (iNOS) is not a normal cellular constitute. It is expressed by cytokines and non-cytokines e.g. fasting, trauma, intravenous glucose, and lipid infusion, which are encountered in surgical operations. Review of current ...

  7. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    -transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  8. High diversity of polyketide synthase genes and the melanin biosynthesis gene cluster in Penicillium marneffei.

    Science.gov (United States)

    Woo, Patrick C Y; Tam, Emily W T; Chong, Ken T K; Cai, James J; Tung, Edward T K; Ngan, Antonio H Y; Lau, Susanna K P; Yuen, Kwok-Yung

    2010-09-01

    Despite the unique phenotypic properties and clinical importance of Penicillium marneffei, the polyketide synthase genes in its genome have never been characterized. Twenty-three putative polyketide synthase genes and two putative polyketide synthase nonribosomal peptide-synthase hybrid genes were identified in the P. marneffei genome, a diversity much higher than found in other pathogenic thermal dimorphic fungi, such as Histoplasma capsulatum (one polyketide synthase gene) and Coccidioides immitis (10 polyketide synthase genes). These genes were evenly distributed on the phylogenetic tree with polyketide synthase genes of Aspergillus and other fungi, indicating that the high diversity was not a result of lineage-specific gene expansion through recent gene duplication. The melanin-biosynthesis gene cluster had gene order and orientations identical to those in the Talaromyces stipitatus (a teleomorph of Penicillium emmonsii) genome. Phylogenetically, all six genes of the melanin-biosynthesis gene cluster in P. marneffei were also most closely related to those in T. stipitatus, with high bootstrap supports. The polyketide synthase gene of the melanin-biosynthesis gene cluster (alb1) in P. marneffei was knocked down, which was accompanied by loss of melanin pigment production and reduced ornamentation in conidia. The survival of mice challenged with the alb1 knockdown mutant was significantly better than those challenged with wild-type P. marneffei (P melanin-biosynthesis gene cluster contributed to virulence through decreased susceptibility to killing by hydrogen peroxide. © 2010 The Authors Journal compilation © 2010 FEBS.

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  10. Selectivity of the surface binding site (SBS) on barley starch synthase I

    DEFF Research Database (Denmark)

    Wilkens, Casper; Cuesta-Seijo, Jose A.; Palcic, Monica

    2014-01-01

    Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was ...

  11. The polyketide components of waxes and the Cer-cqu gene cluster encoding a novel polyketide synthase, the β-diketone synthase, DKS

    DEFF Research Database (Denmark)

    von Wettstein, Penny

    2017-01-01

    Cer-cqu gene cluster encoding a novel polyketide synthase-the β-diketone synthase (DKS), a lipase/carboxyl transferase, and a P450 hydroxylase, respectively, establishes a new, major pathway for the synthesis of plant waxes. The major product is a β-diketone (14,16-hentriacontane) aliphatic that forms...... long, thin crystalline tubes. A pathway branch leads to the formation of esterified alkan-2-ols....

  12. Acyl-ACP substrate recognition in Burkholderia mallei BmaI1 acyl-homoserine lactone synthase.

    Science.gov (United States)

    Montebello, Aubrey N; Brecht, Ryan M; Turner, Remington D; Ghali, Miranda; Pu, Xinzhu; Nagarajan, Rajesh

    2014-10-07

    The acyl-homoserine lactone (AHL) autoinducer mediated quorum sensing regulates virulence in several pathogenic bacteria. The hallmark of an efficient quorum sensing system relies on the tight specificity in the signal generated by each bacterium. Since AHL signal specificity is derived from the acyl-chain of the acyl-ACP (ACP = acyl carrier protein) substrate, AHL synthase enzymes must recognize and react with the native acyl-ACP with high catalytic efficiency while keeping reaction rates with non-native acyl-ACPs low. The mechanism of acyl-ACP substrate recognition in these enzymes, however, remains elusive. In this study, we investigated differences in catalytic efficiencies for shorter and longer chain acyl-ACP substrates reacting with an octanoyl-homoserine lactone synthase Burkholderia mallei BmaI1. With the exception of two-carbon shorter hexanoyl-ACP, the catalytic efficiencies of butyryl-ACP, decanoyl-ACP, and octanoyl-CoA reacting with BmaI1 decreased by greater than 20-fold compared to the native octanoyl-ACP substrate. Furthermore, we also noticed kinetic cooperativity when BmaI1 reacted with non-native acyl-donor substrates. Our kinetic data suggest that non-native acyl-ACP substrates are unable to form a stable and productive BmaI1·acyl-ACP·SAM ternary complex and are thus effectively discriminated by the enzyme. These results offer insights into the molecular basis of substrate recognition for the BmaI1 enzyme.

  13. Emission and accumulation of monoterpene and the key terpene synthase (TPS associated with monoterpene biosynthesis in Osmanthus fragrans Lour.

    Directory of Open Access Journals (Sweden)

    Xaingling eZeng

    2016-01-01

    Full Text Available Osmanthus fragrans is an ornamental and economically important plant known for its magnificent aroma, and the most important aroma-active compounds in flowers are monoterpenes, mainly β-ocimene, linalool and linalool derivatives. To understand the molecular mechanism of monoterpene production, we analyzed the emission and accumulation patterns of these compounds and the transcript levels of the genes involved in their biosynthesis in two O. fragrans cultivars during flowering stages. The results showed that both emission and accumulation of monoterpenes varied with flower development and glycosylation had an important impact on floral linalool emission during this process. Gene expression demonstrated that the transcript levels of terpene synthase (TPS genes probably played a key role in monoterpene production, compared to the genes in the MEP pathway. Phylogenetic analysis showed that OfTPS1 and OfTPS2 belonged to a TPS-g subfamily, and OfTPS3 and OfTPS4 clustered into a TPS-b subfamily. Their transient and stable expression in tobacco leaves suggested that OfTPS1 and OfTPS2 exclusively produced β-linalool, and trans-β-ocimene was the sole product from OfTPS3, while OfTPS4, a predictive sesquiterpene synthase, produced α-farnesene. These results indicate that OfTPS1, OfTPS2 and OfTPS3 could account for the major floral monoterpenes, linalool and trans-β-ocimene, produced in O. fragrans flowers.

  14. Feline acute intermittent porphyria: a phenocopy masquerading as an erythropoietic porphyria due to dominant and recessive hydroxymethylbilane synthase mutations

    National Research Council Canada - National Science Library

    Clavero, Sonia; Bishop, David F; Haskins, Mark E; Giger, Urs; Kauppinen, Raili; Desnick, Robert J

    2010-01-01

    Human acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal dominant inborn error of heme biosynthesis due to the half-normal activity of hydroxymethylbilane synthase (HMB-synthase...

  15. Structure of dimeric mitochondrial ATP synthase: Novel F0 bridging features and the structural basis of mitochondrial cristae biogenesis

    National Research Council Canada - National Science Library

    Fernando Minauro-Sanmiguel; Stephan Wilkens; José J. García

    2005-01-01

    .... How two ATP synthase complexes dimerize to promote cristae formation is unknown. Here we resolved the structure of the dimeric F 1 F 0 ATP synthase complex isolated from bovine heart mitochondria by transmission electron microscopy...

  16. Cloning and characterization of isoprenyl diphosphate synthases with farnesyl diphosphate and geranylgeranyl diphosphate synthase activity from Norway spruce (Picea abies) and their relation to induced oleoresin formation.

    Science.gov (United States)

    Schmidt, Axel; Gershenzon, Jonathan

    2007-11-01

    The conifer Picea abies (Norway spruce) employs terpenoid-based oleoresins as part of its constitutive and induced defense responses to herbivores and pathogens. The isoprenyl diphosphate synthases are branch-point enzymes of terpenoid biosynthesis leading to the various terpene classes. We isolated three genes encoding isoprenyl diphosphate synthases from P. abies cDNA libraries prepared from the bark and wood of methyl jasmonate-treated saplings and screened via a homology-based PCR approach using degenerate primers. Enzyme assays of the purified recombinant proteins expressed in Escherichia coli demonstrated that one gene (PaIDS 4) encodes a farnesyl diphosphate synthase and the other two (PaIDS 5 and PaIDS 6) encode geranylgeranyl diphosphate synthases. The sequences have moderate similarity to those of farnesyl diphosphate and geranylgeranyl diphosphate synthases already known from plants, and the kinetic properties of the enzymes are not unlike those of other isoprenyl diphosphate synthases. Of the three genes, only PaIDS 5 displayed a significant increase in transcript level in response to methyl jasmonate spraying, suggesting its involvement in induced oleoresin biosynthesis.

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

    Science.gov (United States)

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

    2016-01-01

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

  18. Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank

    2011-01-01

    CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer...... completely dominates the oligomeric state of the enzyme. Furthermore, phosphorylation has been shown to regulate the oligomeric states of the enzymes from yeast and human. The crystal structure of a dimeric form of CTP synthase from Sulfolobus solfataricus has been determined at 2.5 Å resolution....... A comparison of the dimeric interface with the intermolecular interfaces in the tetrameric structures of Thermus thermophilus CTP synthase and Escherichia coli CTP synthase shows that the dimeric interfaces are almost identical in the three systems. Residues that are involved in the tetramerization of S...

  19. 5,10-Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) gene polymorphisms and adult meningioma risk.

    Science.gov (United States)

    Zhang, Jun; Zhou, Yan-Wen; Shi, Hua-Ping; Wang, Yan-Zhong; Li, Gui-Ling; Yu, Hai-Tao; Xie, Xin-You

    2013-11-01

    The causes of meningiomas are not well understood. Folate metabolism gene polymorphisms have been shown to be associated with various human cancers. It is still controversial and ambiguous between the functional polymorphisms of folate metabolism genes 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) and risk of adult meningioma. A population-based case–control study involving 600 meningioma patients (World Health Organization [WHO] Grade I, 391 cases; WHO Grade II, 167 cases; WHO Grade III, 42 cases) and 600 controls was done for the MTHFR C677T and A1298C, MTRR A66G, and MTR A2756G variants in Chinese Han population. The folate metabolism gene polymorphisms were determined by using a polymerase chain reaction–restriction fragment length polymorphism assay. Meningioma cases had a significantly lower frequency of MTHFR 677 TT genotype [odds ratio (OR) = 0.49, 95 % confidence interval (CI) 0.33–0.74; P = 0.001] and T allele (OR = 0.80, 95 % CI 0.67–0.95; P = 0.01) than controls. A significant association between risk of meningioma and MTRR 66 GG (OR = 1.41, 95 % CI 1.02–1.96; P = 0.04) was also observed. When stratifying by the WHO grade of meningioma, no association was found. Our study suggested that MTHFR C677T and MTRR A66G variants may affect the risk of adult meningioma in Chinese Han population.

  20. Eplerenone promotes alternative activation in human monocyte-derived macrophages.

    Science.gov (United States)

    Łabuzek, Krzysztof; Liber, Sebastian; Bułdak, Łukasz; Machnik, Grzegorz; Liber, Justyna; Okopień, Bogusław

    2013-01-01

    In this study, we have analyzed the response of human monocyte-derived macrophages to mineralocorticoid axis modulators. Human monocyte-derived macrophages were incubated with aldosterone alone, eplerenone alone, and the combination of aldosterone and eplerenone. The analyzed variables were nitric oxide and reactive oxygen species production, and the gene and protein expression of inducible nitric oxide synthase, arginase I, and mannose receptor. We showed that aldosterone promotes a classic inflammatory response in macrophages, whereas its antagonist, eplerenone, attenuates aldosterone-induced activity. Eplerenone did not quantitatively weaken the response of macrophages to aldosterone but instead qualitatively changed their behavior.

  1. Altering the expression of two chitin synthase genes differentially affects the growth and morphology of Aspergillus oryzae

    DEFF Research Database (Denmark)

    Müller, Christian; Hjort, C.M.; Hansen, K.

    2002-01-01

    In Aspergillus oryzae, one full-length chitin synthase (chsB) and fragments of two other chitin synthases (csmA and chsC) were identified. The deduced amino acid sequence of chsB was similar (87% identity) to chsB from Aspergillus nidulans, which encodes a class III chitin synthase. The sequence...

  2. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  3. ATP synthase from slow and fast growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  4. Cloning and heterologous expression of a novel subgroup of class IV polyhydroxyalkanoate synthase genes from the genus Bacillus.

    Science.gov (United States)

    Mizuno, Kouhei; Kihara, Takahiro; Tsuge, Takeharu; Lundgren, Benjamin R; Sarwar, Zaara; Pinto, Atahualpa; Nomura, Christopher T

    2017-01-01

    Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinant Escherichia coli.

  5. Arginase Inhibitor 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-Glucoside Activates Endothelial Nitric Oxide Synthase and Improves Vascular Function.

    Science.gov (United States)

    Yi, Bonggu; Nguyen, Minh Cong; Won, Moo-Ho; Kim, Young Myeong; Ryoo, Sungwoo

    2017-02-01

    Endothelial arginase constrains the activity of endothelial nitric oxide synthase by reducing nitric oxide bioavailability, which contributes to vascular diseases. During screening, we identified a novel compound from the rhizome of Polygonum multiflorum (Polygonaceae), 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (THSG), which inhibited arginase activity. THSG exhibited noncompetitive inhibition of arginase II and inhibited both arginases I and II in a dose-dependent manner. THSG-dependent arginase inhibition reciprocally increased nitric oxide production and decreased reactive oxygen species generation in aortic endothelia. These effects were associated with increased dimerization of endothelial nitric oxide synthase without changes in the protein expression levels of arginase I, arginase II, or endothelial nitric oxide synthase. In vascular tension assays, when aortic vessels from wild-type mice are incubated with THSG, responses to the nitric oxide-dependent vasorelaxant acetylcholine were augmented, but responses to an nitric oxide donor, sodium nitroprusside, were not affected. On the other hand, phenylephrine-dependent vasoconstriction was significantly retarded in THSG-treated vessels. In a high-cholesterol diet-fed atherogenic model mice (ApoE-/-), THSG improved endothelial function by enhancement of the nitric oxide-cGMP pathway. Taken together, these results suggest that THSG may exert vasoprotective effects through augmentation of nitric oxide signaling by inhibiting arginase. Therefore, THSG may be useful in the treatment of vascular diseases that are derived from endothelial dysfunction, such as atherosclerosis. Georg Thieme Verlag KG Stuttgart · New York.

  6. Regulation of C. elegans fat uptake and storage by acyl-CoA synthase-3 is dependent on NR5A family nuclear hormone receptor nhr-25.

    Science.gov (United States)

    Mullaney, Brendan C; Blind, Raymond D; Lemieux, George A; Perez, Carissa L; Elle, Ida C; Faergeman, Nils J; Van Gilst, Marc R; Ingraham, Holly A; Ashrafi, Kaveh

    2010-10-06

    Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3, a long-chain acyl-CoA synthase, causes enhanced intestinal lipid uptake, de novo fat synthesis, and accumulation of enlarged, neutral lipid-rich intestinal depots. Here, we show that ACS-3 functions in seam cells, epidermal cells anatomically distinct from sites of fat uptake and storage, and that acs-3 mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. Sequential expression of endothelial nitric oxide synthase, inducible nitric oxide synthase, and nitrotyrosine in odontoblasts and pulp cells during dentin repair after tooth preparation in rat molars.

    Science.gov (United States)

    Mei, Yu Feng; Yamaza, Takayoshi; Atsuta, Ikiru; Danjo, Atsushi; Yamashita, Yoshio; Kido, Mizuho A; Goto, Masaaki; Akamine, Akifumi; Tanaka, Teruo

    2007-04-01

    Nitric oxide (NO) stimulates osteoblast differentiation, but whether NO contributes to odontoblast differentiation during dentin repair is unknown. By using reverse transcription/polymerase chain reaction and immunostaining, we investigated the gene expression and/or immunolocalization of endothelial NO synthase (eNOS), inducible NOS (iNOS), and nitrotyrosine (a biomarker for NO-derived peroxinitrite), and alkaline phosphatase (ALP) and osteocalcin (early and terminal differentiation markers of odontoblasts, respectively) in dental pulp tissue after rat tooth preparation. At the early stage (1-3 days) post-preparation, markedly increased expression of iNOS and nitrotyrosine was found in odontoblasts and pulp cells beneath the cavity, whereas eNOS expression was significantly decreased. ALP mRNA expression was significantly increased after 1 day but decreased after 3 days, whereas ALP activity was weak in the dentin-pulp interface under the cavity after 1 day but strong after 3 days. Osteocalcin mRNA expression was significantly increased at this stage. At 7 days post-preparation, tertiary dentin was formed under the cavity. All the molecules studied were expressed at control levels in odontoblasts/pulp cells beneath the cavity. These findings show that abundant NO is released from odontoblasts and pulp cells at an early stage after tooth preparation and indicate that, after tooth preparation, the up-regulation of iNOS and nitrotyrosine in odontoblasts is synchronized with increased cellular expression of ALP and osteocalcin. Therefore, the NO synthesized by iNOS after tooth preparation probably participates in regulating odontoblast differentiation during tertiary dentinogenesis.

  8. Two Distinct Cyclodipeptide Synthases from a Marine Actinomycete Catalyze Biosynthesis of the Same Diketopiperazine Natural Product.

    Science.gov (United States)

    James, Elle D; Knuckley, Bryan; Alqahtani, Norah; Porwal, Suheel; Ban, Jisun; Karty, Jonathan A; Viswanathan, Rajesh; Lane, Amy L

    2016-07-15

    Diketopiperazine natural products are structurally diverse and offer many biological activities. Cyclodipeptide synthases (CDPSs) were recently unveiled as a novel enzyme family that employs aminoacyl-tRNAs as substrates for 2,5-diketopiperazine assembly. Here, the Nocardiopsis sp. CMB-M0232 genome is predicted to encode two CDPSs, NozA and NcdA. Metabolite profiles from E. coli expressing these genes and assays with purified recombinant enzymes revealed that NozA and NcdA catalyze cyclo(l-Trp-l-Trp) (1) biosynthesis from tryptophanyl-tRNA and do not accept other aromatic aminoacyl-tRNA substrates. Fidelity is uncommon among characterized CDPSs, making NozA and NcdA important CDPS family additions. Further, 1 was previously supported as a biosynthetic precursor of the nocardioazines; the current study suggests that Nocardiopsis sp. may derive this precursor from both NozA and NcdA. This study offers a rare example of a single bacterium encoding multiple phylogenetically distinct enzymes that yield the same secondary metabolite and provides tools for chemoenzymatic syntheses of indole alkaloid diketopiperazines.

  9. Fatty acid synthase inhibitors from plants: isolation, structure elucidation, and SAR studies.

    Science.gov (United States)

    Li, Xing-Cong; Joshi, Alpana S; ElSohly, Hala N; Khan, Shabana I; Jacob, Melissa R; Zhang, Zhizheng; Khan, Ikhlas A; Ferreira, Daneel; Walker, Larry A; Broedel, Sheldon E; Raulli, Robert E; Cihlar, Ronald L

    2002-12-01

    Fatty acid synthase (FAS) has been identified as a potential antifungal target. FAS prepared from Saccharomyces cerevisiae was employed for bioactivity-guided fractionation of Chlorophora tinctoria,Paspalum conjugatum, Symphonia globulifera, Buchenavia parviflora, and Miconia pilgeriana. Thirteen compounds (1-13), including three new natural products (1, 4, 12), were isolated and their structures identified by spectroscopic interpretation. They represented five chemotypes, namely, isoflavones, flavones, biflavonoids, hydrolyzable tannin-related derivatives, and triterpenoids. 3'-Formylgenistein (1) and ellagic acid 4-O-alpha-l-rhamnopyranoside (9) were the most potent compounds against FAS, with IC(50) values of 2.3 and 7.5 microg/mL, respectively. Furthermore, 43 (14-56) analogues of the five chemotypes from our natural product repository and commercial sources were tested for their FAS inhibitory activity. Structure-activity relationships for some chemotypes were investigated. All these compounds were further evaluated for antifungal activity against Candida albicans and Cryptococcus neoformans. Although there were several antifungal compounds in the set, correlation between the FAS inhibitory activity and antifungal activity could not be defined.

  10. Post-Translational Modification of Constitutive Nitric Oxide Synthase in the Penis

    Science.gov (United States)

    Musicki, Biljana; Ross, Ashley E.; Champion, Hunter C.; Burnett, Arthur L.; Bivalacqua, Trinity J.

    2009-01-01

    Erectile dysfunction (ED) is a common men's health problem characterized by the consistent inability to sustain an erection sufficient for sexual intercourse. Basic science research on erectile physiology has been devoted to investigating the pathogenesis of ED and has led to the conclusion that ED is predominately a disease of vascular origin and/or neurogenic dysfunction. The constitutive forms of nitric oxide synthase [NOS; endothelial NOS (eNOS) and neuronal NOS (nNOS)] are important enzymes involved in the production of nitric oxide (NO) and thus regulate penile vascular homeostasis. Given the impact of endothelial- and neuronal-derived NO in penile vascular biology, a great deal of research over the past decade has focused on the role of NO synthesis from the endothelium and nitrergic nerve terminal in normal erectile physiology as well as in disease states. Loss of the functional integrity of the endothelium and subsequent endothelial dysfunction plays an integral role in the occurrence of ED. Therefore, molecular mechanisms involved in dysregulation of these NOS isoforms in the development of ED are essential to discovering the pathogenesis of ED in various disease states. This communication reviews the role of eNOS and nNOS in erectile physiology and discusses the alterations in eNOS and nNOS via post-translation modification in various vascular diseases of the penis. PMID:19342700

  11. Inhibition of platelet activation by lachrymatory factor synthase (LFS)-silenced (tearless) onion juice.

    Science.gov (United States)

    Thomson, Susan J; Rippon, Paula; Butts, Chrissie; Olsen, Sarah; Shaw, Martin; Joyce, Nigel I; Eady, Colin C

    2013-11-06

    Onion and garlic are renowned for their roles as functional foods. The health benefits of garlic are attributed to di-2-propenyl thiosulfinate (allicin), a sulfur compound found in disrupted garlic but not found in disrupted onion. Recently, onions have been grown with repressed lachrymatory factor synthase (LFS) activity, which causes these onions to produce increased amounts of di-1-propenyl thiosulfinate, an isomer of allicin. This investigation into the key health attributes of LFS-silenced (tearless) onions demonstrates that they have some attributes more similar to garlic and that this is likely due to the production of novel thiosulfinate or metabolites. The key finding was that collagen-induced in vitro platelet aggregation was significantly reduced by tearless onion extract over normal onion extract. Thiosulfinate or derived compounds were shown not to be responsible for the observed changes in the inflammatory response of AGS (stomach adenocarcinoma) cells to tumor necrosis factor alpha (TNFα) when pretreated with model onion juices. A preliminary rat feeding trial indicated that the tearless onions may also play a key role in reducing weight gain.

  12. Arginine-Based Inhibitors of Nitric Oxide Synthase: Therapeutic Potential and Challenges

    Science.gov (United States)

    Víteček, Jan; Lojek, Antonín; Valacchi, Giuseppe; Kubala, Lukáš

    2012-01-01

    In the past three decades, nitric oxide has been well established as an important bioactive molecule implicated in regulation of cardiovascular, nervous, and immune systems. Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric oxide synthases (NOS), the enzymes responsible for production of nitric oxide. Among the many NOS inhibitors developed to date, inhibitors based on derivatives and analogues of arginine are of special interest, as this category includes a relatively high number of compounds with good potential for experimental as well as clinical application. Though this group of inhibitors covers early nonspecific compounds, modern drug design strategies such as biochemical screening and computer-aided drug design have provided NOS-isoform-specific inhibitors. With an emphasis on major advances in this field, a comprehensive list of inhibitors based on their structural characteristics is discussed in this paper. We provide a summary of their biochemical properties as well as their observed effects both in vitro and in vivo. Furthermore, we focus in particular on their pharmacology and use in recent clinical studies. The potential of newly designed specific NOS inhibitors developed by means of modern drug development strategies is highlighted. PMID:22988346

  13. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

    2017-07-03

    New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB a–b-subunit interface and affects multiple steps in the enzyme’s overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

  14. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

    2017-07-03

    New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α–β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

  15. Leptin action through hypothalamic nitric oxide synthase-1-expressing neurons controls energy balance.

    Science.gov (United States)

    Leshan, Rebecca L; Greenwald-Yarnell, Megan; Patterson, Christa M; Gonzalez, Ian E; Myers, Martin G

    2012-05-01

    Few effective measures exist to combat the worldwide obesity epidemic(1), and the identification of potential therapeutic targets requires a deeper understanding of the mechanisms that control energy balance. Leptin, an adipocyte-derived hormone that signals the long-term status of bodily energy stores, acts through multiple types of leptin receptor long isoform (LepRb)-expressing neurons (called here LepRb neurons) in the brain to control feeding, energy expenditure and endocrine function(2-4). The modest contributions to energy balance that are attributable to leptin action in many LepRb populations(5-9) suggest that other previously unidentified hypothalamic LepRb neurons have key roles in energy balance. Here we examine the role of LepRb in neuronal nitric oxide synthase (NOS1)-expressing LebRb (LepRb(NOS1)) neurons that comprise approximately 20% of the total hypothalamic LepRb neurons. Nos1(cre)-mediated genetic ablation of LepRb (Lepr(Nos1KO)) in mice produces hyperphagic obesity, decreased energy expenditure and hyperglycemia approaching that seen in whole-body LepRb-null mice. In contrast, the endocrine functions in Lepr(Nos1KO) mice are only modestly affected by the genetic ablation of LepRb in these neurons. Thus, hypothalamic LepRb(NOS1) neurons are a key site of action of the leptin-mediated control of systemic energy balance.

  16. Fatty acid synthase inhibitors from the hulls of Nephelium lappaceum L.

    Science.gov (United States)

    Zhao, You-Xing; Liang, Wen-Juan; Fan, Hui-Jin; Ma, Qing-Yun; Tian, Wei-Xi; Dai, Hao-Fu; Jiang, He-Zhong; Li, Ning; Ma, Xiao-Feng

    2011-08-16

    Natural products inhibiting fatty acid synthase (FAS) are appearing as potential therapeutic agents to treat cancer and obesity. The bioassay-guided chemical investigation of the hulls of Nephelium lappaceum L. resulted in the isolation of ten compounds (1-10) mainly including flavonoids and oleane-type triterpene oligoglycosides, in which all of the compounds were isolated from this plant for the first time. Additionally, compounds 8 and 9 were new hederagenin derivatives and were elucidated as hederagenin 3-O-(2,3-di-O-acetyl-α-l-arabinofuranosyl)-(1→3)-[α-l-rhamnopyranosyl(1→2)]-β-l-arabinopyranoside and hederagenin 3-O-(3-O-acetyl-α-l-arabinofuranosyl)-(1→3)-[α-l-rhamnopyranosyl-(1→2)]-β-l-arabinopyranoside, respectively. All these isolates were evaluated for inhibitory activities of FAS, which showed these isolates had inhibitory activity against FAS with IC(50) values ranging from 6.69 to 204.40 μM, comparable to the known FAS inhibitor EGCG (IC(50)=51.97 μM). The study indicates that the hulls of Nephelium lappaceum L. could be considered as potential sources of promising FAS inhibitors and the oleane-type triterpene oligoglycosides could be considered as another type of natural FAS inhibitors. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. It takes two to tango: defining an essential second active site in pyridoxal 5'-phosphate synthase.

    Directory of Open Access Journals (Sweden)

    Cyril Moccand

    Full Text Available The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2 that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5'-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5'-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.

  18. Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways.

    Science.gov (United States)

    Piérard, A; Glansdorff, N; Gigot, D; Crabeel, M; Halleux, P; Thiry, L

    1976-07-01

    Cumulative repression of Escherichia coli carbamoylphosphate synthase (CPSase; EC 2.7.2.9) by arginine and pyrimidine was analyzed in relation to control enzyme synthesis in the arginine and pyrimidine pathways. The expression of carA and carB, the adjacent genes that specify the two subunits of the enzyme, was estimated by means of an in vitro complementation assay. The synthesis of each gene product was found to be under repression control. Coordinate expression of the two genes was observed under most conditions investigated. They might thus form an operon. The preparation of strains blocked in the degradation of cytidine and harboring leaky mutations affecting several steps of pyrimidine nucleotide synthesis made it possible to distinguish between the effects of cytidine and uridine compounds in the repression of the pyrimidine pathway enzymes. The data obtained suggest that derivatives of both cytidine and uridine participate in the repression of CPSase. In addition, repression of CPSase by arginine did not appear to occur unless pyrimidines were present at a significant intracellular concentration. This observation, together with our previous report that argR mutations impair the cumulative repression of CPSase, suggests that this control is mediated through the concerted effects of regulatory elements specific for the arginine and pyrimidine pathways.

  19. Evolution of the key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase

    Science.gov (United States)

    Junker, Anne; Fischer, Juliane; Sichhart, Yvonne; Brandt, Wolfgang; Dräger, Birgit

    2013-01-01

    Putrescine N-methyltransferases (PMTs) are the first specific enzymes of the biosynthesis of nicotine and tropane alkaloids. PMTs transfer a methyl group onto the diamine putrescine from S-adenosyl-l-methionine (SAM) as coenzyme. PMT proteins have presumably evolved from spermidine synthases (SPDSs), which are ubiquitous enzymes of polyamine metabolism. SPDSs use decarboxylated SAM as coenzyme to transfer an aminopropyl group onto putrescine. In an attempt to identify possible and necessary steps in the evolution of PMT from SPDS, homology based modeling of Datura stramonium SPDS1 and PMT was employed to gain deeper insight in the preferred binding positions and conformations of the substrate and the alternative coenzymes. Based on predictions of amino acids responsible for the change of enzyme specificities, sites of mutagenesis were derived. PMT activity was generated in D. stramonium SPDS1 after few amino acid exchanges. Concordantly, Arabidopsis thaliana SPDS1 was mutated and yielded enzymes with both, PMT and SPDS activities. Kinetic parameters were measured for enzymatic characterization. The switch from aminopropyl to methyl transfer depends on conformational changes of the methionine part of the coenzyme in the binding cavity of the enzyme. The rapid generation of PMT activity in SPDS proteins and the wide-spread occurrence of putative products of N-methylputrescine suggest that PMT activity is present frequently in the plant kingdom. PMID:23908659

  20. Evolution of the key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase.

    Directory of Open Access Journals (Sweden)

    Anne eJunker

    2013-07-01

    Full Text Available Putrescine N-methyltransferases (PMTs are the first specific enzymes of the biosynthesis of nicotine and tropane alkaloids. PMTs transfer a methyl group onto the diamine putrescine from S-adenosyl-L-methionine (SAM as coenzyme. PMT proteins have presumably evolved from spermidine synthases (SPDSs, which are ubiquitous enzymes of polyamine metabolism. SPDS use decarboxylated SAM as coenzyme to transfer an aminopropyl group onto putrescine. In an attempt to identify possible and necessary steps in the evolution of PMT from SPDS, homology based modeling of Datura stramonium SPDS1 and PMT was employed to gain deeper insight in the preferred binding positions and conformations of the substrate and the alternative coenzymes. Based on predictions of amino acids responsible for the change of enzyme specificities, sites of mutagenesis were derived. PMT activity was generated in Datura stramonium SPDS1 after few amino acid exchanges. Concordantly, Arabidopsis thaliana SPDS1 was mutated and yielded enzymes with both, PMT and SPDS activities. Kinetic parameters were measured for enzymatic characterization. The switch from aminopropyl to methyl transfer depends on conformational changes of the methionine part of the coenzyme in the binding cavity of the enzyme. The rapid generation of PMT activity in SPDS proteins and the wide-spread occurrence of putative products of N-methylputrescine suggest that PMT activity is present frequently in the plant kingdom.

  1. Nitric Oxide Synthase Type III Overexpression By Gene Therapy Exerts Antitumoral Activity In Mouse Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Raúl González

    2015-08-01

    Full Text Available Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO synthase type III (NOS-3 overexpression induces cell death in hepatoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. Hepa 1-6 cells were used for in vitro and in vivo experiments. The first generation adenovirus was designed to overexpress NOS-3 (or GFP and luciferase cDNA under the regulation of murine alpha-fetoprotein (AFP and Rous Sarcoma Virus (RSV promoters, respectively. Both adenoviruses were administered through the tail vein two weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-Luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8 activity in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by l-NAME or p53 siRNA. The tail vein infusion of AFP-NOS- 3/RSV-Luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

  2. Genes encoding chavicol/eugenol synthase from the creosote bush Larrea tridentata

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Norman G.; Davin, Laurence B.; Kim, Sung -Jin; Vassao, Daniel Giddings; Patten, Ann M.; Eichinger, Dietmar

    2015-09-15

    Particular aspects provide novel methods for redirecting carbon allocation in plants or cell culture from lignification to inherently more useful and tractable materials, and to facilitate the generation of, e.g., biofuels from the remaining plant ro culture biomass. Particular aspects provided novel methods for converting monolignols into allyl/propenyl phenols, and for chavicol/eugenol formation or production. Additional aspects relate to the discovery of novel chavicol/eugenol synthases that convert p-coumaryl/coniferyl alcohol esters into chavicol/eugenol, and to novel compositions (e.g., novel proteins and nucleic acids encoding same), and novel methods using same for producing or forming chavicol/eugenol and other derivatives in cell culture and/or genetically modified plants, and for re-engineering the composition of plant biomass. Particular aspects provide novel methods for generation in culture or in planta of liquid/combustible allyl/propenyl phenols, and these phenolic products are utilized for (non-ethanol) biofuel/bioenergy purposes, while the remaining plant biomass facilitates the generation of other biofuels.

  3. Mechanism-Based Post-Translational Modification and Inactivation in Terpene Synthases.

    Science.gov (United States)

    Kersten, Roland D; Diedrich, Jolene K; Yates, John R; Noel, Joseph P

    2015-11-20

    Terpenes are ubiquitous natural chemicals with diverse biological functions spanning all three domains of life. In specialized metabolism, the active sites of terpene synthases (TPSs) evolve in shape and reactivity to direct the biosynthesis of a myriad of chemotypes for organismal fitness. As most terpene biosynthesis mechanistically involves highly reactive carbocationic intermediates, the protein surfaces catalyzing these cascade reactions possess reactive regions possibly prone to premature carbocation capture and potentially enzyme inactivation. Here, we show using proteomic and X-ray crystallographic analyses that cationic intermediates undergo capture by conserved active site residues leading to inhibitory self-alkylation. Moreover, the level of cation-mediated inactivation increases with mutation of the active site, upon changes in the size and structure of isoprenoid diphosphate substrates, and alongside increases in reaction temperatures. TPSs that individually synthesize multiple products are less prone to self-alkylation then TPSs possessing relatively high product specificity. In total, the results presented suggest that mechanism-based alkylation represents an overlooked mechanistic pressure during the evolution of cation-derived terpene biosynthesis.

  4. Identification of a Polyketide Synthase Involved in Sorbicillin Biosynthesis by Penicillium chrysogenum.

    Science.gov (United States)

    Salo, Oleksandr; Guzmán-Chávez, Fernando; Ries, Marco I; Lankhorst, Peter P; Bovenberg, Roel A L; Vreeken, Rob J; Driessen, Arnold J M

    2016-07-01

    Secondary metabolism in Penicillium chrysogenum was intensively subjected to classical strain improvement (CSI), the resulting industrial strains producing high levels of β-lactams. During this process, the production of yellow pigments, including sorbicillinoids, was eliminated as part of a strategy to enable the rapid purification of β-lactams. Here we report the identification of the polyketide synthase (PKS) gene essential for sorbicillinoid biosynthesis in P. chrysogenum We demonstrate that the production of polyketide precursors like sorbicillinol and dihydrosorbicillinol as well as their derivatives bisorbicillinoids requires the function of a highly reducing PKS encoded by the gene Pc21g05080 (pks13). This gene belongs to the cluster that was mutated and transcriptionally silenced during the strain improvement program. Using an improved β-lactam-producing strain, repair of the mutation in pks13 led to the restoration of sorbicillinoid production. This now enables genetic studies on the mechanism of sorbicillinoid biosynthesis in P. chrysogenum and opens new perspectives for pathway engineering. Sorbicillinoids are secondary metabolites with antiviral, anti-inflammatory, and antimicrobial activities produced by filamentous fungi. This study identified the gene cluster responsible for sorbicillinoid formation in Penicillium chrysogenum, which now allows engineering of this diverse group of compounds. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Gene expression and characterization of isoprene synthase from Populus alba.

    Science.gov (United States)

    Sasaki, Kanako; Ohara, Kazuaki; Yazaki, Kazufumi

    2005-04-25

    Isoprene synthase cDNA from Populus alba (PaIspS) was isolated by RT-PCR. This PaIspS mRNA, which was predominantly observed in the leaves, was strongly induced by heat stress and continuous light irradiation, and was substantially decreased in the dark, suggesting that isoprene emission was regulated at the transcriptional level. The subcellular localization of PaIspS protein with green fluorescent protein fusion was shown to be in plastids. PaIspS expressed in Escherichia coli was characterized enzymatically: it had an optimum pH of approximately 8.0, and an optimum temperature 40 degrees C. Its preference for divalent cations for its activity was also studied.

  6. Inducible nitric oxide synthase immunoreactivity in healthy rat pancreas.

    Science.gov (United States)

    Keklikoglu, Nurullah

    2008-01-01

    Nitric oxide (NO) is produced by NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). It is believed that, while nNOS and eNOS are effective in regulation of normal physiological processes, iNOS is expressed at an increasing rate especially in inflammatory process. The aim of this study was to determine the presence of iNOS immunoreactivity (iNOS-IR) and, to compare the iNOS-IR in islet of Langerhans cells (LC), acinar cells (AC), centroacinar cells (CC) and ductal cells (DC) by immunohistochemical (IHC) method in healthy rat pancreata. This study revealed the presence of iNOS-IR in all cell types except AC. Statistical analysis revealed a highly significant difference (preseach related to diabetes, it should not be disregarded that iNOS may be constitutively present in pancreatic islets.

  7. Human blood platelets lack nitric oxide synthase activity.

    Science.gov (United States)

    Böhmer, Anke; Gambaryan, Stepan; Tsikas, Dimitrios

    2015-01-01

    Reports on expression and functionality of nitric oxide synthase (NOS) activity in human blood platelets and erythrocytes are contradictory. We used a specific gas chromatography-mass spectrometry (GC-MS) method to detect NOS activity in human platelets. The method measures simultaneously [(15)N]nitrite and [(15)N]nitrate formed from oxidized (15)N-labeled nitric oxide ((15)NO) upon its NOS-catalyzed formation from the substrate l-[guanidino-(15)N2]-arginine. Using this GC-MS assay, we did not detect functional NOS in non-stimulated platelets and in intact platelets activated by various agonists (adenosine diphosphate, collagen, thrombin, or von Willebrand factor) or lysed platelets. l-[guanidino-nitro]-Arginine-inhibitable NOS activity was measured after addition of recombinant human endothelial NOS to lysed platelets. Previous and recent studies from our group challenge expression and functionality of NOS in human platelets and erythrocytes.

  8. Conformational Flexibility of Metazoan Fatty Acid Synthase Enables Catalysis

    Science.gov (United States)

    Brignole, Edward J.; Smith, Stuart; Asturias, Francisco J.

    2008-01-01

    The metazoan cytosolic fatty acid synthase (FAS) contains all of the enzymes required for de novo fatty acid biosynthesis covalently linked around two reaction chambers. While the 3D architecture of FAS has been mostly defined, it is unclear how reaction intermediates can transfer between distant catalytic domains. Using single-particle electron microscopy we have identified a near continuum of conformations consistent with remarkable flexibility of FAS. The distribution of conformations was influenced by the presence of substrates and altered by different catalytic mutations suggesting a direct correlation between conformation and specific enzymatic activities. 3D reconstructions were interpreted by docking high-resolution structures of individual domains and illustrate that the substrate loading and condensation domains dramatically swing and swivel to access substrates within either reaction chamber. Concomitant rearrangement of the β-carbon processing domains synchronizes acyl-chain reduction in one chamber with acyl-chain elongation in the other. PMID:19151726

  9. Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance

    DEFF Research Database (Denmark)

    Huang, Laurence; Crothers, Kristina; Atzori, Chiara

    2004-01-01

    Pneumocystis pneumonia (PCP) remains a major cause of illness and death in HIV-infected persons. Sulfa drugs, trimethoprim-sulfamethoxazole (TMP-SMX) and dapsone are mainstays of PCP treatment and prophylaxis. While prophylaxis has reduced the incidence of PCP, its use has raised concerns about...... in the dihydropteroate synthase (DHPS) gene. Similar mutations have been observed in P. jirovecii. Studies have consistently demonstrated a significant association between the use of sulfa drugs for PCP prophylaxis and DHPS gene mutations. Whether these mutations confer resistance to TMP-SMX or dapsone plus trimethoprim...... for PCP treatment remains unclear. We review studies of DHPS mutations in P. jirovecii and summarize the evidence for resistance to sulfamethoxazole and dapsone....

  10. Catalysis and Sulfa Drug Resistance in Dihydropteroate Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Mi-Kyung; Wu, Yinan; Li, Zhenmei; Zhao, Ying; Waddell, M. Brett; Ferreira, Antonio M.; Lee, Richard E.; Bashford, Donald; White, Stephen W. (SJCH)

    2013-04-08

    The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S{sub N}1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

  11. The glamour and gloom of glycogen synthase kinase-3.

    Science.gov (United States)

    Jope, Richard S; Johnson, Gail V W

    2004-02-01

    Glycogen synthase kinase-3 (GSK3) is now recognized as a key component of a surprisingly large number of cellular processes and diseases. Several mechanisms play a part in controlling the actions of GSK3, including phosphorylation, protein complex formation, and subcellular distribution. These are used to control and direct the far-reaching influences of GSK3 on cellular structure, growth, motility and apoptosis. Dysregulation of GSK3 is linked to several prevalent pathological conditions, such as diabetes and/or insulin resistance, and Alzheimer's disease. Therefore, much effort is currently directed towards understanding the functions and control of GSK3, and identifying methods capable of diminishing the deleterious impact of GSK3 in pathological conditions.

  12. Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity.

    Science.gov (United States)

    Buller, Andrew R; van Roye, Paul; Murciano-Calles, Javier; Arnold, Frances H

    2016-12-27

    Tryptophan synthase (TrpS) catalyzes the final steps in the biosynthesis of l-tryptophan from l-serine (Ser) and indole-3-glycerol phosphate (IGP). We report that native TrpS can also catalyze a productive reaction with l-threonine (Thr), leading to (2S,3S)-β-methyltryptophan. Surprisingly, β-substitution occurs in vitro with a 3.4-fold higher catalytic efficiency for Ser over Thr using saturating indole, despite a >82000-fold preference for Ser in direct competition using IGP. Structural data identify a novel product binding site, and kinetic experiments clarify the atypical mechanism of specificity: Thr binds efficiently but decreases the affinity for indole and disrupts the allosteric signaling that regulates the catalytic cycle.

  13. Identification and characterization of the Populus sucrose synthase gene family.

    Science.gov (United States)

    An, Xinmin; Chen, Zhong; Wang, Jingcheng; Ye, Meixia; Ji, Lexiang; Wang, Jia; Liao, Weihua; Ma, Huandi

    2014-04-10

    In this study, we indentified 15 sucrose synthase (SS) genes in Populus and the results of RT-qPCR revealed that their expression patterns were constitutive and partially overlapping but diverse. The release of the most recent Populus genomic data in Phytozome v9.1 has revealed the largest SS gene family described to date, comprising 15 distinct members. This information will now enable the analysis of transcript expression profiles for those that have not been previously reported. Here, we performed a comprehensive analysis of SS genes in Populus by describing the gene structure, chromosomal location and phylogenetic relationship of each family member. A total of 15 putative SS gene members were identified in the Populus trichocarpa (Torr. & Gray) genome using the SS domain and amino acid sequences from Arabidopsis thaliana as a probe. A phylogenetic analysis indicated that the 15 members could be classified into four groups that fall into three major categories: dicots, monocots & dicots 1 (M & D 1), and monocots & dicots 2 (M & D 2). In addition, the 15 SS genes were found to be unevenly distributed on seven chromosomes. The two conserved domains (sucrose synthase and glycosyl transferase) were found in this family. Meanwhile, the expression profiles of all 15 gene members in seven different organs were investigated in Populus tomentosa (Carr.) by using RT-qPCR. Additional analysis indicated that the poplar SS gene family is also involved in response to water-deficit. The current study provides basic information that will assist in elucidating the functions of poplar SS family. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Investigation of aldosterone-synthase inhibition in rats.

    Science.gov (United States)

    Ménard, Joël; Gonzalez, Marie-Françoise; Guyene, Thanh-Tam; Bissery, Alvine

    2006-06-01

    In-vivo investigation of aldosterone-synthase inhibitors requires experimental models to characterize the biological effects of these compounds. Seven successive experiments were performed in groups of 2-month-old male spontaneously hypertensive rats. Urinary free aldosterone was the main end-point measured during two contrasted diets: low sodium-high potassium (LS), inducing high urinary aldosterone (839 pmol/24 h, 95% confidence interval 654-1077), and high sodium-normal potassium (HS), inducing low urinary aldosterone (38.1 pmol/24 h; 95% confidence interval, 32.4-44.9). FAD 286 A (10 and 30 mg/kg) decreased urinary free aldosterone by 53 and 87% on the LS diet, and 50 and 75% on the HS. Plasma renin concentration increased three-fold after a 4-week treatment of 30 mg/kg FAD 286 A on the LS diet and did not change on the HS. The combination of FAD 286 A (30 mg/kg) and spironolactone (30 mg/kg) on the LS diet induced a biological picture of severe hypoaldosteronism and was not tolerated, whereas the HS diet prevented these abnormalities. The combination of FAD 286 A (30 mg/kg) and furosemide (30 mg/kg) on the HS diet corrected the diuretic-induced hypokalemia (4.1 +/- 0.2 versus 3.7 +/- 2.2 mEq/l, P < 0.033). This experimental model will be useful to screen future aldosterone-synthase inhibitors and study their biological effects in various experimental conditions.

  15. Significance of differential expression of thymidylate synthase in normal and primary tumor tissues from patients with colorectal cancer

    Directory of Open Access Journals (Sweden)

    Hua Yawei

    2011-08-01

    Full Text Available Abstract The role of thymidylate synthase (TS is essential as a key rate-limiting enzyme in DNA synthesis. It is the primary target of fluorouracil and its derivates in colorectal cancer. In this study, TS mRNA expression was examined in primary tumor and normal tissues from 76 patients with high- risk stage II/III colorectal cancer by laser capture microdissection and polymerase chain reaction. Thirty (39.47% patients were found to have higher TS expression in primary tumors with earlier stage (P = 0.018, lower histological grades (P = 0.001 and high frequency microsatellite instability (P = 0.000. Multivariate analysis showed that microsatellite instability, histological grade and number of lymph nodes examined are independent prognostic markers.

  16. Allele specific CAPS marker development and characterization of chalcone synthase gene in Indian mulberry (Morus spp., family Moraceae.

    Directory of Open Access Journals (Sweden)

    Vivek Arora

    Full Text Available Chalcone synthase (CHS is an essential enzyme in the phenylpropanoid pathway that catalyzes the first step in flavonoid biosynthesis in plants under diverse environmental stress. We have used CHS as a candidate gene in mulberry and developed Single Nucleotide Polymorphism (SNP based co-dominant Cleaved Amplified Polymorphic Sequence (CAPS marker associated with the CHS locus. The segregation pattern of the marker was studied in an F1 population derived from a hybridization program between two mulberry genotypes showing polymorphism for the CHS locus. Differential CHS activity of the recombinants has been correlated with the segregation pattern of the marker. Homology modelling and docking studies are performed for both the identified CHS alleles and correlated with respective CHS activity. Phenotyping of Powdery Mildew infected F1 population indicated a probable association with the CAPS marker.

  17. Exploring geometric properties of gold nanoparticles using TEM images to explain their chaperone like activity for citrate synthase.

    Science.gov (United States)

    Kaushik, Vikas; Lahiri, Tapobrata; Singha, Shantiswaroop; Dasgupta, Anjan Kumar; Mishra, Hrishikesh; Kumar, Upendra; Kumar, Rajeev

    2011-01-01

    Study on geometric properties of nanoparticles and their relation with biomolecular activities, especially protein is quite a new field to explore. This work was carried out towards this direction where images of gold nanoparticles obtained from transmission electron microscopy were processed to extract their size and area profile at different experimental conditions including and excluding a protein, citrate synthase. Since the images were ill-posed, texture of a context-window for each pixel was used as input to a back-propagation network architecture to obtain decision on its membership as nanoparticle. The segmented images were further analysed by k-means clustering to derive geometric properties of individual nanoparticles even from their assembled form. The extracted geometric information was found to be crucial to give a model featuring porous cage like configuration of nanoparticle assembly using which the chaperone like activity of gold nanoparticles can be explained.

  18. Crowding interactions perturb structure and stability by destabilizing the stable core of the α-subunit of tryptophan synthase.

    Science.gov (United States)

    Kadumuri, Rajashekar Varma; Gullipalli, Jagadeesh; Subramanian, SriVidya; Jaipuria, Garima; Atreya, Hanudatta S; Vadrevu, Ramakrishna

    2016-07-01

    The consequences of crowding derived from relatively small and intrinsically disordered proteins are not clear yet. We report the effect of ficoll-70 on the structure and stability of native and partially folded states of the 29 kDa alpha subunit of tryptophan synthase (αTS). Overall, combining the changes in the circular dichroism and fluorescence spectra, in conjunction with the gradual loss of cooperativity under urea denaturation in the presence of increasing amounts of ficoll, it may be concluded that the crowding agent perturbs not only the native state but also the partially folded state of αTS. Importantly, NMR data indicate that ficoll interacts with the residues that constitute the stable core of the protein thus shedding light on the origin of the observed perturbation. © 2016 Federation of European Biochemical Societies.

  19. N-Acetylaspartate Synthase Deficiency Corrects the Myelin Phenotype in a Canavan Disease Mouse Model But Does Not Affect Survival Time.

    Science.gov (United States)

    Maier, Helena; Wang-Eckhardt, Lihua; Hartmann, Dieter; Gieselmann, Volkmar; Eckhardt, Matthias

    2015-10-28

    Canavan disease (CD) is a severe, lethal leukodystrophy caused by deficiency in aspartoacylase (ASPA), which hydrolyzes N-acetylaspartate (NAA). In the brains of CD patients, NAA accumulates to high millimolar concentrations. The pathology of the disease is characterized by loss of oligodendrocytes and spongy myelin degeneration in the CNS. Whether accumulating NAA, absence of NAA-derived acetate, or absence of any unknown functions of the ASPA enzyme is responsible for the pathology of the disease is not fully understood. We generated ASPA-deficient (Aspa(nur7/nur7)) mice that are also deficient for NAA synthase Nat8L (Nat8L(-/-)/Aspa(nur7/nur7)). These mice have no detectable NAA. Nevertheless, they exhibited normal myelin content, myelin sphingolipid composition, and full reversal of spongy myelin and axonal degeneration. Surprisingly, although pathology was fully reversed, the survival time of the mice was not prolonged. In contrast, Aspa(nur7/nur7) mice with only one intact Nat8L allele accumulated less NAA, developed a less severe pathology, phenotypic improvements, and, importantly, an almost normal survival time. Therefore, inhibition of NAA synthase is a promising therapeutic option for CD. The reduced survival rate of Nat8L(-/-)/Aspa(nur7/nur7) mice, however, indicates that complete inhibition of NAA synthase may bear unforeseeable risks for the patient. Furthermore, we demonstrate that acetate derived from NAA is not essential for myelin lipid synthesis and that loss of NAA-derived acetate does not cause the myelin phenotype of Aspa(nur7/nur7) mice. Our data clearly support the hypothesis that NAA accumulation is the major factor in the development of CD. Copyright © 2015 the authors 0270-6474/15/3514501-16$15.00/0.

  20. Isolation and characterization of chalcone synthase gene isolated from Dendrobium Sonia Earsakul.

    Science.gov (United States)

    Pitakdantham, W; Sutabutra, T; Chiemsombat, P; Pitaksutheepong, C

    2010-10-15

    To isolate and characterize chalcone synthase gene in anthocyanin biosynthetic pathway during flower development of Dendrobium Sonia Earsakul. The gene was isolated from floral tissues of the orchid by reverse transcriptase polymerase chain reaction. Characterization of the gene considered to its relatedness to chalcone synthase gene in other orchid plants elucidated by construction of a neighbor-joining phylogenetic tree. Gene expression pattern related to flower development and pigmentation was investigated by relative quantification real time polymerase chain reaction. A complete coding sequence was obtained and sequence analysis revealed that the gene of Dendrobium Sonia Earsakul consisted of 1,188 bp. Blast analysis and multiple alignments showed that the chalcone synthase gene of Dendrobium Sonia Earsakul shares high homology to chalcone synthase gene of Dendrobium genus particularly Dendrobium hybrid Uniwai prince. Phylogenetic tree revealed that chalcone synthase of Dendrobium genus are highly conserved. The chalcone synthase gene of Dendrobium Sonia Earsakul was highly expressed in young flower bud with no pigmentation and the expression was sharply decreased when young flower bud started accumulation of pigments. Expression of chalcone synthase gene was then maintained at the same level until young bud developed into fully opened flowers.

  1. Nitric oxide signalling and neuronal nitric oxide synthase in the heart under stress

    Science.gov (United States)

    Zhang, Yin Hua

    2017-01-01

    Nitric oxide (NO) is an imperative regulator of the cardiovascular system and is a critical mechanism in preventing the pathogenesis and progression of the diseased heart. The scenario of bioavailable NO in the myocardium is complex: 1) NO is derived from both endogenous NO synthases (endothelial, neuronal, and/or inducible NOSs [eNOS, nNOS, and/or iNOS]) and exogenous sources (entero-salivary NO pathway) and the amount of NO from exogenous sources varies significantly; 2) NOSs are located at discrete compartments of cardiac myocytes and are regulated by distinctive mechanisms under stress; 3) NO regulates diverse target proteins through different modes of post-transcriptional modification (soluble guanylate cyclase [sGC]/cyclic guanosine monophosphate [cGMP]/protein kinase G [PKG]-dependent phosphorylation, S-nitrosylation, and transnitrosylation); 4) the downstream effectors of NO are multidimensional and vary from ion channels in the plasma membrane to signalling proteins and enzymes in the mitochondria, cytosol, nucleus, and myofilament; 5) NOS produces several radicals in addition to NO (e.g. superoxide, hydrogen peroxide, peroxynitrite, and different NO-related derivatives) and triggers redox-dependent responses. However, nNOS inhibits cardiac oxidases to reduce the sources of oxidative stress in diseased hearts. Recent consensus indicates the importance of nNOS protein in cardiac protection under pathological stress. In addition, a dietary regime with high nitrate intake from fruit and vegetables together with unsaturated fatty acids is strongly associated with reduced cardiovascular events. Collectively, NO-dependent mechanisms in healthy and diseased hearts are better understood and shed light on the therapeutic prospects for NO and NOSs in clinical applications for fatal human heart diseases. PMID:28649367

  2. Molecular mechanisms of neuronal nitric oxide synthase in cardiac function and pathophysiology

    Science.gov (United States)

    Zhang, Yin Hua; Jin, Chun Zi; Jang, Ji Hyun; Wang, Yue

    2014-01-01

    Neuronal nitric oxide synthase (nNOS or NOS1) is the major endogenous source of myocardial nitric oxide (NO), which facilitates cardiac relaxation and modulates contraction. In the healthy heart it regulates intracellular Ca2+, signalling pathways and oxidative homeostasis and is upregulated from early phases upon pathogenic insult. nNOS plays pivotal roles in protecting the myocardium from increased oxidative stress, systolic/diastolic dysfunction, adverse structural remodelling and arrhythmias in the failing heart. Here, we show that the downstream target proteins of nNOS and underlying post-transcriptional modifications are shifted during disease progression from Ca2+-handling proteins [e.g. PKA-dependent phospholamban phosphorylation (PLN-Ser16)] in the healthy heart to cGMP/PKG-dependent PLN-Ser16 with acute angiotensin II (Ang II) treatment. In early hypertension, nNOS-derived NO is involved in increases of cGMP/PKG-dependent troponin I (TnI-Ser23/24) and cardiac myosin binding protein C (cMBP-C-Ser273). However, nNOS-derived NO is shown to increase S-nitrosylation of various Ca2+-handling proteins in failing myocardium. The spatial compartmentation of nNOS and its translocation for diverse binding partners in the diseased heart or various nNOS splicing variants and regulation in response to pathological stress may be responsible for varied underlying mechanisms and functions. In this review, we endeavour to outline recent advances in knowledge of the molecular mechanisms mediating the functions of nNOS in the myocardium in both normal and diseased hearts. Insights into nNOS gene regulation in various tissues are discussed. Overall, nNOS is an important cardiac protector in the diseased heart. The dynamic localization and various mediating mechanisms of nNOS ensure that it is able to regulate functions effectively in the heart under stress. PMID:24756636

  3. Bacterial Nitric Oxide Synthase Is Required for the Staphylococcus aureus Response to Heme Stress.

    Science.gov (United States)

    Surdel, Matthew C; Dutter, Brendan F; Sulikowski, Gary A; Skaar, Eric P

    2016-08-12

    Staphylococcus aureus is a pathogen that causes significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for multiple cellular processes. Although required for pathogenesis, excess heme is toxic. S. aureus employs a two-component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for the pathogenesis of numerous Gram-positive organisms, yet the mechanism of heme sensing remains unknown. Compound '3981 was identified in a high-throughput screen as an activator of staphylococcal HssRS that triggers HssRS independently of heme accumulation. '3981 is toxic to S. aureus; however, derivatives of '3981 were synthesized that lack toxicity while retaining HssRS activation, enabling the interrogation of the heme stress response without confounding toxic effects of the parent molecule. Using '3981 derivatives as probes of the heme stress response, numerous genes required for '3981-induced activation of HssRS were uncovered. Specifically, multiple genes involved in the production of nitric oxide were identified, including the gene encoding bacterial nitric oxide synthase (bNOS). bNOS protects S. aureus from oxidative stress imposed by heme. Taken together, this work identifies bNOS as crucial for the S. aureus heme stress response, providing evidence that nitric oxide synthesis and heme sensing are intertwined.

  4. Effects of nitric oxide synthase inhibition on sympathetically-mediated tachycardia

    Science.gov (United States)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The aim of the present study was to determine whether inhibition of nitric oxide (NO) synthesis directly alters the tachycardia produced by sympathetically-derived norepinephrine. The NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 50 micromol/kg, i.v.), produced a marked rise in mean arterial blood pressure. This pressor response was associated with a fall in heart rate which involved the withdrawal of cardiac sympathetic nerve activity. The NO-donor, sodium nitroprusside (5 microg/kg, i.v.), produced a pronounced fall in mean arterial blood pressure but only a minor increase in heart rate. The beta-adrenoceptor agonist, isoproterenol (0.5 micromol/kg, i.v.), and the membrane-permeable cAMP analogue, 8-(4-chlorophenylthiol)-cAMP (10 micromol/kg, i.v.), produced falls in mean arterial blood pressure and pronounced increases in heart rate. The indirectly acting sympathomimetic agent, tyramine (0.5 mg/kg, i.v.), produced a pressor response and a tachycardia. The effects of sodium nitroprusside, tyramine, isoproterenol and 8-(4-chlorophenylthiol)-cAMP on mean arterial blood pressure were not markedly affected by L-NAME. However, the tachycardia produced by these agents was considerably exaggerated in the presence of this NO synthesis inhibitor. These findings suggest that L-NAME potentiates the tachycardia produced by sympathetically-derived norepinephrine. The increased responsiveness to norepinephrine may involve (i) a rapid up-regulation of cardiac beta1-adrenoceptors and cAMP signaling in cardiac pacemaker cells due to the loss of the inhibitory influence of cardiac NO, and (ii) the up-regulation of beta1-adrenoceptor-mediated signal transduction processes in response to the L-NAME-induced withdrawal of cardiac sympathetic nerve activity.

  5. Identification of sesquiterpene synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120.

    Science.gov (United States)

    Agger, Sean A; Lopez-Gallego, Fernando; Hoye, Thomas R; Schmidt-Dannert, Claudia

    2008-09-01

    Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-alpha-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-alpha-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.

  6. Characterization of three novel isoprenyl diphosphate synthases from the terpenoid rich mango fruit.

    Science.gov (United States)

    Kulkarni, Ram; Pandit, Sagar; Chidley, Hemangi; Nagel, Raimund; Schmidt, Axel; Gershenzon, Jonathan; Pujari, Keshav; Giri, Ashok; Gupta, Vidya

    2013-10-01

    Mango (cv. Alphonso) is popular due to its highly attractive, terpenoid-rich flavor. Although Alphonso is clonally propagated, its fruit-flavor composition varies when plants are grown in different geo-climatic zones. Isoprenyl diphosphate synthases catalyze important branch-point reactions in terpenoid biosynthesis, providing precursors for common terpenoids such as volatile terpenes, sterols and carotenoids. Two geranyl diphosphate synthases and a farnesyl diphosphate synthase were isolated from Alphonso fruits, cloned for recombinant expression and found to produce the respective products. Although, one of the geranyl diphosphate synthases showed high sequence similarity to the geranylgeranyl diphosphate synthases, it did not exhibit geranylgeranyl diphosphate synthesizing activity. When modeled, this geranyl diphosphate synthase and farnesyl diphosphate synthase structures were found to be homologous with the reference structures, having all the catalytic side chains appropriately oriented. The optimum temperature for both the geranyl diphosphate synthases was 40 °C and that for farnesyl diphosphate synthase was 25 °C. This finding correlated well with the dominance of monoterpenes in comparison to sesquiterpenes in the fruits of Alphonso mango in which the mesocarp temperature is higher during ripening than development. The absence of activity of these enzymes with the divalent metal ion other than Mg(2+) indicated their adaptation to the Mg(2+) rich mesocarp. The typical expression pattern of these genes through the ripening stages of fruits from different cultivation localities depicting the highest transcript levels of these genes in the stage preceding the maximum terpene accumulation indicated the involvement of these genes in the biosynthesis of volatile terpenes. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  7. Fractional Derivative as Fractional Power of Derivative

    OpenAIRE

    Tarasov, Vasily E.

    2007-01-01

    Definitions of fractional derivatives as fractional powers of derivative operators are suggested. The Taylor series and Fourier series are used to define fractional power of self-adjoint derivative operator. The Fourier integrals and Weyl quantization procedure are applied to derive the definition of fractional derivative operator. Fractional generalization of concept of stability is considered.

  8. The small subunit of snapdragon geranyl diphosphate synthase modifies the chain length specificity of tobacco geranylgeranyl diphosphate synthase in planta.

    Science.gov (United States)

    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

    2009-12-01

    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.

  9. The role of 1-deoxy-d-xylulose-5-phosphate synthase and phytoene synthase gene family in citrus carotenoid accumulation.

    Science.gov (United States)

    Peng, Gang; Wang, Chunyan; Song, Song; Fu, Xiumin; Azam, Muhammad; Grierson, Don; Xu, Changjie

    2013-10-01

    Three 1-deoxy-D-xylulose-5-phosphate synthases (DXS) and three phytoene synthases (PSY) were identified in citrus, from Affymetrix GeneChip Citrus Genome Array, GenBank and public orange genome databases. Tissue-specific expression analysis of these genes was carried out on fruit peel and flesh, flower and leaf of Satsuma mandarin (Citrus unshiu Marc.) in order to determine their roles in carotenoid accumulation in different tissues. Expression of CitDXS1 and CitPSY1 was highest in all test tissues, while that of CitDXS2 and CitPSY2 was lower, and that of CitDXS3 and CitPSY3 undetectable. The transcript profiles of CitDXS1 and CitPSY1 paralleled carotenoid accumulation in flesh of Satsuma mandarin and orange (Citrus sinensis Osbeck) during fruit development, and CitPSY1 expression was also associated with carotenoid accumulation in peel, while the CitDXS1 transcript level was only weakly correlated with carotenoid accumulation in peel. Similar results were obtained following correlation analysis between expression of CitDXS1 and CitPSY1 and carotenoid accumulation in peel and flesh of 16 citrus cultivars. These findings identify CitPSY1 and CitDXS1 as the main gene members controlling carotenoid biosynthesis in citrus fruit. Furthermore, chromoplasts were extracted from flesh tissue of these citrus, and chromoplasts of different shape (spindle or globular), different size, and color depth were observed in different cultivars, indicating chromoplast abundance, number per gram tissue, size and color depth were closely correlated with carotenoid content in most cultivars. The relationship between carotenoid biosynthesis and chromoplast development was discussed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  10. Microsatellite instability and the association with plasma homocysteine and thymidylate synthase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Lindebjerg, Jan; Crüger, Dorthe G.

    2008-01-01

    The possible associations between microsatellite instability, homocysteine and thymidylate synthase were investigated in tumors and plasma from 130 patients with colorectal cancer. Other analyses included thymidylate synthase and 5,10-methylene-tetrahydrofolate reductase gene polymorphisms......, carcinoembryonic antigen, vitamin B12, and folate. Microsatellite instability of tumors was associated with higher levels of plasma homocysteine (p = 0.008) and higher protein expression of thymidylate synthase (p ... factors. CEA was not associated with neither homocysteine nor microsatellite instability. The data suggests that there is a more pronounced methyl unit deficiency in microsatellite instable tumors....

  11. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  12. Fatty acid synthase cooperates with glyoxalase 1 to protect against sugar toxicity.

    Directory of Open Access Journals (Sweden)

    Damien Garrido

    2015-02-01

    Full Text Available Fatty acid (FA metabolism is deregulated in several human diseases including metabolic syndrome, type 2 diabetes and cancers. Therefore, FA-metabolic enzymes are potential targets for drug therapy, although the consequence of these treatments must be precisely evaluated at the organismal and cellular levels. In healthy organism, synthesis of triacylglycerols (TAGs-composed of three FA units esterified to a glycerol backbone-is increased in response to dietary sugar. Saturation in the storage and synthesis capacity of TAGs is associated with type 2 diabetes progression. Sugar toxicity likely depends on advanced-glycation-end-products (AGEs that form through covalent bounding between amine groups and carbonyl groups of sugar or their derivatives α-oxoaldehydes. Methylglyoxal (MG is a highly reactive α-oxoaldehyde that is derived from glycolysis through a non-enzymatic reaction. Glyoxalase 1 (Glo1 works to neutralize MG, reducing its deleterious effects. Here, we have used the power of Drosophila genetics to generate Fatty acid synthase (FASN mutants, allowing us to investigate the consequence of this deficiency upon sugar-supplemented diets. We found that FASN mutants are lethal but can be rescued by an appropriate lipid diet. Rescued animals do not exhibit insulin resistance, are dramatically sensitive to dietary sugar and accumulate AGEs. We show that FASN and Glo1 cooperate at systemic and cell-autonomous levels to protect against sugar toxicity. We observed that the size of FASN mutant cells decreases as dietary sucrose increases. Genetic interactions at the cell-autonomous level, where glycolytic enzymes or Glo1 were manipulated in FASN mutant cells, revealed that this sugar-dependent size reduction is a direct consequence of MG-derived-AGE accumulation. In summary, our findings indicate that FASN is dispensable for cell growth if extracellular lipids are available. In contrast, FA-synthesis appears to be required to limit a cell

  13. On $n$-derivations

    Directory of Open Access Journals (Sweden)

    Mohammad Hossein Sattari

    2016-06-01

    Full Text Available In this article, the notion of $n-$derivation is introduced for all integers $ngeq 2$. Although all derivations are $n-$derivations,  in general these notions are not equivalent. Some properties of ordinary derivations are  investigated for $n-$derivations. Also, we show that under certain mild condition  $n-$derivations are derivations.

  14. In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression

    Energy Technology Data Exchange (ETDEWEB)

    Rimando A. M.; Liu C.; Pan, Z.; Polashock, J. J.; Dayan, F. E., Mizuno, C. S.; Snook, M. E.; Baerson, S. R.

    2012-04-01

    Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

  15. Exploring second coordination sphere effects in nitric oxide synthase.

    Science.gov (United States)

    McQuarters, Ashley B; Speelman, Amy L; Chen, Li; Elmore, Bradley O; Fan, Weihong; Feng, Changjian; Lehnert, Nicolai

    2016-12-01

    Second coordination sphere (SCS) effects in proteins are modulated by active site residues and include hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic residues. In Cyt P450s, extended H-bonding networks are located around the proximal cysteinate ligand of the heme, referred to as the 'Cys pocket'. These hydrogen bonding networks are generally believed to regulate the Fe-S interaction. Previous work identified the S(Cys) → Fe σ CT transition in the high-spin (hs) ferric form of Cyt P450cam and corresponding Cys pocket mutants by low-temperature (LT) MCD spectroscopy [Biochemistry 50:1053, 2011]. In this work, we have investigated the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in the hs ferric state (with H4B and L-Arg bound) of rat neuronal nitric oxide synthase oxygenase construct (nNOSoxy) using MCD spectroscopy. For this purpose, wt enzyme and W409 mutants were investigated where the H-bonding network with the axial Cys ligand is perturbed. Overall, the results are similar to Cyt P450cam and show the intense S(Cys) → Fe σ CT band in the LT MCD spectrum at about 27,800 cm-1, indicating that this feature is a hallmark of {heme-thiolate} active sites. The discovery of this MCD feature could constitute a new approach to classify {heme-thiolate} sites in hs ferric proteins. Finally, the W409 mutants show that the hydrogen bond from this group only has a small effect on the Fe-S(Cys) bond strength, at least in the hs ferric form of the protein studied here. Low-temperature MCD spectroscopy is used to investigate the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in neuronal nitric oxide synthase. The intense S(Cys) → Fe σ-CT band is monitored to identify changes in the Fe-S(Cys) bond in wild-type protein and W409 mutants.

  16. Flavin-dependent thymidylate synthase: N5 of flavin as a Methylene carrier.

    Science.gov (United States)

    Karunaratne, Kalani; Luedtke, Nicholas; Quinn, Daniel M; Kohen, Amnon

    2017-10-15

    Thymidylate is synthesized de novo in all living organisms for replication of genomes. The chemical transformation is reductive methylation of deoxyuridylate at C5 to form deoxythymidylate. All eukaryotes including humans complete this well-understood transformation with thymidylate synthase utilizing 6R-N 5 -N 10 -methylene-5,6,7,8-tetrahydrofolate as both a source of methylene and a reducing hydride. In 2002, flavin-dependent thymidylate synthase was discovered as a new pathway for de novo thymidylate synthesis. The flavin-dependent catalytic mechanism is different than thymidylate synthase because it requires flavin as a reducing agent and methylene transporter. This catalytic mechanism is not well-understood, but since it is known to be very different from thymidylate synthase, there is potential for mechanism-based inhibitors that can selectively inhibit the flavin-dependent enzyme to target many human pathogens with low host toxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

    Czech Academy of Sciences Publication Activity Database

    Benoni, Roberto; De Bei, O.; Paredi, G.; Hayes, C. S.; Franko, N.; Mozzarelli, A.; Bettati, S.; Campanini, B.

    2017-01-01

    Roč. 591, č. 9 (2017), s. 1212-1224 ISSN 0014-5793 Institutional support: RVO:61388963 Keywords : cysteine synthase * protein-protein interaction * serine acetyltransferase Subject RIV: CE - Biochemistry Impact factor: 3.623, year: 2016

  18. (SNP) of endothelial nitric oxide synthase gene and serum level of ...

    African Journals Online (AJOL)

    T-786C single-nucleotide polymorphism (SNP) of endothelial nitric oxide synthase gene and serum level of vascular endothelial relaxant factor (VERF) in non-diabetic patients with coronary artery disease.

  19. Inhibition of a multiproduct terpene synthase from Medicago truncatula by 3-bromoprenyl diphosphates.

    Science.gov (United States)

    Vattekkatte, Abith; Gatto, Nathalie; Schulze, Eva; Brandt, Wolfgang; Boland, Wilhelm

    2015-04-28

    The multiproduct sesquiterpene synthase MtTPS5 from Medicago truncatula catalyzes the conversion of farnesyl diphosphate (FDP) into a complex mixture of 27 terpenoids. 3-Bromo substrate analogues of geranyl diphosphate (3-BrGDP) and farnesyl diphosphate (3-BrFDP) were evaluated as substrates of MTPS5 enzyme. Kinetic studies demonstrated that these compounds were highly potent competitive inhibitors of the MtTPS5 enzyme with fast binding and slow reversibility. Since there is a lack of knowledge about the crystal structure of multiproduct terpene synthases, these molecules might be ideal candidates for obtaining a co-crystal structure with multiproduct terpene synthases. Due to the structural and mechanistic similarity between various terpene synthases we expect these 3-bromo isoprenoids to be ideal probes for crystal structure studies.

  20. Immunobiology of Nitric Oxide and Regulation of Inducible Nitric Oxide Synthase.

    Science.gov (United States)

    Lee, Martin; Rey, Kevin; Besler, Katrina; Wang, Christine; Choy, Jonathan

    Nitric oxide (NO) is a bioactive gas that has multiple roles in innate and adaptive immune responses. In macrophages, nitric oxide is produced by inducible nitric oxide synthase upon microbial and cytokine stimulation. It is needed for host defense against pathogens and for immune regulation. This review will summarize the role of NO and iNOS in inflammatory and immune responses and will discuss the regulatory mechanisms that control inducible nitric oxide synthase expression and activity.

  1. Identification and Characterization of a Novel Deoxyhypusine Synthase in Leishmania donovani*

    OpenAIRE

    Chawla, Bhavna; Jhingran, Anupam; Singh, Sushma; Tyagi, Nidhi; Park, Myung Hee; Srinivasan, N.; Roberts, Sigrid C.; Madhubala, Rentala

    2009-01-01

    Deoxyhypusine synthase, an NAD+-dependent enzyme, catalyzes the first step in the post-translational synthesis of an unusual amino acid, hypusine (Nϵ-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic initiation factor 5A precursor protein. Two putative deoxyhypusine synthase (DHS) sequences have been identified in the Leishmania donovani genome, which are present on chromosomes 20: DHSL20 (DHS-like gene from chromosome 20) and DHS34 (DHS from chromosome 34). Although both sequences exhibit a...

  2. Probing the Mechanism of 1,4-Conjugate Elimination Reactions Catalyzed by Terpene Synthases

    OpenAIRE

    Faraldos, Juan A.; Gonzalez, Veronica; Li, Amang; Yu, Fanglei; Köksal, Mustafa; Christianson, David W.; Allemann, Rudolf K.

    2012-01-01

    The reaction mechanisms of (E)-β-farnesene synthase (EBFS) and isoprene synthase (ISPS), enzymes that catalyze a formal regioespecific 1,4-conjugate elimination of hydrogen-diphosphate from (E, E)-farnesyl and dimethylallyl diphosphate (FDP and DMADP) to generate the semiochemicals (E)-β-farnesene and isoprene, respectively, were probed with substrate analogs and kinetic measurements. The results support stepwise reaction mechanisms through analogous enzyme-bound allylic cationic intermediate...

  3. Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

    Science.gov (United States)

    Göpfert, Jens C; MacNevin, Gillian; Ro, Dae-Kyun; Spring, Otmar

    2009-01-01

    Background Sesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae) which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development. Results Glandular trichomes of sunflower (Helianthus annuus L.) were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of in vitro and in vivo characterization of sesquiterpene synthase gene products in Escherichia coli and Saccharomyces cerevisiae, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low in vitro activity, the third enzyme was expressed in vivo in yeast as a thioredoxin-fusion protein for functional characterization. In in vivo assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon δ-cadinene as one of the two main products with α-muuorlene, β-caryophyllene, α-humulene and α-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes as well. Conclusion This

  4. Alterations of inducible and constitutive nitric oxide synthase after hippocampal injury in rats.

    Science.gov (United States)

    Safari, M; Ghahari, L

    2009-08-15

    The aim of this study was to study the changes of inducible and constitutive Nitric Oxide Synthase (NOS) after brain injury. In order to brain injury 42 wistar rats were submitted and divided in 7 groups. Nitric oxide synthase activities were assayed at different times after injury. Present results showed that a significant increase of iNOS and cNOS activity 8 h after lesion. In conclusion, both isoformes of NOS increase at different time after brain injury.

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

    Science.gov (United States)

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

    2012-03-01

    Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and β-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/β-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively). Copyright © 2011 Elsevier GmbH. All rights reserved.

  6. Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic Overstimulation: Role of Perivascular Adipose Tissue.

    Science.gov (United States)

    Victorio, Jamaira A; Clerici, Stefano P; Palacios, Roberto; Alonso, María J; Vassallo, Dalton V; Jaffe, Iris Z; Rossoni, Luciana V; Davel, Ana P

    2016-09-01

    Sustained stimulation of β-adrenoceptors (β-ARs) and activation of renin-angiotensin-aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by β-AR overstimulation. β-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase-derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue-derived corticosterone in association with increased expression of 11β-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by β-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by β-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation. © 2016 The Authors.

  7. Drugs targeting nitric oxide synthase for migraine treatment.

    Science.gov (United States)

    Barbanti, Piero; Egeo, Gabriella; Aurilia, Cinzia; Fofi, Luisa; Della-Morte, David

    2014-08-01

    Ample evidence that nitric oxide (NO) is a causative molecule in migraine has encouraged research to develop drugs that target the NO-cGMP cascade for migraine treatment. NO synthase (NOS) inhibition is an innovative therapeutic principle. This paper reviews the rationale underlying NOS inhibition in migraine treatment. It also provides a review on the efficacy and safety data for NOS inhibitors (nonselective NOS inhibitor L-N(G)-methyl-arginine hydrochloride [L-NMMA], selective inducible NOS [iNOS] inhibitors GW273629 and GW274150, combined neuronal NOS [nNOS] inhibitor and 5-HT1B/1D receptor agonist NXN-188) in acute or preventive migraine treatment. The data highlighted herein, from four placebo-controlled trials and 1 open-labeled clinical trial using 4 different NOS inhibitors on a total of 705 patients, provide convincing efficacy data only for the nonselective NOS inhibitor L-NMMA. Unfortunately, this NOS inhibitor raises cardiovascular safety concerns and has an unfavorable pharmacokinetic profile. As experimental studies predicted, iNOS inhibitors are ineffective in migraine. Still, upcoming selective nNOS inhibitors are a hope for migraine treatment, with the nNOS isoform being most clearly involved in trigeminovascular transmission and central sensitization. Future studies should help to clarify whether NOS inhibition is equally fruitful in acute and preventive treatment. It should also clarify if nNOS inhibition holds promise as a therapeutic tool for the treatment of chronic migraine and other forms of headache.

  8. Arginine and nitric oxide synthase: regulatory mechanisms and cardiovascular aspects.

    Science.gov (United States)

    Lorin, Julie; Zeller, Marianne; Guilland, Jean-Claude; Cottin, Yves; Vergely, Catherine; Rochette, Luc

    2014-01-01

    L-Arginine (L-Arg) is a conditionally essential amino acid in the human diet. The most common dietary sources of L-Arg are meat, poultry and fish. L-Arg is the precursor for the synthesis of nitric oxide (NO); a key signaling molecule via NO synthase (NOS). Endogenous NOS inhibitors such as asymmetric-dimethyl-L-Arg inhibit NO synthesis in vivo by competing with L-Arg at the active site of NOS. In addition, NOS possesses the ability to be "uncoupled" to produce superoxide anion instead of NO. Reduced NO bioavailability may play an essential role in cardiovascular pathologies and metabolic diseases. L-Arg deficiency syndromes in humans involve endothelial inflammation and immune dysfunctions. Exogenous administration of L-Arg restores NO bioavailability, but it has not been possible to demonstrate, that L-Arg supplementation improved endothelial function in cardiovascular disease such as heart failure or hypertension. L-Arg supplementation may be a novel therapy for obesity and metabolic syndrome. The utility of l-Arg supplementation in the treatment of L-Arg deficiency syndromes remains to be established. Clinical trials need to continue to determine the optimal concentrations and combinations of L-Arg, with other protective compounds such as tetrahydrobiopterin (BH4 ), and antioxidants to combat oxidative stress that drives down NO production in humans. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Expression, Purification, and Characterisation of Dehydroquinate Synthase from Pyrococcus furiosus

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    Leonardo Negron

    2011-01-01

    Full Text Available Dehydroquinate synthase (DHQS catalyses the second step of the shikimate pathway to aromatic compounds. DHQS from the archaeal hyperthermophile Pyrococcus furiosus was insoluble when expressed in Escherichia coli but was partially solubilised when KCl was included in the cell lysis buffer. A purification procedure was developed, involving lysis by sonication at 30∘C followed by a heat treatment at 70∘C and anion exchange chromatography. Purified recombinant P. furiosus DHQS is a dimer with a subunit Mr of 37,397 (determined by electrospray ionisation mass spectrometry and is active over broad pH and temperature ranges. The kinetic parameters are KM (3-deoxy-D-arabino-heptulosonate 7-phosphate 3.7 μM and kcat 3.0 sec-1 at 60∘C and pH 6.8. EDTA inactivates the enzyme, and enzyme activity is restored by several divalent metal ions including (in order of decreasing effectiveness Cd2+, Co2+, Zn2+, and Mn2+. High activity of a DHQS in the presence of Cd2+ has not been reported for enzymes from other sources, and may be related to the bioavailability of Cd2+ for P. furiosus. This study is the first biochemical characterisation of a DHQS from a thermophilic source. Furthermore, the characterisation of this hyperthermophilic enzyme was carried out at elevated temperatures using an enzyme-coupled assay.

  10. Structure-Based Discovery of Inhibitors of Thymidylate Synthase

    Science.gov (United States)

    Shoichet, Brian K.; Stroud, Robert M.; Santi, Daniel V.; Kuntz, Irwin D.; Perry, Kathy M.

    1993-03-01

    A molecular docking computer program (DOCK) was used to screen the Fine Chemical Directory, a database of commercially available compounds, for molecules that are complementary to thymidylate synthase (TS), a chemotherapeutic target. Besides retrieving the substrate and several known inhibitors, DOCK proposed putative inhibitors previously unknown to bind to the enzyme. Three of these compounds inhibited Lactobacillus caser TS at submillimolar concentrations. One of these inhibitors, sulisobenzone, crystallized with TS in two configurations that differed from the DOCK-favored geometry: a counterion was bound in the substrate site, which resulted in a 6 to 9 angstrom displacement of the inhibitor. The structure of the complexes suggested another binding region in the active site that could be exploited. This region was probed with molecules sterically similar to sulisobenzone, which led to the identification of a family of phenolphthalein analogs that inhibit TS in the 1 to 30 micromolar range. These inhibitors do not resemble the substrates of the enzyme. A crystal structure of phenolphthalein with TS shows that it binds in the target site in a configuration that resembles the one suggested by DOCK.

  11. Conservation and Role of Electrostatics in Thymidylate Synthase

    Science.gov (United States)

    Garg, Divita; Skouloubris, Stephane; Briffotaux, Julien; Myllykallio, Hannu; Wade, Rebecca C.

    2015-11-01

    Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome.

  12. Analysis of mRNA recognition by human thymidylate synthase.

    Science.gov (United States)

    Brunn, Nicholas D; Dibrov, Sergey M; Kao, Melody B; Ghassemian, Majid; Hermann, Thomas

    2014-12-23

    Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2'-deoxyuridine-5'-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix-loop-helix domain on the protein surface was identified as the putative RNA-binding site.

  13. Tumor cell responses to inhibition of thymidylate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Keyomarsi, K.

    1989-01-01

    The cellular, biochemical and molecular events that occur in tumor cells treated with inhibitors of thymidylate synthase (TS) were studied. 5-Fluorouracil (5-FUra) and fluorodeoxyuridine (FdUrd) are more growth inhibitory to mouse and human tumor cells when grown in medium containing folinate. L1210 cells exposed to folinate and noncytotoxic concentrations of 5-FUra or FdUrd, resulted in a 98% to 99.98% cell kill. Exposure of L1210 cells to folinate resulted in expansion of intracellular pools of 5,10-methylenetetrahydrofolate, delayed the reappearance of catalytically active TS following FdUrd exposure, and stabilized inactive TS complexes over the same concentration range that augmented the cytotoxic effect of FdUrd and 5-FUra. In intact L1210 cells, fluorodeoxyuridylate (FdUMP) behaved as an inhibitor whose complexes with TS dissociate with a biologically significant rate. However, these complexes become functionally irreversible in cells incubated with high levels of folinate. CB 3717 eliminated TS activity in L1210 cells, yet the inactive enzyme retained the ability to bind ({sup 3}H)-FdUMP covalently, suggesting that the binding of one subunit of TS inactivates the catalytic activity of both subunits.

  14. Oxidative Stress and Response to Thymidylate Synthase-Targeted Antimetabolites.

    Science.gov (United States)

    Ozer, Ufuk; Barbour, Karen W; Clinton, Sarah A; Berger, Franklin G

    2015-12-01

    Thymidylate synthase (TYMS; EC 2.1.1.15) catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) by N(5),N(10)-methyhlenetetrahydrofolate, forming dTMP for the maintenance of DNA replication and repair. Inhibitors of TYMS have been widely used in the treatment of neoplastic disease. A number of fluoropyrimidine and folate analogs have been developed that lead to inhibition of the enzyme, resulting in dTMP deficiency and cell death. In the current study, we have examined the role of oxidative stress in response to TYMS inhibitors. We observed that intracellular reactive oxygen species (ROS) concentrations are induced by these inhibitors and promote apoptosis. Activation of the enzyme NADPH oxidase (NOX), which catalyzes one-electron reduction of O2 to generate superoxide (O2 (●-)), is a significant source of increased ROS levels in drug-treated cells. However, gene expression profiling revealed a number of other redox-related genes that may contribute to ROS generation. TYMS inhibitors also induce a protective response, including activation of the transcription factor nuclear factor E2-related factor 2 (NRF2), a critical mediator of defense against oxidative and electrophilic stress. Our results show that exposure to TYMS inhibitors induces oxidative stress that leads to cell death, while simultaneously generating a protective response that may underlie resistance against such death. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  15. Hyperbaric oxygen upregulates cochlear constitutive nitric oxide synthase

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    Kao Ming-Ching

    2011-02-01

    Full Text Available Abstract Background Hyperbaric oxygen therapy (HBOT is a known adjuvant for treating ischemia-related inner ear diseases. Controversies still exist in the role of HBOT in cochlear diseases. Few studies to date have investigated the cellular changes that occur in inner ears after HBOT. Nitric oxide, which is synthesized by nitric oxide synthase (NOS, is an important signaling molecule in cochlear physiology and pathology. Here we investigated the effects of hyperbaric oxygen on eardrum morphology, cochlear function and expression of NOS isoforms in cochlear substructures after repetitive HBOT in guinea pigs. Results Minor changes in the eardrum were observed after repetitive HBOT, which did not result in a significant hearing threshold shift by tone burst auditory brainstem responses. A differential effect of HBOT on the expression of NOS isoforms was identified. Upregulation of constitutive NOS (nNOS and eNOS was found in the substructures of the cochlea after HBOT, but inducible NOS was not found in normal or HBOT animals, as shown by immunohistochemistry. There was no obvious DNA fragmentation present in this HBOT animal model. Conclusions The present evidence indicates that the customary HBOT protocol may increase constitutive NOS expression but such upregulation did not cause cell death in the treated cochlea. The cochlear morphology and auditory function are consequently not changed through the protocol.

  16. Inducible nitric oxide synthase immunoreactivity in healthy rat pancreas.

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    Nurullah Keklikoglu

    2008-06-01

    Full Text Available Nitric oxide (NO is produced by NO synthase (NOS isoforms: neuronal NOS (nNOS, endothelial NOS (eNOS and inducible NOS (iNOS. It is believed that, while nNOS and eNOS are effective in regulation of normal physiological processes, iNOS is expressed at an increasing rate especially in inflammatory process. The aim of this study was to determine the presence of iNOS immunoreactivity (iNOS-IR and, to compare the iNOS-IR in islet of Langerhans cells (LC, acinar cells (AC, centroacinar cells (CC and ductal cells (DC by immunohistochemical (IHC method in healthy rat pancreata. This study revealed the presence of iNOS-IR in all cell types except AC. Statistical analysis revealed a highly significant difference (p<0.001 with respect to iNOS-IR in comparison of all cell types. However, binary comparison of cell types revealed no significant differences between LC and DC (p=0.136, significant differences LC and CC, CC and DC (p=0.001 and 0.022, respectively and a highly significant differences LC and AC, AC and DC (P<0.001. The results of this study indicate that iNOS-IR is present in almost all LC. Thus, especially in reseach related to diabetes, it should not be disregarded that iNOS may be constitutively present in pancreatic islets.

  17. Nitric oxide synthase in the vestibulocochlear system of mice.

    Science.gov (United States)

    Hess, A; Bloch, W; Arnhold, S; Andressen, C; Stennert, E; Addicks, K; Michel, O

    1998-11-30

    The exact distribution of nitric oxide-synthases (NOS) and the NO-target enzyme soluble guanylyl cyclase (sGC) in the cochlea and vestibular organ is an issue of current discussion. The existence of NOS-isoforms in the cochlea of the guinea pig has been described recently, while information about the vestibular system are still rare and non-satisfying. In order to gain more information, immunostaining was performed, using specific antibodies to NOS I-III and to sGC, on paraffin sections of complete temporal bones from mice. NOS III could be detected in cochlea and vestibular ganglion cells, in nerve fibres, in outer hair cells of the cochlear and in the sensory epithelium of the maculae. Also, the spiral ligament and the limbus epithelium was positive to NOS III. NOS I was found in the sensory epithelium of the maculae and cristae ampullares, outer and inner hair cells of the cochlea, in nerve fibres and in ganglion cells. In contrast to that NOS II could not be detected at all. Furthermore, a strong NOS I immunoreaction was displayed on the endosteum of the bone, while the periosteum was lacking of NOS. NOS detection was accompanied by immunoreactivity to sGC. The findings imply that NOS I and III-generated NO is involved in neurotransmission and other regulative processes in the vestibulocochlear system. Copyright 1998 Elsevier Science B.V.

  18. Nitric oxide synthase deficiency and the pathophysiology of muscular dystrophy

    Science.gov (United States)

    Tidball, James G; Wehling-Henricks, Michelle

    2014-01-01

    The secondary loss of neuronal nitric oxide synthase (nNOS) that occurs in dystrophic muscle is the basis of numerous, complex and interacting features of the dystrophic pathology that affect not only muscle itself, but also influence the interaction of muscle with other tissues. Many mechanisms through which nNOS deficiency contributes to misregulation of muscle development, blood flow, fatigue, inflammation and fibrosis in dystrophic muscle have been identified, suggesting that normalization in NO production could greatly attenuate diverse aspects of the pathology of muscular dystrophy through multiple regulatory pathways. However, the relative importance of the loss of nNOS from the sarcolemma versus the importance of loss of total nNOS from dystrophic muscle remains unknown. Although most current evidence indicates that nNOS localization at the sarcolemma is not required to achieve NO-mediated reductions of pathology in muscular dystrophy, the question remains open concerning whether membrane localization would provide a more efficient rescue from features of the dystrophic phenotype. PMID:25194047

  19. Two Distinct Cardiolipin Synthases Operate in Agrobacterium tumefaciens.

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    Simon Czolkoss

    Full Text Available Cardiolipin (CL is a universal component of energy generating membranes. In most bacteria, it is synthesized via the condensation of two molecules phosphatidylglycerol (PG by phospholipase D-type cardiolipin synthases (PLD-type Cls. In the plant pathogen and natural genetic engineer Agrobacterium tumefaciens CL comprises up to 15% of all phospholipids in late stationary growth phase. A. tumefaciens harbors two genes, atu1630 (cls1 and atu2486 (cls2, coding for PLD-type Cls. Heterologous expression of either cls1 or cls2 in Escherichia coli resulted in accumulation of CL supporting involvement of their products in CL synthesis. Expression of cls1 and cls2 in A. tumefaciens is constitutive and irrespective of the growth phase. Membrane lipid profiling of A. tumefaciens mutants suggested that Cls2 is required for CL synthesis at early exponential growth whereas both Cls equally contribute to CL production at later growth stages. Contrary to many bacteria, which suffer from CL depletion, A. tumefaciens tolerates large changes in CL content since the CL-deficient cls1/cls2 double mutant showed no apparent defects in growth, stress tolerance, motility, biofilm formation, UV-stress and tumor formation on plants.

  20. Alternatively spliced neuronal nitric oxide synthase mediates penile erection

    Science.gov (United States)

    Hurt, K. Joseph; Sezen, Sena F.; Champion, Hunter C.; Crone, Julie K.; Palese, Michael A.; Huang, Paul L.; Sawa, Akira; Luo, Xiaojiang; Musicki, Biljana; Snyder, Solomon H.; Burnett, Arthur L.

    2006-01-01

    A key role for nitric oxide (NO) in penile erection is well established, but the relative roles of the neuronal NO synthase (nNOS) versus endothelial forms of NOS are not clear. nNOS- and endothelial NOS-deficient mice maintain erectile function and reproductive capacity, questioning the importance of NO. Alternatively, residual NO produced by shorter transcripts in the nNOS−/− animals might suffice for normal physiologic function. We show that the β splice variant of nNOS elicits normal erection despite a decrease in stimulus-response characteristics and a 5-fold increased sensitivity to the NOS inhibitor, l-NAME. Residual nNOSβ generates only 10% of the normal NO level in vitro but produces citrulline and diaphorase staining reflecting in vivo NOS activity in pelvic ganglion nerves that is comparable to WT animals. Thus, alternatively spliced forms of nNOS are major mediators of penile erection and so may be targets for therapeutic intervention. PMID:16488973

  1. SUCROSE SYNTHASE: ELUCIDATION OF COMPLEX POST-TRANSLATIONAL REGULATORY MECHANISMS

    Energy Technology Data Exchange (ETDEWEB)

    Steven C. Huber

    2009-05-12

    Studies have focused on the enzyme sucrose synthase, which plays an important role in the metabolism of sucrose in seeds and tubers. There are three isoforms of SUS in maize, referred to as SUS1, SUS-SH1, and SUS2. SUS is generally considered to be tetrameric protein but recent evidence suggests that SUS can also occur as a dimeric protein. The formation of tetrameric SUS is regulated by sucrose concentration in vitro and this could also be an important factor in the cellular localization of the protein. We found that high sucrose concentrations, which promote tetramer formation, also inhibit the binding of SUS1 to actin filaments in vitro. Previously, high sucrose concentrations were shown to promote SUS association with the plasma membrane. The specific regions of the SUS molecule involved in oligomerization are not known, but we identified a region of the SUS1 moelcule by bioinformatic analysis that was predicted to form a coiled coil. We demonstrated that this sequence could, in fact, self-associate as predicted for a coiled coil, but truncation analysis with the full-length recombinant protein suggested that it was not responsible for formation of dimers or tetramers. However, the coiled coil may function in binding of other proteins to SUS1. Overall, sugar availability may differentially influence the binding of SUS to cellular structures, and these effects may be mediated by changes in the oligomeric nature of the enzyme.

  2. Computational insights into the mechanism of porphobilinogen synthase.

    Science.gov (United States)

    Erdtman, Edvin; Bushnell, Eric A C; Gauld, James W; Eriksson, Leif A

    2010-12-23

    Porphobilinogen synthase (PBGS) is a key enzyme in heme biosynthesis that catalyzes the formation of porphobilinogen (PBG) from two 5-aminolevulinic acid (5-ALA) molecules via formation of intersubstrate C-N and C-C bonds. The active site consists of several invariant residues, including two lysyl residues (Lys210 and Lys263; yeast numbering) that bind the two substrate moieties as Schiff bases. Based on experimental studies, various reaction mechanisms have been proposed for this enzyme that generally can be classified according to whether the intersubstrate C-C or C-N bond is formed first. However, the detailed catalytic mechanism of PBGS remains unclear. In the present study, we have employed density functional theory methods in combination with chemical models of the two key lysyl residues and two substrate moieties in order to investigate various proposed reaction steps and gain insight into the mechanism of PBGS. Importantly, it is found that mechanisms in which the intersubstrate C-N bond is formed first have a rate-limiting barrier (17.5 kcal/mol) that is lower than those in which the intersubstrate C-C bond is formed first (22.8 kcal/mol).

  3. Two Distinct Cardiolipin Synthases Operate in Agrobacterium tumefaciens.

    Science.gov (United States)

    Czolkoss, Simon; Fritz, Christiane; Hölzl, Georg; Aktas, Meriyem

    2016-01-01

    Cardiolipin (CL) is a universal component of energy generating membranes. In most bacteria, it is synthesized via the condensation of two molecules phosphatidylglycerol (PG) by phospholipase D-type cardiolipin synthases (PLD-type Cls). In the plant pathogen and natural genetic engineer Agrobacterium tumefaciens CL comprises up to 15% of all phospholipids in late stationary growth phase. A. tumefaciens harbors two genes, atu1630 (cls1) and atu2486 (cls2), coding for PLD-type Cls. Heterologous expression of either cls1 or cls2 in Escherichia coli resulted in accumulation of CL supporting involvement of their products in CL synthesis. Expression of cls1 and cls2 in A. tumefaciens is constitutive and irrespective of the growth phase. Membrane lipid profiling of A. tumefaciens mutants suggested that Cls2 is required for CL synthesis at early exponential growth whereas both Cls equally contribute to CL production at later growth stages. Contrary to many bacteria, which suffer from CL depletion, A. tumefaciens tolerates large changes in CL content since the CL-deficient cls1/cls2 double mutant showed no apparent defects in growth, stress tolerance, motility, biofilm formation, UV-stress and tumor formation on plants.

  4. Conservation and Role of Electrostatics in Thymidylate Synthase.

    Science.gov (United States)

    Garg, Divita; Skouloubris, Stephane; Briffotaux, Julien; Myllykallio, Hannu; Wade, Rebecca C

    2015-11-27

    Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome.

  5. Oncogene dependent requirement of fatty acid synthase in hepatocellular carcinoma.

    Science.gov (United States)

    Che, Li; Pilo, Maria G; Cigliano, Antonio; Latte, Gavinella; Simile, Maria M; Ribback, Silvia; Dombrowski, Frank; Evert, Matthias; Chen, Xin; Calvisi, Diego F

    2017-03-19

    Hepatocellular carcinoma (HCC), the most frequent primary tumor of the liver, is an aggressive cancer type with limited treatment options. Cumulating evidence underlines a crucial role of aberrant lipid biosynthesis (a process known as de novo lipogenesis) along carcinogenesis. Previous studies showed that suppression of fatty acid synthase (FASN), the major enzyme responsible for de novo lipogenesis, is highly detrimental for the in vitro growth of HCC cell lines. To assess whether de novo lipogenesis is required for liver carcinogenesis, we have generated various mouse models of liver cancer by stably overexpressing candidate oncogenes in the mouse liver via hydrodynamic gene delivery. We found that overexpression of FASN in the mouse liver is unable to malignantly transform hepatocytes. However, genetic deletion of FASN totally suppresses hepatocarcinogenesis driven by AKT and AKT/c-Met protooncogenes in mice. On the other hand, liver tumor development is completely unaffected by FASN depletion in mice co-expressing β-catenin and c-Met. Our data indicate that tumors might be either addicted to or independent from de novo lipogenesis for their growth depending on the oncogenes involved. Additional investigation is required to unravel the molecular mechanisms whereby some oncogenes render cancer cells resistant to inhibition of de novo lipogenesis.

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

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    Na Shang

    2014-05-01

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

  7. Inhibition studies of Mycobacterium tuberculosis salicylate synthase (MbtI).

    Science.gov (United States)

    Manos-Turvey, Alexandra; Bulloch, Esther M M; Rutledge, Peter J; Baker, Edward N; Lott, J Shaun; Payne, Richard J

    2010-07-05

    Mycobacterium tuberculosis salicylate synthase (MbtI), a member of the chorismate-utilizing enzyme family, catalyses the first committed step in the biosynthesis of the siderophore mycobactin T. This complex secondary metabolite is essential for both virulence and survival of M. tuberculosis, the etiological agent of tuberculosis (TB). It is therefore anticipated that inhibitors of this enzyme may serve as TB therapies with a novel mode of action. Herein we describe the first inhibition study of M. tuberculosis MbtI using a library of functionalized benzoate-based inhibitors designed to mimic the substrate (chorismate) and intermediate (isochorismate) of the MbtI-catalyzed reaction. The most potent inhibitors prepared were those designed to mimic the enzyme intermediate, isochorismate. These compounds, based on a 2,3-dihydroxybenzoate scaffold, proved to be low-micromolar inhibitors of MbtI. The most potent inhibitors in this series possessed hydrophobic enol ether side chains at C3 in place of the enol-pyruvyl side chain found in chorismate and isochorismate.

  8. Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

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    Vinciane Régnier

    Full Text Available The cystathionine β-synthase (CBS gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA metabolism, a pathway important for several brain physiological processes.Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1 expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

  9. Glucosylceramide synthase inhibition alleviates aberrations in synucleinopathy models.

    Science.gov (United States)

    Sardi, S Pablo; Viel, Catherine; Clarke, Jennifer; Treleaven, Christopher M; Richards, Amy M; Park, Hyejung; Olszewski, Maureen A; Dodge, James C; Marshall, John; Makino, Elina; Wang, Bing; Sidman, Richard L; Cheng, Seng H; Shihabuddin, Lamya S

    2017-03-07

    Mutations in the glucocerebrosidase gene (GBA) confer a heightened risk of developing Parkinson's disease (PD) and other synucleinopathies, resulting in a lower age of onset and exacerbating disease progression. However, the precise mechanisms by which mutations in GBA increase PD risk and accelerate its progression remain unclear. Here, we investigated the merits of glucosylceramide synthase (GCS) inhibition as a potential treatment for synucleinopathies. Two murine models of synucleinopathy (a Gaucher-related synucleinopathy model, GbaD409V/D409V and a A53T-α-synuclein overexpressing model harboring wild-type alleles of GBA, A53T-SNCA mouse model) were exposed to a brain-penetrant GCS inhibitor, GZ667161. Treatment of GbaD409V/D409V mice with the GCS inhibitor reduced levels of glucosylceramide and glucosylsphingosine in the central nervous system (CNS), demonstrating target engagement. Remarkably, treatment with GZ667161 slowed the accumulation of hippocampal aggregates of α-synuclein, ubiquitin, and tau, and improved the associated memory deficits. Similarly, prolonged treatment of A53T-SNCA mice with GZ667161 reduced membrane-associated α-synuclein in the CNS and ameliorated cognitive deficits. The data support the contention that prolonged antagonism of GCS in the CNS can affect α-synuclein processing and improve behavioral outcomes. Hence, inhibition of GCS represents a disease-modifying therapeutic strategy for GBA-related synucleinopathies and conceivably for certain forms of sporadic disease.

  10. Marine biomolecules inhibit rat brain nitric oxide synthase activity.

    Science.gov (United States)

    Venkateswara Rao, J; Desaiah, D; Vig, P J; Venkateswarlu, Y

    1998-08-21

    A large number of substances of medical importance have been isolated from marine flora and fauna and their chemical structures were elucidated. Among the many compounds isolated in our laboratories only two compounds were identified as neurotoxins as they produced depolarizing effects in nerve fibers. The Xestospongin D and Araguspongin C, isolated and purified to 100% from sponge, Haliclona exigua were tested for their effects on rat brain nitric oxide synthase (NOS) activity in vitro. The results showed that NOS activity was significantly inhibited in a concentration and time dependent manner with an estimated IC50 of 31.5 and 46.5 microM for Xestospongin D and Araguspongin C, respectively, and the maximum inhibition occurred within 3 min of incubation. To explore the mechanism of action of these compounds on NOS, we have conducted kinetic studies with L-arginine, NADPH and Ca2+ in the presence of IC50 concentrations of these two compounds. The maximum velocity (Vmax) and enzyme constant (Km) were calculated using the Michaelis Menten equation. The results show that both compounds are competitive inhibitors of NOS with the substrate, L-arginine and uncompetitive with NADPH and free Ca2+. The NOS inhibition by these two compounds was similar to N omega-nitro-L-arginine methylester (L-NAME), a known inhibitor of NOS. These results suggest that the marine biomolecules Xestospongin D and Araguspongin C are in vitro modulators of neuronal NOS.

  11. Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene

    Energy Technology Data Exchange (ETDEWEB)

    Feinbaum, R.L.; Ausubel, F.M.

    1988-05-01

    The authors cloned an Arabiodpsis thaliana chalcone synthase (CHS) gene on the basis of cross-hybridization with a Petroselinum hortense CHS cDNA clone. The protein sequence deduced from the A. thaliana CHS DNA sequence is at least 85% homologous to the CHS sequences from P. hortense, Antirrhinum majus, and Petunia hybrida. Southern blot analysis indicated that CHS is a single-copy gene in A. thaliana. High-intensity light treatment of A. thaliana plants for 24 h caused a 50-fold increase in CHS enzyme activity and an accumulation of visibly detectable levels of anthocyanin pigments in the vegetative structures of these plants. A corresponding increase in the steady-state level of CHS mRNA was detected after high-intensity light treatment for the same period of time. The accumulation of CHS mRNA in response to high-intensity light was due, at least in part, to an increased rate of transcription of the CHS gene as demonstrated by nuclear runoff experiment.

  12. ASMPKS: an analysis system for modular polyketide synthases

    Directory of Open Access Journals (Sweden)

    Kong Eun-Bae

    2007-09-01

    Full Text Available Abstract Background Polyketides are secondary metabolites of microorganisms with diverse biological activities, including pharmacological functions such as antibiotic, antitumor and agrochemical properties. Polyketides are synthesized by serialized reactions of a set of enzymes called polyketide synthase(PKSs, which coordinate the elongation of carbon skeletons by the stepwise condensation of short carbon precursors. Due to their importance as drugs, the volume of data on polyketides is rapidly increasing and creating a need for computational analysis methods for efficient polyketide research. Moreover, the increasing use of genetic engineering to research new kinds of polyketides requires genome wide analysis. Results We describe a system named ASMPKS (Analysis System for Modular Polyketide Synthesis for computational analysis of PKSs against genome sequences. It also provides overall management of information on modular PKS, including polyketide database construction, new PKS assembly, and chain visualization. ASMPKS operates on a web interface to construct the database and to analyze PKSs, allowing polyketide researchers to add their data to this database and to use it easily. In addition, the ASMPKS can predict functional modules for a protein sequence submitted by users, estimate the chemical composition of a polyketide synthesized from the modules, and display the carbon chain structure on the web interface. Conclusion ASMPKS has powerful computation features to aid modular PKS research. As various factors, such as starter units and post-processing, are related to polyketide biosynthesis, ASMPKS will be improved through further development for study of the factors.

  13. (-)-Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase.

    Science.gov (United States)

    Ramírez-Sánchez, Israel; Rodríguez, Alonso; Moreno-Ulloa, Aldo; Ceballos, Guillermo; Villarreal, Francisco

    2016-05-01

    (-)-Epicatechin increases indicators associated with mitochondrial biogenesis in endothelial cells and myocardium. We investigated endothelial nitric oxide synthase involvement on (-)-epicatechin-induced increases in indicators associated with mitochondrial biogenesis in human coronary artery endothelial cells cultured in normal-glucose and high-glucose media, as well as to restore indicators of cardiac mitochondria from the effects of simulated diabetes. Here, we demonstrate the role of endothelial nitric oxide synthase on (-)-epicatechin-induced increases in mitochondrial proteins, transcription factors and sirtuin 1 under normal-glucose conditions. In simulated diabetes endothelial nitric oxide synthase function, mitochondrial function-associated and biogenesis-associated indicators were adversely impacted by high glucose, effects that were reverted by (-)-epicatechin. As an animal model of type 2 diabetes, 2-month old C57BL/6 mice were fed a high-fat diet for 16 weeks. Fasting and fed blood glucose levels were increased and NO plasma levels decreased. High-fat-diet-fed mice myocardium revealed endothelial nitric oxide synthase dysfunction, reduced mitochondrial activity and markers of mitochondrial biogenesis. The administration of 1 mg/kg (-)-epicatechin for 15 days by oral gavage shifted these endpoints towards control mice values. Results suggest that endothelial nitric oxide synthase mediates (-)-epicatechin-induced increases of indicators associated with mitochondrial biogenesis in endothelial cells. (-)-Epicatechin also counteracts the negative effects that high glucose or simulated type 2 diabetes has on endothelial nitric oxide synthase function. © The Author(s) 2016.

  14. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  15. Cation-induced formation of a macro-glucan synthase complex

    Energy Technology Data Exchange (ETDEWEB)

    Delmer, D.; Solomon, M.; Andrawis, A.; Amor, Y. (Hebrew Univ., Jerusalem (Israel))

    1990-05-01

    Incubation of Chaps or digitonin-solubilized membrane proteins from cotton fiber with Ca{sup 2+} in combination with Mg{sup 2+}, leads to formation of a complex which can be sedimented within 15 min at 15,000 g. The complex is enriched >10-fold in callose synthase activity and possesses a characteristic pattern of enriched polypeptides when analyzed by SDS-PAGE. Although cation dependent, formation of the complex is not dependent upon the presence of the callose synthase substrate, UDP-glc, indicating that complex formation is not due to entrapment of the enzyme by association with glucan product. The enriched polypeptides include: >200, 50, and 46 kD, all of which have been shown by direct photo-labeling to interact with {sup 92}P-UDP-glc in a Ca{sup 2+} or beta-glucoside dependent reaction are considered likely subunits of callose synthase; a 60-62 kD doublet which is recognized by our MAb 2-1 which can form an immune complex with callose synthase; 74 and 34 kD polypeptides which also interact with UDP-glc, but do not associate with callose synthase in the presence of EDTA. A similar phenomenon is also observed with solubilized membrane proteins from mung beans. Possible functions of each of the enriched polypeptides, the catalytic properties, and ultra-structure of this macro-glucan synthase complex are currently under investigation.

  16. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S. (Univ. of Texas Medical School, Houston (United States))

    1991-03-15

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-({sup 35}S)methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

  17. A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.

    Directory of Open Access Journals (Sweden)

    Maiko Furubayashi

    Full Text Available Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

  18. Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot.

    Science.gov (United States)

    Yahyaa, Mosaab; Matsuba, Yuki; Brandt, Wolfgang; Doron-Faigenboim, Adi; Bar, Einat; McClain, Alan; Davidovich-Rikanati, Rachel; Lewinsohn, Efraim; Pichersky, Eran; Ibdah, Mwafaq

    2015-11-01

    Bay laurel (Laurus nobilis) is an agriculturally and economically important dioecious tree in the basal dicot family Lauraceae used in food and drugs and in the cosmetics industry. Bay leaves, with their abundant monoterpenes and sesquiterpenes, are used to impart flavor and aroma to food, and have also drawn attention in recent years because of their potential pharmaceutical applications. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, we performed RNA sequencing to profile the transcriptome of L. nobilis leaves. Bioinformatic analysis led to the identification of eight TPS complementary DNAs. We characterized the enzymes encoded by three of these complementary DNAs: a monoterpene synthase that belongs to the TPS-b clade catalyzes the formation of mostly 1,8-cineole; a sesquiterpene synthase belonging to the TPS-a clade catalyzes the formation of mainly cadinenes; and a diterpene synthase of the TPS-e/f clade catalyzes the formation of geranyllinalool. Comparison of the sequences of these three TPSs indicated that the TPS-a and TPS-b clades of the TPS gene family evolved early in the evolution of the angiosperm lineage, and that geranyllinalool synthase activity is the likely ancestral function in angiosperms of genes belonging to an ancient TPS-e/f subclade that diverged from the kaurene synthase gene lineages before the split of angiosperms and gymnosperms. © 2015 American Society of Plant Biologists. All Rights Reserved.

  19. Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot1[OPEN

    Science.gov (United States)

    Yahyaa, Mosaab; Matsuba, Yuki; Brandt, Wolfgang; Doron-Faigenboim, Adi; Bar, Einat; McClain, Alan; Davidovich-Rikanati, Rachel; Lewinsohn, Efraim; Pichersky, Eran; Ibdah, Mwafaq

    2015-01-01

    Bay laurel (Laurus nobilis) is an agriculturally and economically important dioecious tree in the basal dicot family Lauraceae used in food and drugs and in the cosmetics industry. Bay leaves, with their abundant monoterpenes and sesquiterpenes, are used to impart flavor and aroma to food, and have also drawn attention in recent years because of their potential pharmaceutical applications. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, we performed RNA sequencing to profile the transcriptome of L. nobilis leaves. Bioinformatic analysis led to the identification of eight TPS complementary DNAs. We characterized the enzymes encoded by three of these complementary DNAs: a monoterpene synthase that belongs to the TPS-b clade catalyzes the formation of mostly 1,8-cineole; a sesquiterpene synthase belonging to the TPS-a clade catalyzes the formation of mainly cadinenes; and a diterpene synthase of the TPS-e/f clade catalyzes the formation of geranyllinalool. Comparison of the sequences of these three TPSs indicated that the TPS-a and TPS-b clades of the TPS gene family evolved early in the evolution of the angiosperm lineage, and that geranyllinalool synthase activity is the likely ancestral function in angiosperms of genes belonging to an ancient TPS-e/f subclade that diverged from the kaurene synthase gene lineages before the split of angiosperms and gymnosperms. PMID:26157114

  20. Understanding the link between antimicrobial properties of dietary olive phenolics and bacterial ATP synthase.

    Science.gov (United States)

    Amini, Amon; Liu, Mason; Ahmad, Zulfiqar

    2017-08-01

    The naturally occurring olive phenolics tyrosol, hydroxytyrosol, dihydroxyphenylglycol (DHPG), and oleuropein are known to have antioxidant, antitumor, and antibacterial properties. In the current study, we examined whether the antimicrobial properties of tyrosol, hydroxytyrosol, DHPG, and oleuropein were linked to the inhibition of bacterial ATP synthase. Tyrosol, hydroxytyrosol, DHPG, and oleuropein inhibited Escherichia coli wild-type and mutant membrane-bound F1Fo ATP synthase to variable degrees. The growth properties of wild-type, null, and mutant strains in presence of above olive phenolics were also abrogated to variable degrees on limiting glucose and succinate. Tyrosol and oleuropein synergistically inhibited the wild-type enzyme. Comparative wild-type and mutant F1Fo ATP synthase inhibitory profiles suggested that αArg-283 is an important residue and olive phenolics bind at the polyphenol binding pocket of ATP synthase. Growth patterns of wild-type, null, and mutant strains in the presence of tyrosol, hydroxytyrosol, DHPG, and oleuropein also hint at the possibility of additional molecular targets. Our results demonstrated that ATP synthase can be used as a molecular target and the antimicrobial properties of olive phenolics in general and tyrosol in particular can be linked to the binding and inhibition of bacterial ATP synthase. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Nishimura, Kohji [Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Jisaka, Mitsuo; Nagaya, Tsutomu [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Shono, Fumiaki [Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima-shi, Tokushima 770-8514 (Japan); Yokota, Kazushige, E-mail: yokotaka@life.shimane-u.ac.jp [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan)

    2012-02-15

    Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD{sub 2} and its non-enzymatic dehydration products, PGJ{sub 2} series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD{sub 2} and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD{sub 2} from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE{sub 2} remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize {Delta}{sup 12}-PGJ{sub 2} increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists including troglitazone and {Delta}{sup 12}-PGJ{sub 2}. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPAR{gamma} signaling pathway without the contribution of endogenous pro-adipogenic prostanoids

  2. Predictive Significance of Thymidylate Synthase Expression in Non-small Cell Lung Cancer.

    Science.gov (United States)

    Kulda, Vlastimil; Hrda, Kristyna; Houdek, Zbynek; Dobra, Jana Kolaja; Vrzakova, Radana; Svaton, Martin; Safranek, Jarmil; Dolezal, Jan; Babuska, Vaclav; Pesek, Milos; Topolcan, Ondrej; Pesta, Martin

    2017-12-01

    To date, many studies have suggested that thymidylate synthase (TS) could be used as a prognostic and predictive marker in non-small cell lung cancer (NSCLC) patients. However, results have been contradictory. The aim of this study was to evaluate TS mRNA levels in tumor tissue of NSCLC patients who underwent complete surgical resection and to analyze its prognostic and predictive potential. The study group consisted of 64 patients who underwent curative lung resection. Paired lung tissue samples were taken directly from the tumor tissue and from adjacent, histologically cancer-free lung tissue. The quantitative estimation of TS expression was performed by reverse transcription real-time polymerase chain reaction (RT-qPCR). The relationship between TS expression level and disease-free interval (DFI) and overall survival (OS) was analyzed. There was significantly higher TS expression in NSCLC tumor tissue comparing to normal lung tissue. In the group of patients who received adjuvant chemotherapy based on platinum derivatives in combination with paclitaxel or gemcitabine, we found shorter DFI (p=0.0473) and OS (p=0.0053) in those with high expression of TS. Our results demonstrated the relationship of high tumor tissue TS levels to adverse prognosis in patients undergoing adjuvant chemotherapy. TS is a non-specific tumor marker with respect to NSCLC, therefore we think that its best use would be as a member of the panel of predictors of adjuvant treatment efficacy. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  3. Chrysanthemum expressing a linalool synthase gene 'smells good', but 'tastes bad' to western flower thrips.

    Science.gov (United States)

    Yang, Ting; Stoopen, Geert; Thoen, Manus; Wiegers, Gerrie; Jongsma, Maarten A

    2013-09-01

    Herbivore-induced plant volatiles are often involved in direct and indirect plant defence against herbivores. Linalool is a common floral scent and found to be released from leaves by many plants after herbivore attack. In this study, a linalool/nerolidol synthase, FaNES1, was overexpressed in the plastids of chrysanthemum plants (Chrysanthemum morifolium). The volatiles of FaNES1 chrysanthemum leaves were strongly dominated by linalool, but they also emitted small amount of the C11-homoterpene, (3E)-4,8-dimethyl-1,3,7-nonatriene, a derivative of nerolidol. Four nonvolatile linalool glycosides in methanolic extracts were found to be significantly increased in the leaves of FaNES1 plants compared to wild-type plants. They were putatively identified by LC-MS-MS as two linalool-malonyl-hexoses, a linalool-pentose-hexose and a glycoside of hydroxy-linalool. A leaf-disc dual-choice assay with western flower thrips (WFT, Frankliniella occidentalis) showed, initially during the first 15 min of WFT release, that FaNES1 plants were significantly preferred. This gradually reversed into significant preference for the control, however, at 20-28 h after WFT release. The initial preference was shown to be based on the linalool odour of FaNES1 plants by olfactory dual-choice assays using paper discs emitting pure linalool at similar rates as leaf discs. The reversal of preference into deterrence could be explained by the initial nonvolatile composition of the FaNES1 plants, as methanolic extracts were less preferred by WFT. Considering the common occurrence of linalool and its glycosides in plant tissues, it suggests that plants may balance attractive fragrance with 'poor taste' using the same precursor compound. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  4. A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase from Rahnella aquatilis with Significantly Reduced Glyphosate Sensitivity

    Science.gov (United States)

    Xiong, Ai-Sheng; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Chen, Chen; Jin, Xiao-Fen; Yao, Quan-Hong

    2012-01-01

    The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroAR.aquatilis, was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroAE.coli), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroAR.aquatilis were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroAE.coli. To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious. PMID:22870190

  5. Increased fatty acid synthase expression and activity during progression of prostate cancer in the TRAMP model.

    Science.gov (United States)

    Pflug, Beth R; Pecher, Stefana M; Brink, Alisa W; Nelson, Joel B; Foster, Barbara A

    2003-11-01

    Fatty acid synthase (FAS) is the major enzyme required to convert carbohydrates to fatty acids. Recent evidence suggests that FAS activity is essential for prostate cancer growth and survival, since blocking the enzyme activity results in cell death. In this study, the role of FAS up-regulation during prostate tumor progression in the transgenic adenocarcinoma of mouse prostate (TRAMP) model was investigated. Sensitivity to FAS anti-metabolites was also analyzed in TRAMP prostate tumor cells and tissue to determine therapeutic potential of FAS inhibition in the treatment of prostate cancer. FAS expression was evaluated by immunohistochemistry of TRAMP tissues, including primary and metastatic lesions in mice of varying ages. FAS pathway activity was studied in vitro using TRAMP-derived cell lines and in vivo in TRAMP tissues. The sensitivity of TRAMP cell lines and tissues to the antimetabolite drugs (2R,3S)-2,3-epoxy-4-oxo-7,10-trans, transdodecadienamide (cerulenin) and C-75, which target FAS, was determined by FAS antimetabolite inhibition of 14C-acetate conversion to fatty acids, cell growth inhibition, and apoptosis analyses. High FAS expression and activity in the TRAMP mouse prostate was evident at 12 weeks of age compared with nontransgenic littermates and further increased with age, tumor progression, and in metastatic lesions. FAS pathway inhibition resulted in a dose-dependent reduction in cell survival and decreased enzyme activity in these models. These data suggest that the up-regulation of FAS expression play a role in tumorigenesis of the prostate in the TRAMP model and hence can provide valuable insight into human prostate cancer. Given the response of tumor cells to FAS antimetabolites, FAS may serve as a novel target for prostate cancer therapy. Copyright 2003 Wiley-Liss, Inc.

  6. Active site topologies and cofactor-mediated conformational changes of nitric-oxide synthases.

    Science.gov (United States)

    Gerber, N C; Rodriguez-Crespo, I; Nishida, C R; Ortiz de Montellano, P R

    1997-03-07

    The active site topologies of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric-oxide synthases heterologously expressed in Escherichia coli have been examined using three aryldiazene (Ar-N=NH) probes. The topological information derives from (a) the rate and extent of aryl-iron complex formation in the presence and absence of tetrahydrobiopterin (H4B), Ca2+-dependent calmodulin (CaM), and L-arginine, and (b) the N-phenylprotoporphyrin IX regioisomer ratios obtained upon migration of the phenyl of the phenyl-iron complex to the heme nitrogen atoms. The N-phenylprotoporphyrin ratios indicate that the three NOS isoforms have related active site topologies with unencumbered space above all four pyrrole rings but particularly above pyrrole ring D. H4B binds directly above the heme pyrrole ring D or causes a conformational change that constricts that region, because H4B markedly decreases phenyl migration to pyrrole ring D. Small CaM-dependent changes in the nNOS N-phenylporphyrin isomer pattern are consistent with a conformational link between the CaM and heme sites in this protein. The ceiling height directly above the heme iron atom differs among the isoforms and is lower than in the P450 enzymes because only nNOS and iNOS react with 2-naphthyldiazene, and none of the isoforms reacts with p-biphenyldiazene. L-Arg blocks access to the heme iron atom in all three NOS isoforms and nearly suppresses the phenyldiazene reaction. The data indicate that topological differences, including differences in the size of the active site, are superimposed on the structural similarities among the NOS active sites.

  7. The cell wall-associated mycolactone polyketide synthases are necessary but not sufficient for mycolactone biosynthesis.

    Directory of Open Access Journals (Sweden)

    Jessica L Porter

    Full Text Available Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb encode the mycolactone type I polyketide synthases (PKS. The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL. Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules.

  8. Alendronate induces gastric damage by reducing nitric oxide synthase expression and NO/cGMP/K(ATP) signaling pathway.

    Science.gov (United States)

    Silva, Renan O; Lucetti, Larisse T; Wong, Deysi V T; Aragão, Karoline S; Junior, Eudmar M A; Soares, Pedro M G; Barbosa, André Luiz R; Ribeiro, Ronaldo A; Souza, Marcellus H L P; Medeiros, Jand-Venes R

    2014-08-31

    Chronic use of alendronate has been linked to gastrointestinal tract problems. Our objective was to evaluate the role of the NO/cGMP/KATP signaling pathway and nitric oxide synthase expression in alendronate-induced gastric damage. Rats were either treated with the NO donor, sodium nitroprusside (SNP; 1, 3, and 10 mg/kg), or the NO synthase (NOS) substrate, L-arginine (L-Arg; 50, 100, and 200 mg/kg). Some rats were pretreated with either ODQ (a guanylate cyclase inhibitor; 10 mg/kg) or glibenclamide (KATP channels blocker; 10 mg/kg). In other experiments, rats were pretreated with L-NAME (non-selective NOS inhibitor; 10 mg/kg), 1400 W (selective inducible NOS [iNOS] inhibitor; 10 mg/kg), or L-NIO (a selective endothelial NOS [eNOS] inhibitor; 30 mg/kg). After 1 h, the rats were treated with alendronate (30 mg/kg) by gavage for 4 days. SNP and L-Arg prevented alendronate-induced gastric damage in a dose-dependent manner. Alendronate reduced nitrite/nitrate levels, an effect that was reversed with SNP or L-Arg treatment. Pretreatment with ODQ or glibenclamide reversed the protective effects of SNP and L-Arg. L-NAME, 1400 W, or L-NIO aggravated the severity of alendronate-induced lesions. In addition, alendronate reduced the expression of iNOS and eNOS in the gastric mucosa. Gastric ulcerogenic responses induced by alendronate were mediated by a decrease in NO derived from both eNOS and iNOS. In addition, our findings support the hypothesis that activation of the NO/cGMP/KATP pathway is of primary importance for protection against alendronate-induced gastric damage. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Folic Acid Promotes Recycling of Tetrahydrobiopterin and Protects Against Hypoxia-Induced Pulmonary Hypertension by Recoupling Endothelial Nitric Oxide Synthase

    Science.gov (United States)

    Chalupsky, Karel; Kračun, Damir; Kanchev, Ivan; Bertram, Katharina

    2015-01-01

    Abstract Aims: Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions. Results: In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH. Innovation: FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH. Conclusion: FA might serve as a novel therapeutic option combating PH. Antioxid. Redox Signal. 23, 1076–1091. PMID:26414244

  10. Gene identification and functional analysis of methylcitrate synthase in citric acid-producing Aspergillus niger WU-2223L.

    Science.gov (United States)

    Kobayashi, Keiichi; Hattori, Takasumi; Honda, Yuki; Kirimura, Kohtaro

    2013-01-01

    Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.

  11. Nitric oxide synthase localized in a subpopulation of vestibular efferents with NADPH diaphorase histochemistry and nitric oxide synthase immunohistochemistry.

    Science.gov (United States)

    Lysakowski, A; Singer, M

    2000-11-27

    Efferent innervation of the vestibular labyrinth is known to be cholinergic. More recent studies have also demonstrated the presence of the neuropeptide calcitonin gene-related peptide in this system. Nitric oxide is one of a new class of neurotransmitters, the gaseous transmitters. It acts as a second messenger and neurotransmitter in diverse physiological systems. We decided to investigate the anatomical distribution of the synthetic enzyme for nitric oxide, nitric oxide synthase (NOS), to clarify the role of nitric oxide in the vestibular periphery. NADPH diaphorase histochemical and NOS I immunohistochemical studies were done in the adult chinchilla and rat vestibular brainstem; diaphorase histochemistry was done in the chinchilla periphery. Retrograde tracing studies to verify the presence of NOS in brainstem efferent neurons were performed in young chinchillas. Our light microscopic results show that NOS I, as defined mainly by the presence of NADPH diaphorase, is present in a subpopulation of both brainstem efferent neurons and peripheral vestibular efferent boutons. Our ultrastructural results confirm these findings in the periphery. NADPH diaphorase is also present in a subpopulation of type I hair cells, suggesting that nitric oxide might be produced in and act locally upon these cells and other elements in the sensory epithelium. A hypothesis about how nitric oxide is produced in the vestibular periphery and how it may interact with other elements in the vestibular sensory apparatus is presented in the discussion. Copyright 2000 Wiley-Liss, Inc.

  12. Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine [beta]-synthase

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Kabil, Omer; Smith, Janet L.; Banerjee, Ruma (Michigan-Med)

    2011-08-17

    The catalytic potential for H{sub 2}S biogenesis and homocysteine clearance converge at the active site of cystathionine {beta}-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes {beta}-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H{sub 2}S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 {angstrom}) and an aminoacrylate intermediate (1.55 {angstrom}) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory 'energy-sensing' CBS domains, named after this protein, suggests a mechanism for allosteric activation by S-adenosylmethionine.

  13. The geranylgeranyl pyrophosphate synthase gene from Ginkgo biloba

    African Journals Online (AJOL)

    derived GGDPSs could be divided into two classes, angiosperm and gymnosperm classes, which might have evolved in parallel from the same ancestor. The homology-based structural modeling showed that GbGGDPS has the typical structure ...

  14. Identification of a Fungal 1,8-Cineole Synthase from Hypoxylon sp. with Specificity Determinants in Common with the Plant Synthases*

    Science.gov (United States)

    Shaw, Jeffrey J.; Berbasova, Tetyana; Sasaki, Tomoaki; Jefferson-George, Kyra; Spakowicz, Daniel J.; Dunican, Brian F.; Portero, Carolina E.; Narváez-Trujillo, Alexandra; Strobel, Scott A.

    2015-01-01

    Terpenes are an important and diverse class of secondary metabolites widely produced by fungi. Volatile compound screening of a fungal endophyte collection revealed a number of isolates in the family Xylariaceae, producing a series of terpene molecules, including 1,8-cineole. This compound is a commercially important component of eucalyptus oil used in pharmaceutical applications and has been explored as a potential biofuel additive. The genes that produce terpene molecules, such as 1,8-cineole, have been little explored in fungi, providing an opportunity to explore the biosynthetic origin of these compounds. Through genome sequencing of cineole-producing isolate E7406B, we were able to identify 11 new terpene synthase genes. Expressing a subset of these genes in Escherichia coli allowed identification of the hyp3 gene, responsible for 1,8-cineole biosynthesis, the first monoterpene synthase discovered in fungi. In a striking example of convergent evolution, mutational analysis of this terpene synthase revealed an active site asparagine critical for water capture and specificity during cineole synthesis, the same mechanism used in an unrelated plant homologue. These studies have provided insight into the evolutionary relationship of fungal terpene synthases to those in plants and bacteria and further established fungi as a relatively untapped source of this important and diverse class of compounds. PMID:25648891

  15. Identification of mouse sphingomyelin synthase 1 as a suppressor of Bax-mediated cell death in yeast.

    Science.gov (United States)

    Yang, Zhao; Khoury, Chamel; Jean-Baptiste, Gaël; Greenwood, Michael T

    2006-08-01

    We have identified mouse sphingomyelin synthase 1 as a novel suppressor of the growth inhibitory effect of heterologously expressed Bax. Yeast cells expressing sphingomyelin synthase 1 were also found to show an increased resistance to a variety of cytotoxic stimuli including hydrogen peroxide, osmotic stress and elevated temperature. Sphingomyelin synthase 1 functions by catalyzing the conversion of ceramide and phosphatidylcholine to sphingomyelin and diacylglycerol. Ceramide is an antiproliferative and proapoptotic sphingolipid whose level increases in response to a variety of stresses. Consistent with its biochemical function, yeast cells expressing sphingomyelin synthase 1 have an enhanced ability to grow in media containing the cell-permeable C2-ceramide analog as well as the ceramide precursor phytosphingosine. We also show that overexpression of AUR1, a potential yeast functional homolog of sphingomyelin synthase, also protects cells from osmotic stress. Taken together, these results suggest that sphingomyelin synthase 1 likely prevents cell death by counteracting stress-mediated accumulation of endogenous sphingolipids.

  16. Platensimycin activity against mycobacterial beta-ketoacyl-ACP synthases.

    Directory of Open Access Journals (Sweden)

    Alistair K Brown

    2009-07-01

    Full Text Available There is an urgent need for the discovery and development of new drugs against Mycobacterium tuberculosis, the causative agent of tuberculosis, especially due to the recent emergence of multi-drug and extensively-drug resistant strains. Herein, we have examined the susceptibility of mycobacteria to the natural product platensimycin.We have demonstrated that platensimycin has bacteriostatic activity against the fast growing Mycobacterium smegmatis (MIC = 14 microg/ml and against Mycobacterium tuberculosis (MIC = 12 microg/ml. Growth in the presence of paltensimycin specifically inhibited the biosynthesis of mycolic acids suggesting that the antibiotic targeted the components of the mycolate biosynthesis complex. Given the inhibitory activity of platensimycin against beta-ketoacyl-ACP synthases from Staphylococcus aureus, M. tuberculosis KasA, KasB or FabH were overexpressed in M. smegmatis to establish whether these mycobacterial KAS enzymes were targets of platensimycin. In M. smegmatis overexpression of kasA or kasB increased the MIC of the strains from 14 microg/ml, to 30 and 124 microg/ml respectively. However, overexpression of fabH on did not affect the MIC. Additionally, consistent with the overexpression data, in vitro assays using purified proteins demonstrated that platensimycin inhibited Mt-KasA and Mt-KasB, but not Mt-FabH.Our results have shown that platensimycin is active against mycobacterial KasA and KasB and is thus an exciting lead compound against M. tuberculosis and the development of new synthetic analogues.

  17. Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis.

    Directory of Open Access Journals (Sweden)

    Roslyn D Noar

    Full Text Available Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that

  18. Hypoxia activates the cyclooxygenase-2-prostaglandin E synthase axis.

    Science.gov (United States)

    Lee, James J; Natsuizaka, Mitsuteru; Ohashi, Shinya; Wong, Gabrielle S; Takaoka, Munenori; Michaylira, Carmen Z; Budo, Daniela; Tobias, John W; Kanai, Michiyuki; Shirakawa, Yasuhiro; Naomoto, Yoshio; Klein-Szanto, Andres J P; Haase, Volker H; Nakagawa, Hiroshi

    2010-03-01

    Hypoxia-inducible factors (HIFs), in particular HIF-1alpha, have been implicated in tumor biology. However, HIF target genes in the esophageal tumor microenvironment remain elusive. Gene expression profiling was performed upon hypoxia-exposed non-transformed immortalized human esophageal epithelial cells, EPC2-hTERT, and comparing with a gene signature of esophageal squamous cell carcinoma (ESCC). In addition to known HIF-1alpha target genes such as carbonic anhydrase 9, insulin-like growth factor binding protein-3 (IGFBP3) and cyclooxygenase (COX)-2, prostaglandin E synthase (PTGES) was identified as a novel target gene among the commonly upregulated genes in ESCC as well as the cells exposed to hypoxia. The PTGES induction was augmented upon stabilization of HIF-1alpha by hypoxia or cobalt chloride under normoxic conditions and suppressed by dominant-negative HIF-1alpha. Whereas PTGES messenger RNA (mRNA) was negatively regulated by normoxia, PTGES protein remained stable upon reoxygenation. Prostaglandin E(2) (PGE(2)) biosynthesis was documented in transformed human esophageal cells by ectopic expression of PTGES as well as RNA interference directed against PTGES. Moreover, hypoxia stimulated PGE(2) production in a HIF-1alpha-dependent manner. In ESCC, PTGES was overexpressed frequently at the mRNA and protein levels. Finally, COX-2 and PTGES were colocalized in primary tumors along with HIF-1alpha and IGFBP3. Activation of the COX-2-PTGES axis in primary tumors was further corroborated by concomitant upregulation of interleukin-1beta and downregulation of hydroxylprostaglandin dehydrogenase. Thus, PTGES is a novel HIF-1alpha target gene, involved in prostaglandin E biosynthesis in the esophageal tumor hypoxic microenvironment, and this has implications in diverse tumors types, especially of squamous origin.

  19. Vasomotor control in mice overexpressing human endothelial nitric oxide synthase.

    Science.gov (United States)

    van Deel, Elza D; Merkus, Daphne; van Haperen, Rien; de Waard, Monique C; de Crom, Rini; Duncker, Dirk J

    2007-08-01

    Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase [eNOS-transgenic (Tg)] have a 20% lower systemic vascular resistance (SVR) than wild-type (WT) mice. However, because eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by alterations in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg mice compared with WT mice, consistent with increased NO synthesis. Vasodilation to sodium nitroprusside (SNP) was reduced, whereas the vasodilator responses to phosphodiesterase-5 blockade and 8-bromo-cGMP (8-Br-cGMP) were maintained in eNOS-Tg compared with WT mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, because scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, whereas after eNOS inhibition the vasodilator response to ROS scavenging was similar in WT and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone [including cyclooxygenase, cytochrome P-450 2C9, endothelin, adenosine, and Ca-activated K(+) channels] did not significantly affect SVR in either eNOS-Tg or WT mice, whereas the marked vasoconstrictor responses to ATP-sensitive K(+) and voltage-dependent K(+) channel blockade were similar in WT and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways.

  20. Insights into the reactivation of cobalamin-dependent methionine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Datta, Supratim; Pattridge, Katherine A.; Smith, Janet L.; Matthews, Rowena G.; (Michigan)

    2009-12-10

    Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every {approx}2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from S-adenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetH{sup CT}) revealed a reactivation conformation that juxtaposes the cobalamin- and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized MetH{sup CT} ({sub s-s}MetH{sup CT}) that offer further insight into the reactivation of MetH. The structure of {sub s-s}MetH{sup CT} with cob(II)alamin and S-adenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of {sub s-s}MetH{sub CT} with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation.

  1. Topoisomerase 2α and thymidylate synthase expression in adrenocortical cancer.

    Science.gov (United States)

    Roca, Elisa; Berruti, Alfredo; Sbiera, Silviu; Rapa, Ida; Oneda, Ester; Sperone, Paola; Ronchi, Cristina L; Ferrari, Laura; Grisanti, Salvatore; Germano, Antonina; Zaggia, Barbara; Scagliotti, Giorgio Vittorio; Fassnacht, Martin; Volante, Marco; Terzolo, Massimo; Papotti, Mauro

    2017-07-01

    Topoisomerase II alpha (TOP2A) and thymidylate synthase (TS) are known prognostic parameters in several tumors and also predictors of efficacy of anthracyclines, topoisomerase inhibitors and fluoropirimidines, respectively. Expression of TOP2A and TS mRNA was assessed in 98 patients with adrenocortical carcinoma (ACC) and protein expression was assessed by immunohistochemistry in a subset of 39 tumors. Ninety-two patients were radically resected for stage II-III disease and 38 of them received adjuvant mitotane. Twenty-six patients with metastatic disease received the EDP-M (etoposide, doxorubicin, Adriamycin, cisplatin plus mitotane). TOP2A and TS expression in ACC tissue was directly correlated with the clinical data. Both markers were not associated with either disease free survival (DFS) or overall survival (OS) in multivariate analyses and failed to be associated to mitotane efficacy. Disease response or stabilization to EDP-M treatment was observed in 12/17 (71%) and 1/9 (11%) patients with high and low TOP2A expressing tumors (P = 0.0039) and 9/13 (69%) and 4/13 (31%) patients with high and low TS expressing ACC, respectively (P = 0.049). High TOP2A expression was significantly associated with longer time to progression (TTP) after EDP-M. TOP2A and TS proteins assessed by immunohistochemistry significantly correlated with mRNA expression. Immunohistochemical TOP2A expression was associated with a non-significant better response and longer TTP after EDP-M. TOP2A and TS were neither prognostic nor predictive of mitotane efficacy in ACC patients. The predictive role of TOP2A expression of EDP-M activity suggests a significant contribution of Adriamycin and etoposide for the efficacy of the EDP scheme. © 2017 Society for Endocrinology.

  2. Thymidylate synthase expression and molecular alterations in adenosquamous carcinoma of the lung.

    Science.gov (United States)

    Shu, Catherine; Cheng, Haiying; Wang, Antai; Mansukhani, Mahesh M; Powell, Charles A; Halmos, Balazs; Borczuk, Alain C

    2013-02-01

    Thymidylate synthase expression is known to be higher in squamous cell carcinoma than in adenocarcinoma of the lung. It is thought that this is the reason for the poor efficacy of pemetrexed in squamous cell carcinoma. However, there is limited data on thymidylate synthase expression in adenosquamous carcinoma, a distinct subtype of lung cancer containing both squamous and glandular differentiation. Furthermore, molecular alterations like epidermal growth factor receptor and Kirsten rat sarcoma 2 viral oncogene homolog mutations, which are seen in adenocarcinomas, are not well understood in mixed histology tumors such as adenosquamous carcinoma. In our study, we sought to better characterize adenosquamous tumors of the lung. Using immunohistochemistry to evaluate thymidylate synthase protein levels, we found that the expression of thymidylate synthase in these mixed tumors roughly parallel that of squamous cell carcinoma, instead of falling in between squamous cell and adenocarcinoma. Of note, in adenosquamous samples, the expression of thymidylate synthase was more closely correlated within the two components than would be expected by random chance alone. Also, we had a relatively high rate of epidermal growth factor receptor (11%) and Kirsten rat sarcoma 2 viral oncogene homolog (33%) mutations in these specimens, with the mutations showing convergence in both the glandular and squamous components upon microdissection. Our results indicate that adenosquamous carcinomas are not simple mixtures of their two histological components; they rather behave as their own entity, and it is important to further understand their behavior. Given the similarity of thymidylate synthase expression between squamous cell and adenosquamous carcinoma, and that thymidylate synthase is the main target of pemetrexed, we extrapolate that pemetrexed may also have inferior clinical activity in adenosquamous carcinoma.

  3. [Cloning and tissue expression pattern analysis of the human citrate synthase cDNA].

    Science.gov (United States)

    Liu, Q; Yu, L; Han, X F; Fu, Q; Zhang, J X; Tang, H; Zhao, S Y

    2000-09-01

    Tricarboxylic acid (TCA) cycle is an important way to generate ATP, which is widely distributed in the cells of animal, plant or microorganism. It catalyses the catabolism of sugar as well as protein and fat. Citrate synthase plays a key role in regulating TCA cycle and is responsible for catalysing the synthesis of citrate from oxaloacetate and acetyl CoA. Screening of genomic informatics was performed by using pig citrate synthase cDNA as a probe and a contig which is 1636 bp long and has highly homologous to the pig citrate synthase cDNA was obtained from selected ESTs with the ASSEMBLY program. According to the sequence of this contig, a pair of primers was designed and used to amplify cDNA libraries. A 1492 bp cDNA containing an open reading frame encoding 466 amino acids was cloned from human testis and skeletal muscle cDNA libraries. The deduced amino acid sequence of the cDNA showed 95%, 92% and 60.9% identity to pig, chicken and yeast citrate synthase respectively. Because the deduced amino acids sequence contains a highly conserved motif of citrate synthase from three different species, it is believed that this cDNA may be a transcript of human citrate synthase gene. Northern analysis showed that the human citrate synthase was expressed at high level in heart and muscle, at middle level in brain, kidney and pancreas tissues, not detectable in thymus and small intestine tissues, and at low level in other nine tested human tissues.

  4. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium.

    Directory of Open Access Journals (Sweden)

    Roberta d'Emmanuele di Villa Bianca

    Full Text Available Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity.

  6. Identification of Sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. Strain PCC 7120▿ †

    OpenAIRE

    Agger, Sean A.; Lopez-Gallego, Fernando; Hoye, Thomas R.; Schmidt-Dannert, Claudia

    2008-01-01

    Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene...

  7. Structural Analysis of Thymidylate Synthase from Kaposi?s Sarcoma-Associated Herpesvirus with the Anticancer Drug Raltitrexed

    OpenAIRE

    Choi, Yong Mi; Yeo, Hyun Ku; Park, Young Woo; Lee, Jae Young

    2016-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a highly infectious human herpesvirus that causes Kaposi's sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2'-deoxyuridine-5'-monophosphate (dUMP) to thymidine-5'-monophosphate (dTMP) using 5,10-methylenetetrahydrofolate (mTHF) as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo), co...

  8. Triterpenoic Acids from Apple Pomace Enhance the Activity of the Endothelial Nitric Oxide Synthase (eNOS).

    Science.gov (United States)

    Waldbauer, Katharina; Seiringer, Günter; Nguyen, Dieu Linh; Winkler, Johannes; Blaschke, Michael; McKinnon, Ruxandra; Urban, Ernst; Ladurner, Angela; Dirsch, Verena M; Zehl, Martin; Kopp, Brigitte

    2016-01-13

    Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the (14)C-L-arginine to (14)C-L-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components.

  9. REVIEW: Epistasis and dominance in the emergence of catalytic function as exemplified by the evolution of plant terpene synthases.

    Science.gov (United States)

    Cheema, Jitender; Faraldos, Juan A; O'Maille, Paul E

    2017-02-01

    Epistasis, the interaction between mutations and the genetic background, is a pervasive force in evolution that is difficult to predict yet derives from a simple principle - biological systems are interconnected. Therefore, one effect may be intimately linked to another, hence interdependent. Untangling epistatic interactions between and within genes is a vibrant area of research. Deriving a mechanistic understanding of epistasis is a major challenge. Particularly, elucidating how epistasis can attenuate the effects of otherwise dominant mutations that control phenotypes. Using the emergence of terpene cyclization in specialized metabolism as an excellent example, this review describes the process of discovery and interpretation of dominance and epistasis in relation to current efforts. Specifically, we outline experimental approaches to isolating epistatic networks of mutations in protein structure, formally quantifying epistatic interactions, then building biochemical models with chemical mechanisms in efforts to achieve an understanding of the physical basis for epistasis. From these models we describe informed conjectures about past evolutionary events that underlie the emergence, divergence and specialization of terpene synthases to illustrate key principles of the constraining forces of epistasis in enzyme function. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. Computational design of novel inhibitors to overcome weed resistance associated with acetohydroxyacid synthase (AHAS) P197L mutant.

    Science.gov (United States)

    Qu, Ren-Yu; Yang, Jing-Fang; Liu, Yu-Chao; Chen, Qiong; Hao, Ge-Fei; Niu, Cong-Wei; Xi, Zhen; Yang, Guang-Fu

    2017-07-01

    Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is the first common enzyme in the biosynthetic pathway leading to the branched-chain amino acids in plants and a wide range of microorganisms. With the long-term and wide application of AHAS inhibitors, weed resistance is becoming a global problem, which leads to an urgent demand for novel inhibitors to antagonize both wild-type and resistant AHAS. Pyrimidinyl salicylic acid derivatives, as one of the main classes of commercial AHAS herbicides, show potential anti-resistant bioactivity to wild-type and P197L mutant. In current work, a series of novel 2-benzoyloxy-6-pyrimidinyl salicylic acid derivatives were designed through fragment-based drug discovery. Fortunately, the newly synthesized compounds showed good inhibitory activity against both wild-type and P197L mutant. Some compounds not only had a lower resistance factor value but also showed excellent inhibitory activity against wild-type AHAS and P197L mutant. Furthermore, greenhouse experiments showed compound 11m displayed almost 100% inhibition against both wild-type and high-resistant Descurainia sophia at a dosage of 150 g a.i. ha-1 . The present work indicated that the 2-benzoyloxy-6-pyrimidinyl salicylic acid motif was well worth further optimization. Also, compound 11m could be used as a potential anti-resistant AHAS herbicide, which requires further research. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  11. The Lc3-synthase gene B3gnt5 is essential to pre-implantation development of the murine embryo

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    Cinelli Paolo

    2008-11-01

    Full Text Available Abstract Background Glycosphingolipids (GSL are integral components of mammalian cell membranes that are involved in cell adhesion and cell signaling processes. GSL are subdivided into structural series, like ganglio-, lacto/neolacto-, globo- and isoglo-series, which are defined by distinct trisaccharide cores. The β1,3 N-acetylglucosaminyltransferase-V (B3gnt5 enzyme catalyzes the formation of the Lc3 structure, which is the core of lactoseries derived GSL. Results The biological significance of the glycoconjugates produced by the B3gnt5 enzyme was investigated by inactivating the B3gnt5 gene in the mouse germline. The disruption of the B3gnt5 protein-coding region in mouse embryonic stem cells resulted in reduced Lc3-synthase activity, supporting its specific contribution to lactoseries derived GSL synthesis. Breeding of heterozygous mutant mice failed to produce any viable progeny homozygous for the B3gnt5-null allele. The genotypic examination of embryos from heterozygous crosses showed that the disruption of the B3gnt5 gene leads to pre-implantation lethality. This finding was compatible with the expression pattern of the B3gnt5 gene in the pre-implantation embryo as shown by in situ hybridization. The analysis of GSL profiles in embryonic stem cells heterozygous for the B3gnt5-null allele confirmed the reduced levels of lactoseries derived GSL levels and of other GSL species. Conclusion The disruption of the B3gnt5 gene in mice affected the expression of lactoseries derived GLS and possibly of protein-bound β3GlcNAc-linked glycans, thereby demonstrating an essential contribution of these glycoconjugates in early embryonic development, and supporting the importance of these glycoconjugates in cell differentiation and adhesion processes.

  12. Role of Polymorphisms of Inducible Nitric Oxide Synthase and Endothelial Nitric Oxide Synthase in Idiopathic Environmental Intolerances

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    Chiara De Luca

    2015-01-01

    Full Text Available Oxidative stress and inflammation play a pathogenetic role in idiopathic environmental intolerances (IEI, namely, multiple chemical sensitivity (MCS, fibromyalgia (FM, and chronic fatigue syndrome (CFS. Given the reported association of nitric oxide synthase (NOS gene polymorphisms with inflammatory disorders, we aimed to investigate the distribution of NOS2A −2.5 kb (CCTTTn as well as Ser608Leu and NOS3 −786T>C variants and their correlation with nitrite/nitrate levels, in a study cohort including 170 MCS, 108 suspected MCS (SMCS, 89 FM/CFS, and 196 healthy subjects. Patients and controls had similar distributions of NOS2A Ser608Leu and NOS3 −786T>C polymorphisms. Interestingly, the NOS3 −786TT genotype was associated with increased nitrite/nitrate levels only in IEI patients. We also found that the NOS2A −2.5 kb (CCTTT11 allele represents a genetic determinant for FM/CFS, and the (CCTTT16 allele discriminates MCS from SMCS patients. Instead, the (CCTTT8 allele reduces by three-, six-, and tenfold, respectively, the risk for MCS, SMCS, and FM/CFS. Moreover, a short number of (CCTTT repeats is associated with higher concentrations of nitrites/nitrates. Here, we first demonstrate that NOS3 −786T>C variant affects nitrite/nitrate levels in IEI patients and that screening for NOS2A −2.5 kb (CCTTTn polymorphism may be useful for differential diagnosis of various IEI.

  13. Biochemical and Structural Studies of RNA Modification and Repair

    Science.gov (United States)

    Chan, Chio Mui

    2009-01-01

    RNA modification, RNA interference, and RNA repair are important events in the cell. This thesis presents three projects related to these three fields. By using both biochemical and structural methods, we characterized enzymatic activities of pseudouridine synthase TruD, solved the structure of "A. aeolicus" GidA, and reconstituted a novel…

  14. Sequence Classification: 891367 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|6321608|ref|NP_011685.1| Pseudouridine synthase responsi...ble for modification of cytoplasmic and mitochondrial tRNAs at position 31; mutatio

  15. Analysis in Escherichia coli of Plasmodium falciparum dihydropteroate synthase (DHPS) alleles implicated in resistance to sulfadoxine.

    Science.gov (United States)

    Berglez, Janette; Iliades, Peter; Sirawaraporn, Worachart; Coloe, Peter; Macreadie, Ian

    2004-01-01

    Mutations in Plasmodium falciparum dihydropteroate synthase have been linked to resistance to the antimalarial drug, sulfadoxine, which competes with the dihydropteroate synthase substrate, p-aminobenzoate. In an effort to evaluate the role of these mutations in a simple model system, we have expressed six relevant alleles of the P. falciparum dihydropteroate synthase gene in Escherichia coli. When each construct was produced in a dihydropteroate synthase disrupted E. coli strain that required thymidine, the thymidine requirement was lost, indicating heterologous complementation had occurred. In the presence of sulfadoxine, the growth of the strain with the wild-type dihydropteroate synthase allele was inhibited while those containing each of the five mutant alleles grew, indicating that these mutations can confer sulfadoxine resistance in E. coli. When tested against twelve additional 'sulfa' drugs a variety of responses were obtained. All strains were resistant to sulfadiazine, but the wild-type allele conferred sensitivity to all other sulfa drugs. Three alleles conferred resistance to dapsone, a drug that is to be targetted for a new regime of malaria treatment in Africa. All mutant alleles remained sensitive to sulfachloropyridazine and sulfacetamide. These results suggest new drugs that could be tried for effective malaria treatment.

  16. High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Zylberman, V.; Craig, P.O.; Klinke, S.; Cauerhff, A.; Goldbaum, F.A. [Instituto Leloir, Buenos Aires (Argentina); Braden, B.C. [Bowie State Univ., Maryland (United States)

    2004-07-01

    The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18 kDa monomers, each extensively contacting neighboring monomers. The icosahedral structure consists of 60 LS monomers arranged as twelve pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella spp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedral enzymes. This unusual divergence prompted to further investigate on its quaternary arrangement. In the present work, we demonstrate by means of solution Light Scattering and X-ray structural analyses that BLS assembles as a very stable dimer of pentamers representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein, and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 deg C compared to pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. (author)

  17. Association of a Soybean Raffinose Synthase Gene with Low Raffinose and Stachyose Seed Phenotype

    Directory of Open Access Journals (Sweden)

    Emily C. Dierking

    2008-11-01

    Full Text Available Oligosaccharides are an important component of soybean [ (L. Merr.] meal in terms of metabolizable energy for monogastric animals. Sucrose, raffinose, and stachyose are the three main oligosaccharides present in soybean meal. Of the three, only sucrose is nutritionally useful. When raffinose and stachyose are fermented by microbes present in the gut, the results are flatulence and discomfort, which ultimately lead to poor weight gain. The long term objective of this research is ultimately to increase the nutritional value of soybean meal by elevating the metabolizable energy at the expense of raffinose and stachyose through the manipulation of soybean raffinose synthase, the key enzyme for raffinose and stachyose biosynthesis. The objectives of this work were to develop molecular genetic information about soybean raffinose synthases and to evaluate the candidate raffinose synthase genes in a soybean germplasm accession (PI 200508 that contains low levels of raffinose and stachyose. Our results indicate the soybean genome contains at least two expressed genes similar to other characterized raffinose synthases. A novel allele of one of these putative soybean raffinose synthase genes was discovered from the PI 200508 that completely associates with the low raffinose and stachyose phenotype. Molecular marker assays specific for the PI 200508 allele were developed to allow direct selection for the low raffinose and low stachyose phenotype.

  18. Inhibition of nitric oxide synthases abrogates pregnancy-induced uterine vascular expansive remodeling.

    Science.gov (United States)

    Osol, George; Barron, Carolyn; Gokina, Natalia; Mandala, Maurizio

    2009-01-01

    It was the aim of this study to test the hypothesis that hypertension and/or inhibition of nitric oxide (NO) synthases alters uterine vascular remodeling during pregnancy. Using a model of hypertension (NO synthase inhibition with L-NAME) in nonpregnant and pregnant rats, comparisons were made with age-matched controls, as well as with animals receiving hydralazine along with L-NAME to maintain normotension in the presence of NO synthase inhibition. Circumferential and axial remodeling of large (main uterine, MUA) and small (premyometrial radial) arteries were quantified and compared. L-NAME treatment prevented expansive circumferential remodeling of the MUA; cotreatment with hydralazine was without effect. Circumferential remodeling of smaller premyometrial radial arteries was also significantly attenuated in hypertensive pregnant animals, while premyometrial radial arteries from rats receiving hydralazine with L-NAME were of intermediate diameter. Neither hypertension nor NO synthase inhibition had any effect on the substantial (200-300%) axial growth of MUA or premyometrial radial arteries. NO plays a major role in facilitating pregnancy-induced expansive remodeling in the uterine circulation, particularly in larger arteries. Some beneficial effects of hydralazine on expansive circumferential remodeling were noted in smaller radial vessels, and these may be linked to its prevention of systemic hypertension and/or to local effects on the arterial wall. Neither NO synthase inhibition nor hypertension had any effect on arterial longitudinal growth.

  19. Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution.

    Science.gov (United States)

    Lee, Pyung Cheon; Mijts, Benjamin N; Petri, Ralf; Watts, Kevin T; Schmidt-Dannert, Claudia

    2004-11-01

    Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.

  20. S-Adenosylmethionine modulates inducible nitric oxide synthase gene expression in rat liver and isolated hepatocytes.

    Science.gov (United States)

    Majano, P L; García-Monzón, C; García-Trevijano, E R; Corrales, F J; Cámara, J; Ortiz, P; Mato, J M; Avila, M A; Moreno-Otero, R

    2001-12-01

    Hepatocellular availability of S-adenosylmethionine, the principal biological methyl donor, is compromised in situations of liver damage. S-Adenosylmethionine administration alleviates experimental liver injury and increases survival in cirrhotic patients. The mechanisms behind these beneficial effects of S-adenosylmethionine are not completely known. An inflammatory component is common to many of the pathological conditions in which S-adenosylmethionine grants protection to the liver. This notion led us to study the effect of S-adenosylmethionine administration on hepatic nitric oxide synthase-2 induction in response to bacterial lipopolysaccharide and proinflammatory cytokines. The effect of S-adenosylmethionine on nitric oxide synthase-2 expression was assessed in rats challenged with bacterial lipopolysaccharide and in isolated rat hepatocytes treated with proinflammatory cytokines. Interactions between S-adenosylmethionine and cytokines on nuclear factor kappa B activation and nitric oxide synthase-2 promoter transactivation were studied in isolated rat hepatocytes and HepG2 cells, respectively. S-Adenosylmethionine attenuated the induction of nitric oxide synthase-2 in the liver of lipopolysaccharide-treated rats and in cytokine-treated hepatocytes. S-Adenosylmethionine accelerated the resynthesis of inhibitor kappa B alpha, blunted the activation of nuclear factor kappa B and reduced the transactivation of nitric oxide synthase-2 promoter. Our findings indicate that the hepatoprotective actions of S-adenosylmethionine may be mediated in part through the modulation of nitric oxide production.

  1. Molecular Cloning, Expression, Purification, and Functional Characterization of Dammarenediol Synthase from Panax ginseng

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    Wei Hu

    2013-01-01

    Full Text Available The objective of this study is to clone and charecterize the expression of dammarenediol synthase gene and then to determine the relationship between the expression of dammarenediol synthase gene that is involved in the ginsenoside biosynthetic pathway and the ginsenoside content. A cDNA phage library was constructed from a five-year-old ginseng root. The cDNA library was screened for the dammarenediol synthase gene by using its specific primers. It was further cloned and expressed in pET-30a vector. The recombinant plasmid pET-30a-DS was expressed in Rosetta E. coli. The recombinant DS protein was purified by affinity chromatography. The production of dammarenediol was detected by liquid chromatography-mass spectrometry (LC-MS. Results showed that dammarenediol synthase gene was cloned from the cDNA library and was expressed in Rosetta E. coli and the SDS-PAGE analysis showed the presence of purified DS protein. LS-MS showed the activity of DS protein, as the protein content increases the dammarenediol increases. Our results indicate that the recombinant dammarenediol synthase protein could increase the production of dammarenediol and the expression of DS played a vital role in the biosynthesis of ginsenosides in P. ginseng.

  2. Haplotype analysis of sucrose synthase gene family in three Saccharum species

    Science.gov (United States)

    2013-01-01

    Background Sugarcane is an economically important crop contributing about 80% and 40% to the world sugar and ethanol production, respectively. The complicated genetics consequential to its complex polyploid genome, however, have impeded efforts to improve sugar yield and related important agronomic traits. Modern sugarcane cultivars are complex hybrids derived mainly from crosses among its progenitor species, S. officinarum and S. spontanuem, and to a lesser degree, S. robustom. Atypical of higher plants, sugarcane stores its photoassimilates as sucrose rather than as starch in its parenchymous stalk cells. In the sugar biosynthesis pathway, sucrose synthase (SuSy, UDP-glucose: D-fructose 2-a-D-glucosyltransferase, EC 2.4.1.13) is a key enzyme in the regulation of sucrose accumulation and partitioning by catalyzing the reversible conversion of sucrose and UDP into UDP-glucose and fructose. However, little is known about the sugarcane SuSy gene family members and hence no definitive studies have been reported regarding allelic diversity of SuSy gene families in Saccharum species. Results We identified and characterized a total of five sucrose synthase genes in the three sugarcane progenitor species through gene annotation and PCR haplotype analysis by analyzing 70 to 119 PCR fragments amplified from intron-containing target regions. We detected all but one (i.e. ScSuSy5) of ScSuSy transcripts in five tissue types of three Saccharum species. The average SNP frequency was one SNP per 108 bp, 81 bp, and 72 bp in S. officinarum, S. robustom, and S. spontanuem respectively. The average shared SNP is 15 between S. officinarum and S. robustom, 7 between S. officinarum and S. spontanuem , and 11 between S. robustom and S. spontanuem. We identified 27, 35, and 32 haplotypes from the five ScSuSy genes in S. officinarum, S. robustom, and S. spontanuem respectively. Also, 12, 11, and 9 protein sequences were translated from the haplotypes in S. officinarum, S. robustom, S

  3. Bifunctional activity of deoxyhypusine synthase/hydroxylase from Trichomonas vaginalis.

    Science.gov (United States)

    Quintas-Granados, Laura Itzel; Carvajal Gamez, Bertha Isabel; Villalpando, Jose Luis; Ortega-Lopez, Jaime; Arroyo, Rossana; Azuara-Liceaga, Elisa; Álvarez-Sánchez, María Elizbeth

    2016-04-01

    The Trichomonas vaginalis genome analysis suggested the presence of a putative deoxyhypusine synthase (TvDHS) that catalyzes the posttranslational modification of eIF-5A. Herein, we expressed and purified the recombinant TvDHS (rTvDHS) protein (43 kDa) and the recombinant TveIF-5A (rTveIF-5A) precursor protein (46 kDa). A 41 kDa band of the native TvDHS was recognized by western blot analysis in T. vaginalis total protein extract by a mouse polyclonal anti-rTvDHS antibody. The enzymatic activity of rTvDHS was determined by in vitro rTveIF-5A precursor modification. The modification reaction was performed by using ((3)H)-spermidine, and the biochemical analysis showed that rTvDHS exhibited Km value of 0.6 μM. The rTvDHS activity was inhibited by the spermidine analog, N″-guanyl-1,7-diamino-heptane (GC7). Native gel electrophoresis analysis showed two bands corresponding to an rTvDHS-rTveIF-5A complex and an intermediate form of rTveIF-5A. The two forms were subsequently separated by ion exchange chromatography to identify the hypusine residue by MS/MS analysis. Moreover, mutations in TvDHS showed that the putative HE motif present in this enzyme is involved in the hydroxylation of TveIF-5A. We observed that only hypusine-containing TveIF-5A was bound to an RNA hairpin ERE structure from the cox-2 gene, which contains the AAAUGUCACAC consensus sequence. Interestingly, 2DE-WB assays, using parasites that were grown in DAB-culture conditions and transferred to exogenous putrescine, showed the new isoform of TveIF-5A. In summary, our results indicate that T. vaginalis contains an active TvDHS capable of modifying the precursor TveIF-5A protein, which subsequently exhibits RNA binding activity. Copyright © 2015. Published by Elsevier B.V.

  4. Modulation of inducible nitric oxide synthase expression by sumoylation

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    Feinstein Douglas L

    2009-03-01

    Full Text Available Abstract Background In astrocytes, the inflammatory induction of Nitric Oxide Synthase type 2 (NOS2 is inhibited by noradrenaline (NA at the transcriptional level however its effects on specific transcription factors are not fully known. Recent studies show that the activity of several transcription factors including C/EBPβ, which is needed for maximal NOS2 expression, is modulated by conjugation of the small molecular weight protein SUMO. We examined whether the expression of SUMO Related Genes (SRGs: SUMO-1, the conjugating enzyme Ubc9, and the protease SENP1 are affected by inflammatory conditions or NA and whether SUMO-1 regulates NOS2 through interaction with C/EBPβ. Methods Bacterial endotoxin lipopolysaccharide (LPS was used to induce inflammatory responses including NOS2 expression in primary astrocytes. The mRNA levels of SRGs were determined by QPCR. A functional role for SUMOylation was evaluated by determining effects of over-expressing SRGs on NOS2 promoter and NFκB binding-element reporter constructs. Interactions of SUMO-1 and C/EBPβ with the NOS2 promoter were examined by chromatin immunoprecipitation assays. Interactions of SUMO-1 with C/EBPβ were examined by immunoprecipitation and Western blot analysis and by fluorescence resonance energy transfer (FRET assays. Results LPS decreased mRNA levels of SUMO-1, Ubc9 and SENP1 in primary astrocytes and a similar decrease occurred during normal aging in brain. NA attenuated the LPS-induced reductions and increased SUMO-1 above basal levels. Over-expression of SUMO-1, Ubc9, or SENP1 reduced the activation of a NOS2 promoter, whereas activation of a 4 × NFκB binding-element reporter was only reduced by SUMO-1. ChIP studies revealed interactions of SUMO-1 and C/EBPβ with C/EBP binding sites on the NOS2 promoter that were modulated by LPS and NA. SUMO-1 co-precipitated with C/EBPβ and a close proximity was confirmed by FRET analysis. Conclusion Our results demonstrate that

  5. Dihydropteroate Synthase of Mycobacterium leprae and Dapsone Resistance

    Science.gov (United States)

    Williams, Diana L.; Spring, Laynette; Harris, Eugene; Roche, Paul; Gillis, Thomas P.

    2000-01-01

    Two Mycobacterium leprae genes, folP1 and folP2, encoding putative dihydropteroate synthases (DHPS), were studied for enzymatic activity and for the presence of mutations associated with dapsone resistance. Each gene was cloned and expressed in a folP knockout mutant of Escherichia coli (C600ΔfolP::Kmr). Expression of M. leprae folP1 in C600ΔfolP::Kmr conferred growth on a folate-deficient medium, and bacterial lysates exhibited DHPS activity. This recombinant displayed a 256-fold-greater sensitivity to dapsone (measured by the MIC) than wild-type E. coli C600, and 50-fold less dapsone was required to block (expressed as the 50% inhibitory concentration [IC50]) the DHPS activity of this recombinant. When the folP1 genes of several dapsone-resistant M. leprae clinical isolates were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting an isoleucine for a threonine residue (T53I) in the DHPS-1, and a second mutation occurred in codon 55, substituting an arginine for a proline residue (P55R). Transformation of the C600ΔfolP::Kmr knockout with plasmids carrying either the T53I or the P55R mutant allele did not substantially alter the DHPS activity compared to levels produced by recombinants containing wild-type M. leprae folP1. However, both mutations increased dapsone resistance, with P55R having the greatest affect on dapsone resistance by increasing the MIC 64-fold and the IC50 68-fold. These results prove that the folP1 of M. leprae encodes a functional DHPS and that mutations within this gene are associated with the development of dapsone resistance in clinical isolates of M. leprae. Transformants created with M. leprae folP2 did not confer growth on the C600ΔfolP::Kmr knockout strain, and DNA sequences of folP2 from dapsone-susceptible and -resistant M. leprae strains were identical, indicating that this gene does not encode a functional DHPS and is not involved in dapsone resistance in M. leprae. PMID:10817704

  6. Intermediacy of eudesmane cation during catalysis by aristolochene synthase.

    Science.gov (United States)

    Faraldos, Juan A; Kariuki, Benson; Allemann, Rudolf K

    2010-02-19

    Aristolochene synthase from Penicillium roqueforti (PR-AS) catalyzes the formation of the bicyclic sesquiterpene (+)-aristolochene (5) from farnesyl diphosphate (1, FDP) in two mechanistically distinct cyclization reactions. The first reaction transforms farnesyl diphosphate to the uncharged intermediate (S)-(-)-germacrene A (3) through a macrocyclization process that links C1 and C10 upon magnesium ion-assisted diphosphate ester activation. In the second reaction mediated by PR-AS, a protonation induced cyclization has been suggested to generate the highly reactive trans-fused eudesmane cation 4 as a consequence of the precise folding of the enzyme-bound germacrene A intermediate. This contribution describes the use of the transition state analogue inhibitor 4-aza-eudesm-11-ene to explore the intermediacy of cation 4 as an on-path intermediate in the biosynthesis of aristolochene. 4-Aza-eudesm-11-ene as the hydrochloride salt 6 was stereospecifically synthesized in seven steps and 37% overall yield starting from chiral enamine 9. The synthetic sequence featured a highly regio- and stereoselective deracemization reaction of 9 that gave rise to the corresponding Michael adduct in >95% diastereomeric excess as evidenced by optical rotation and NMR measurements. 6 acts as a potent competitive inhibitor of PR-AS (K(i) = 0.35 +/- 0.12 microM) independent of the presence of diphosphate (K(i) = 0.24 +/- 0.09 microM). The failure of exogenous PP(i) to enhance the binding affinity of 6 for PR-AS could be interpreted against an eudesmyl cation/diphosphate anion pair mechanism as the enzymatic strategy to stabilize the highly reactive eudesmane cation 4. In addition, these observations seem to rule out simple favorable electrostatic and/or hydrogen bonding interactions between the active site anchored diphosphate ion and the ammonium ion 6 as the binding mode. Ammonium ion 6 seems to act as a genuine mimic of eudesmane cation (4) that most likely binds the active site of PR

  7. Structure and reaction mechanism of basil eugenol synthase.

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    Gordon V Louie

    2007-10-01

    Full Text Available Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs, and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H and a mixed competitive inhibitor EMDF ((7S,8S-ethyl (7,8-methylene-dihydroferulate provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent

  8. Identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene (crtE) and phytoene synthase gene (crtB) for carotenoid biosynthesis in Euglena gracilis.

    Science.gov (United States)

    Kato, Shota; Takaichi, Shinichi; Ishikawa, Takahiro; Asahina, Masashi; Takahashi, Senji; Shinomura, Tomoko

    2016-01-05

    Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin, diatoxanthin, diadinoxanthin and 9'-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated. To clarify the carotenoid biosynthetic pathway in E. gracilis, we searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E. gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene synthase, respectively. Phylogenetic analyses indicated that the predicted proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP synthase and phytoene synthase proteins, respectively, of algae and higher plants. In addition, we investigated the effects of light stress on the expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920 μmol m(-2) s(-1) at 25 °C decreased the E. gracilis cell concentration by 28-40 % and 13-91 %, respectively, relative to the control light intensity (55 μmol m(-2) s(-1)). When grown under continuous light at 920 μmol m(-2) s(-1), the algal cells turned reddish-orange and showed a 1.3-fold increase in the crtB expression. In contrast, EgcrtE expression was not significantly affected by the light-stress treatments examined. We identified genes

  9. Chalcone synthase genes from milk thistle (Silybum marianum ...

    Indian Academy of Sciences (India)

    of products from two phenylpropanoid branch pathways, monolignol and flavonoid biosynthesis. The monolignol component, coniferyl alcohol, derives from the two-step side-chain reduction of feruloyl-CoA, itself formed from the hydroxylation and O-methylation of p- coumaroyl-CoA. The flavonoid component, taxifolin ...

  10. Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins

    DEFF Research Database (Denmark)

    Vestergaard, Martin; Nøhr-Meldgaard, Katrine; Bojer, Martin Saxtorph

    2017-01-01

    Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened......G or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, gallidermin, bacitracin, vancomycin, ciprofloxacin......, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards...

  11. Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis.

    Science.gov (United States)

    Begley, Darren W; Edwards, Thomas E; Raymond, Amy C; Smith, Eric R; Hartley, Robert C; Abendroth, Jan; Sankaran, Banumathi; Lorimer, Donald D; Myler, Peter J; Staker, Bart L; Stewart, Lance J

    2011-09-01

    Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research.

  12. Molecular cloning, functional expression and characterization of d-limonene synthase from Agastache rugosa.

    Science.gov (United States)

    Maruyama, Takuro; Saeki, Daisuke; Ito, Michiho; Honda, Gisho

    2002-05-01

    We cloned the gene of d-limonene synthase (ArLMS) from Agastache rugosa (Labiatae). The function of ArLMS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. ArLMS consisted of 2077 nucleotides including 1839 bp of coding sequence that encodes a protein of 613 amino acids. This protein has a 60 kDa molecular weight, which is identical to that of d-limonene synthase from Schizonepeta tenuifolia (Labiatae). The deduced amino acid sequence of ArLMS shows high homology with the known d- and l-limonene synthases from Labiatae plants. Here, we discussed the amino acid residues responsible for the stereochemical regulation in limonene biosynthesis.

  13. Crystallization and preliminary crystallographic analysis of an octaketide-producing plant type III polyketide synthase

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Hiroyuki [Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511 (Japan); Kondo, Shin; Kato, Ryohei [Innovation Center Yokohama, Mitsubishi Chemical Corporation, 1000 Kamoshida, Aoba, Yokohama, Kanagawa 227-8502 (Japan); Wanibuchi, Kiyofumi; Noguchi, Hiroshi [School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526 (Japan); Sugio, Shigetoshi, E-mail: sugio.shigetoshi@mw.m-kagaku.co.jp [Innovation Center Yokohama, Mitsubishi Chemical Corporation, 1000 Kamoshida, Aoba, Yokohama, Kanagawa 227-8502 (Japan); Abe, Ikuro, E-mail: sugio.shigetoshi@mw.m-kagaku.co.jp [School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526 (Japan); PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kohno, Toshiyuki, E-mail: sugio.shigetoshi@mw.m-kagaku.co.jp [Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511 (Japan)

    2007-11-01

    Octaketide synthase from A. arborescens has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 2.6 Å. Octaketide synthase (OKS) from Aloe arborescens is a plant-specific type III polyketide synthase that produces SEK4 and SEK4b from eight molecules of malonyl-CoA. Recombinant OKS expressed in Escherichia coli was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group I422, with unit-cell parameters a = b = 110.2, c = 281.4 Å, α = β = γ = 90.0°. Diffraction data were collected to 2.6 Å resolution using synchrotron radiation at BL24XU of SPring-8.

  14. Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

    Science.gov (United States)

    Cheng, Jiujun; Charles, Trevor C

    2016-09-01

    Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

  15. 6-MSAS-like polyketide synthase genes occur in lichenized ascomycetes.

    Science.gov (United States)

    Schmitt, Imke; Kautz, Stefanie; Lumbsch, H Thorsten

    2008-02-01

    Lichenized and non-lichenized filamentous ascomycetes produce a great variety of polyketide secondary metabolites. Some polyketide synthase (PKS) genes from non-lichenized fungi have been characterized, but the function of PKS genes from lichenized species remains unknown. Phylogenetic analysis of keto synthase (KS) domains allows prediction of the presence or absence of particular domains in the PKS gene. In the current study we screened genomic DNA from lichenized fungi for the presence of non-reducing and 6-methylsalicylic acid synthase (6-MSAS)-type PKS genes. We developed new degenerate primers in the acyl transferase (AT) region to amplify a PKS fragment spanning most of the KS region, the entire linker between KS and AT, and half of the AT region. Phylogenetic analysis shows that lichenized taxa possess PKS genes of the 6-MSAS-type. The extended alignment confirms overall phylogenetic relationships between fungal non-reducing, 6-MSAS-type and bacterial type I PKS genes.

  16. Identification of novel isoprene synthases through genome mining and expression in Escherichia coli.

    Science.gov (United States)

    Ilmén, Marja; Oja, Merja; Huuskonen, Anne; Lee, Sangmin; Ruohonen, Laura; Jung, Simon

    2015-09-01

    Isoprene is a naturally produced hydrocarbon emitted into the atmosphere by green plants. It is also a constituent of synthetic rubber and a potential biofuel. Microbial production of isoprene can become a sustainable alternative to the prevailing chemical production of isoprene from petroleum. In this work, sequence homology searches were conducted to find novel isoprene synthases. Candidate sequences were functionally expressed in Escherichia coli and the desired enzymes were identified based on an isoprene production assay. The activity of three enzymes was shown for the first time: expression of the candidate genes from Ipomoea batatas, Mangifera indica, and Elaeocarpus photiniifolius resulted in isoprene formation. The Ipomoea batatas isoprene synthase produced the highest amounts of isoprene in all experiments, exceeding the isoprene levels obtained by the previously known Populus alba and Pueraria montana isoprene synthases that were studied in parallel as controls. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  17. Novel terpenes generated by heterologous expression of bacterial terpene synthase genes in an engineered Streptomyces host.

    Science.gov (United States)

    Yamada, Yuuki; Arima, Shiho; Nagamitsu, Tohru; Johmoto, Kohei; Uekusa, Hidehiro; Eguchi, Tadashi; Shin-ya, Kazuo; Cane, David E; Ikeda, Haruo

    2015-06-01

    Mining of bacterial genome data has revealed numerous presumptive terpene synthases. Heterologous expression of several putative terpene synthase genes in an engineered Streptomyces host has revealed 13 newly discovered terpenes whose GC-MS and NMR data did not match with any known compounds in spectroscopic databases. Each of the genes encoding the corresponding terpene synthases were silent in their parent microorganisms. Heterologous expression and detailed NMR spectroscopic analysis allowed assignment of the structures of 13 new cyclic terpenes. Among these newly identified compounds, two were found to be linear triquinane sesquiterpenes that have never previously been isolated from bacteria or any other source. The remaining 11 new compounds were shown to be diterpene hydrocarbons and alcohol, including hydropyrene (1), hydropyrenol (2), tsukubadiene (11) and odyverdienes A (12) and B (13) each displaying a novel diterpene skeleton that had not previously been reported.

  18. Multiple phosphorylation sites in the beta subunit of thylakoid ATP synthase.

    Science.gov (United States)

    del Riego, Guillermo; Casano, Leonardo M; Martín, Mercedes; Sabater, Bartolomé

    2006-07-01

    Proteomic analyses of the beta subunit of the plastid ATP synthase of barley (Hordeum vulgare L.) revealed that mature protein was not carboxy terminus processed and suggested the correction of the 274 codon (GAT to AAT) in the data bank that was confirmed by DNA sequencing. Six isoforms of the ATP synthase beta subunit with pI ranging from 4.95 to 5.14 were resolved by two-dimensional electrophoresis (2-DE). Mass spectrometry analyses indicated that the six isoforms differ in their phosphorylation degree, which was confirmed by the disappearance of more acidic forms after incubation with the protein phosphatase calcineurin. Six Ser and/or Thr were detected as phosphorylated, among them the conserved Thr-179 that is also phosphorylated in the beta subunit of human mitochondria. The results are discussed in relation with the proposed regulation of the ATP synthase by phosphorylation and 14-3-3 proteins.

  19. Broad Substrate Specificity of the Loading Didomain of the Lipomycin Polyketide Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Yuzawa, S; Eng, CH; Katz, L; Keasling, JD

    2013-06-04

    LipPks1, a polyketide synthase subunit of the lipomycin synthase, is believed to catalyze the polyketide chain initiation reaction using isobutyryl-CoA as a substrate, followed by an elongation reaction with methylmalonyl-CoA to start the biosynthesis of antibiotic alpha-lipomycin in Streptomyces aureofaciens Tu117. Recombinant LipPks1, containing the thioesterase domain from the 6-deoxyerythronolide B synthase, was produced in Escherichia coli, and its substrate specificity was investigated in vitro. Surprisingly, several different acyl-CoAs, including isobutyryl-CoA, were accepted as the starter substrates, while no product was observed with acetyl-CoA. These results demonstrate the broad substrate specificity of LipPks1 and may be applied to producing new antibiotics.

  20. Characterization of a sabinene synthase gene from rough lemon (Citrus jambhiri).

    Science.gov (United States)

    Kohzaki, Keisuke; Gomi, Kenji; Yamasaki-Kokudo, Yumiko; Ozawa, Rika; Takabayashi, Junji; Akimitsu, Kazuya

    2009-10-15

    We previously isolated two putative monoterpene synthase genes, RlemTPS1 and RlemTPS2, from rough lemon (Citrus jambhiri) and showed that gene expression of RlemTPS2 was induced by microbial attack. The protein product of RlemTPS2 was obtained using a prokaryotic expression system, and GC and GC-MS of monoterpene synthesis by RlemTPS2 determined that RlemTPS2 encodes a sabinene synthase. Sabinene has antifungal activity toward Alternaria alternata. Furthermore, site-directed mutagenesis identified one amino acid, Ile, located at the front of the metal ion binding motif as an important residue for the product specificity of sabinene synthase.

  1. Functional Characterization of Nine Norway Spruce TPS Genes and Evolution of Gymnosperm Terpene Synthases of the TPS-d Subfamily1[w

    Science.gov (United States)

    Martin, Diane M.; Fäldt, Jenny; Bohlmann, Jörg

    2004-01-01

    Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (−)-limonene synthase, (−)-α/β-pinene synthase, and (−)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-α-farnesene synthase, and E-α-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed. PMID:15310829

  2. Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms With Acute Rejection in Liver Transplant Recipients.

    Science.gov (United States)

    Azarpira, Negar; Namazi, Soha; Malahi, Sayan; Kazemi, Kourosh

    2016-06-01

    Polymorphisms of the endothelial nitric oxide synthase gene have been associated with altered endothelial nitric oxide synthase activity. The purpose of this study was to investigate the relation between endothelial nitric oxide synthase -786T/C and 894G/T polymorphism and their haplotypes on the occurrence of acute rejection episodes in liver transplant recipients. We conducted a case control study in which 100 liver transplant recipients and 100 healthy controls were recruited from Shiraz Transplant Center. The patients used triple therapy including tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression maintenance. DNA was extracted from peripheral blood and endothelial nitric oxide synthase polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism. Patients included 60 men and 40 women (mean age, 32.35 ± 10.2 y). There was a significant association of endothelial nitric oxide synthase 894G/T and acute rejection episode. The GT* gen-otype and acute rejection episodes had a significant association (odds ratio, 2.42; 95% confidence interval, 0.97-6.15; P = .03). The GG and GT* genotype and T* allele frequency were significantly different between patients and control subjects (P = .001). Haplotype TT* was higher in recipients than control subjects (odds ratio, 2.17; 95% confidence interval, 1.12-4.25; P = .01). Haplotype TG was higher in the control group (odds ratio, 0.62; 95% confidence interval, 0.40-0.96; P = .02). Our results suggest a relation between different endothelial nitric oxide synthase geno-types and risk of acute rejection episodes. However, further study is necessary to determine genetic susceptibility for transplant patients.

  3. A bifunctional geranyl and geranylgeranyl diphosphate synthase is involved in terpene oleoresin formation in Picea abies.

    Science.gov (United States)

    Schmidt, Axel; Wächtler, Betty; Temp, Ulrike; Krekling, Trygve; Séguin, Armand; Gershenzon, Jonathan

    2010-02-01

    The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin composed chiefly of monoterpenes (C(10)) and diterpenes (C(20)). An important group of enzymes in oleoresin biosynthesis are the short-chain isoprenyl diphosphate synthases that produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of different terpenoid classes. We isolated a gene from P. abies via a homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl diphosphate synthase making an unusual mixture of two products, geranyl diphosphate (C(10)) and geranylgeranyl diphosphate (C(20)). This bifunctionality was confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this isoprenyl diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to single-product conifer geranyl diphosphate and geranylgeranyl diphosphate synthases. Its catalytic properties and reaction mechanism resemble those of conifer geranylgeranyl diphosphate synthases, except that significant quantities of the intermediate geranyl diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product geranyl diphosphate and geranylgeranyl diphosphate synthases, specific amino acid residues were identified that alter the relative composition of geranyl to geranylgeranyl diphosphate.

  4. Identification and characterization of a second isogene encoding γ-terpinene synthase in Thymus caespititius.

    Science.gov (United States)

    Mendes, Marta D; Barroso, José G; Oliveira, M Margarida; Trindade, Helena

    2014-07-15

    Thymus caespititius Brot. is an Iberian endemic species, whose essential oils possess high polymorphism. They consist mostly of mono- and sesquiterpene, some of them with interest for the pharmaceutical and food industries. The search for terpene synthase genes was performed in three in vitro T. caespititius genotypes. For these plants, the expression of a previously described γ-terpinene synthase gene, Tctps2, was confirmed, occurring concomitantly with a new gene encoding an enzyme with similar activity, named Thymus caespititius terpene synthase 4 (Tctps4). The two isogenes were isolated and functionally characterized in the three plant genotypes. Alignment of the two Tctps revealed a transit peptide much shorter in Tctps4 than in Tctps2 (3-4 amino acids instead of 47). The Tctps4 open reading frame is shorter than Tctps2 (1665 bp versus 1794 bp). The amino acid sequence of both γ-terpinene synthases shared an 88% pairwise identity. The fact that T. caespititius carries two isogenes for γ-terpinene synthases, suggests gene duplication along the evolutionary process, followed by mutations leading to the differentiation of both genes. These mutations didn't compromise protein activity. A high accumulation of transcripts from both genes was found in shoots of in vitro plantlets, while in roots they could not be detected. Still, γ-terpinene levels in aerial parts were reduced, probably due to fast conversion into carvacrol and thymol, the main components from T. caespititius essential oils. This study is a contribution to the identification of terpene synthase genes in Lamiaceae. Copyright © 2014 Elsevier GmbH. All rights reserved.

  5. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase.

    OpenAIRE

    Nishida, K; Harrison, D.G.; Navas, J P; Fisher, A.A.; Dockery, S P; Uematsu, M; Nerem, R M; Alexander, R W; Murphy, T. J.

    1992-01-01

    The constitutive endothelial cell nitric oxide synthase (NOS) importantly regulates vascular homeostasis. To gain understanding of this enzyme, a pEF BOS cDNA library of 5 x 10(5) clones was prepared from bovine aortic endothelial cells (BAEC) and screened with a 2.8-kb cDNA BamHI fragment of rat brain NOS. Clone pBOS13 was found to express NO synthase activity when transfected into COS-7 cells. Sequence analysis revealed sequences compatible with binding domains for calcium/calmodulin, flavi...

  6. Studies on the chalcone synthase gene of two higher plants: petroselinum hortense and matthiola incana

    Energy Technology Data Exchange (ETDEWEB)

    Hemleben, V.; Frey, M.; Rall, S.; Koch, M.; Kittel, M.; Kreuzaler, F.; Ragg, H.; Fautz, E.; Hahlbrock, K.

    1982-01-01

    Two higher plant systems are presented which allow to study coordinated gene expression of the light-induced metabolic pathway of flavonoid biosynthesis: tissue culture cells of Petroselinum hortense (Apiaceae) and different developmental stages of various genotypes of Matthiola incana (Brassicaceae). The gene structure of the chalcone synthase is mainly studied. A cDNA clone (pLF56) of parsley has been constructed and characterized conferring the chalcone synthase gene sequence. Strong cross hybridization between the parsley cDNA and Matthiola DNA allowed to identify a HindIII fragment (6000 bp) identical in size for parsley and different Matthiola wild type lines and a mutant line.

  7. Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

    Directory of Open Access Journals (Sweden)

    Ro Dae-Kyun

    2009-07-01

    Full Text Available Abstract Background Sesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development. Results Glandular trichomes of sunflower (Helianthus annuus L. were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of in vitro and in vivo characterization of sesquiterpene synthase gene products in Escherichia coli and Saccharomyces cerevisiae, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low in vitro activity, the third enzyme was expressed in vivo in yeast as a thioredoxin-fusion protein for functional characterization. In in vivo assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon δ-cadinene as one of the two main products with α-muuorlene, β-caryophyllene, α-humulene and α-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes

  8. Reviewing Ligand-Based Rational Drug Design: The Search for an ATP Synthase Inhibitor

    Directory of Open Access Journals (Sweden)

    Hsueh-Fen Juan

    2011-08-01

    Full Text Available Following major advances in the field of medicinal chemistry, novel drugs can now be designed systematically, instead of relying on old trial and error approaches. Current drug design strategies can be classified as being either ligand- or structure-based depending on the design process. In this paper, by describing the search for an ATP synthase inhibitor, we review two frequently used approaches in ligand-based drug design: The pharmacophore model and the quantitative structure-activity relationship (QSAR method. Moreover, since ATP synthase ligands are potentially useful drugs in cancer therapy, pharmacophore models were constructed to pave the way for novel inhibitor designs.

  9. The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

    Directory of Open Access Journals (Sweden)

    Kaori Kohzuma

    2017-07-01

    Full Text Available The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP

  10. Caractérisation d'inhibiteurs potentiels des NO-synthases

    OpenAIRE

    Pijeaud, Sandra; Bottin, Olivier; Kouoi, Rémy; Berthat, Virginie; Besson, Valérie,; Curis, Emmanuel; Desaulle, Dorota; Lerouet, Dominique

    2016-01-01

    Parmi les mécanismes physiopathologiques du traumatisme crânien figure notamment la voie du monoxyde d'azote (NO), principalement produit par les NO-synthases de type 1 (NOS1) et 2 (NOS2). Le monoxyde d'azote forme des peroxynitrites, qui entraînent diverses altérations fonctionnelles au niveau cérébral. Alors qu'il n'existe aucun traitement préventif des complications du traumatisme crânien, une stratégie thérapeutique potentiellement intéressante serait d'inhiber les NO-synthases. Dans cett...

  11. Studies on the chalcone synthase gene of two higher plants: petroselinum hortense and matthiola incana.

    Science.gov (United States)

    Hemleben, V; Frey, M; Rall, S; Koch, M; Kittel, M; Kreuzaler, F; Ragg, H; Fautz, E; Hahlbrock, K

    1982-01-01

    Two higher plant systems are presented which allow to study coordinated gene expression of the light-induced metabolic pathway of flavonoid biosynthesis: tissue culture cells of Petroselinum hortense (Apiaceae) and different developmental stages of various genotypes of Matthiola incana (Brassicaceae). The gene structure of the chalcone synthase is mainly studied. A cDNA clone (pLF56) of parsley has been constructed and characterized conferring the chalcone synthase gene sequence. Strong cross hybridization between the parsley cDNA and Matthiola DNA allowed to identify a HindIII fragment (6000 bp) identical in size for parsley and different Matthiola wild type lines and a mutant line.

  12. Development of intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase for discriminating Curcuma species.

    Science.gov (United States)

    Kita, Tomoko; Komatsu, Katsuko; Zhu, Shu; Iida, Osamu; Sugimura, Koji; Kawahara, Nobuo; Taguchi, Hiromu; Masamura, Noriya; Cai, Shao-Qing

    2016-03-01

    Various Curcuma rhizomes have been used as medicines or spices in Asia since ancient times. It is very difficult to distinguish them morphologically, especially when they are boiled and dried, which causes misidentification leading to a loss of efficacy. We developed a method for discriminating Curcuma species by intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase. This method could apply to identification of not only fresh plants but also samples of crude drugs or edible spices. By applying this method to Curcuma specimens and samples, and constructing a dendrogram based on these markers, seven Curcuma species were clearly distinguishable. Moreover, Curcuma longa specimens were geographically distinguishable. On the other hand, Curcuma kwangsiensis (gl type) specimens also showed intraspecies polymorphism, which may have occurred as a result of hybridization with other Curcuma species. The molecular method we developed is a potential tool for global classification of the genus Curcuma. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Biallelic variants in WARS2 encoding mitochondrial tryptophanyl-tRNA synthase in six individuals with mitochondrial encephalopathy

    NARCIS (Netherlands)

    Wortmann, Saskia B.; Timal, Sharita; Venselaar, Hanka; Wintjes, Liesbeth T.; Kopajtich, Robert; Feichtinger, Rene G.; Onnekink, Carla; Muhlmeister, Mareike; Brandt, Ulrich; Smeitink, Jan A.; Veltman, Joris A.; Sperl, Wolfgang; Lefeber, Dirk; Pruijn, Ger; Stojanovic, Vesna; Freisinger, Peter; von Spronsen, Francjan; Derks, Terry G. J.; Veenstra-Knol, Hermine E.; Mayr, Johannes A.; Rotig, Agnes; Tarnopolsky, Mark; Prokisch, Holger; Rodenburg, Richard J.

    2017-01-01

    Mitochondrial protein synthesis involves an intricate interplay between mitochondrial DNA encoded RNAs and nuclear DNA encoded proteins, such as ribosomal proteins and aminoacyl-tRNA synthases. Eukaryotic cells contain 17 mitochondria-specific aminoacyl-tRNA synthases. WARS2 encodes mitochondrial

  14. Structure of dimeric ATP synthase from mitochondria : An angular association of monomers induces the strong curvature of the inner membrane

    NARCIS (Netherlands)

    Dudkina, Natalya V.; Heinemeyer, Jesco; Keegstra, Wilko; Boekema, Egbert J.; Braun, Hans-Peter

    2005-01-01

    Respiration in all cells depends upon synthesis of ATP by the ATP synthase complex, a rotary motor enzyme. The structure of the catalytic moiety of ATP synthase, the so-called F1 headpiece, is well established. F1 is connected to the membrane-bound and ion translocating F0 subcomplex by a central

  15. Organization and sequences of genes for the subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716

    NARCIS (Netherlands)

    van Walraven, H. S.; Lutter, R.; Walker, J. E.

    1993-01-01

    The sequences of the genes for the nine subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716 have been determined. The genes were identified by comparison of the encoded proteins with sequences of ATP synthase subunits in other species, and confirmed for subunits alpha,

  16. Predicting the Functions and Specificity of Triterpenoid Synthases: A Mechanism-Based Multi-intermediate Docking Approach

    Science.gov (United States)

    Tian, Bo-Xue; Wallrapp, Frank H.; Holiday, Gemma L.; Chow, Jeng-Yeong; Babbitt, Patricia C.; Poulter, C. Dale; Jacobson, Matthew P.

    2014-01-01

    Terpenoid synthases construct the carbon skeletons of tens of thousands of natural products. To predict functions and specificity of triterpenoid synthases, a mechanism-based, multi-intermediate docking approach is proposed. In addition to enzyme function prediction, other potential applications of the current approach, such as enzyme mechanistic studies and enzyme redesign by mutagenesis, are discussed. PMID:25299649

  17. Translocation of the potato 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase into isolated spinach chloroplasts

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Jianmin; Weaver, L.M.; Herrmann, K.M. (Purdue Univ., West Lafayette, IN (USA))

    1990-05-01

    A cDNA for potato (Solanum tuberosum L.) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, encodes a 56 KD polypeptide whose amino terminus resembles a chloroplast transit sequence. The cDNA was placed downstream of the phage T7 polymerase recognition sequence in plasmid pGEM-3Z. DNA of the resulting plasmid pGEM-DWZ directed T7 polymerase to synthesize potato DAHP synthase mRNA in vitro. The mRNA was used in wheat germ and rabbit reticulocyte lysates for the synthesis of {sup 35}S-labeled pro-DAHP synthase. The predominant translation product is a 59 KD polypeptide that can be immunoprecipitated by rabbit polyclonal antibodies raised against the 53 KD DAHP synthase purified from potato tubers. Isolated spinach chloroplasts process the 59 KD pro-DAHP synthase to a 50 KD polypeptide. The processed polypeptide is protected from protease degradation, suggesting uptake of the enzyme into the cell organelle. Fractionation of reisolated chloroplasts after import of pro-DAHP synthase showed mature enzyme in the stroma. The uptake and processing of DAHP synthase is inhibited by antibodies raised against the mature enzyme. Our results are consistent with the assumption that potato contains a nuclear DNA encoded DAHP synthase that is synthesized as a proenzyme and whose mature form resides in the chloroplasts. Our data provide further evidence that green plants synthesize aromatic amino acids in plastids.

  18. Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

    Science.gov (United States)

    Souza-Moreira, Tatiana M.; Alves, Thaís B.; Pinheiro, Karina A.; Felippe, Lidiane G.; de Lima, Gustavo M. A.; Watanabe, Tatiana F.; Barbosa, Cristina C.; Santos, Vânia A. F. F. M.; Lopes, Norberto P.; Valentini, Sandro R.; Guido, Rafael V. C.; Furlan, Maysa; Zanelli, Cleslei F.

    2016-11-01

    Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

  19. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Petri, Marcelo H. [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Tellier, Céline; Michiels, Carine [NARILIS, URBC, University of Namur, Namur (Belgium); Ellertsen, Ingvill [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Dogné, Jean-Michel [Department of Pharmacy, Namur Thrombosis and Hemostasis Center, University of Namur, Namur (Belgium); Bäck, Magnus, E-mail: Magnus.Back@ki.se [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)

    2013-11-15

    Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

  20. Suppression by Ghrelin of Porphyromonas gingivalis-Induced Constitutive Nitric Oxide Synthase S-Nitrosylation and Apoptosis in Salivary Gland Acinar Cells

    Directory of Open Access Journals (Sweden)

    Bronislaw L. Slomiany

    2010-01-01

    Full Text Available Oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, and its key virulence factor, LPS, are characterized by a massive rise in epithelial cell apoptosis and the disturbances in NO signaling pathways. Here, we report that the LPS-induced enhancement in rat sublingual salivary gland acinar cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS activity and a marked increase in the activity of inducible nitric oxide synthase (iNOS. We demonstrate that the detrimental effect of the LPS on cNOS was manifested by the enzyme protein S-nitrosylation, that was susceptible to inhibition by iNOS inhibitor, 1400 W. Further, we show that a peptide hormone, ghrelin, countered the LPS-induced changes in apoptosis and cNOS activity. This effect of ghrelin was reflected in the decrease in cNOS S-nitrosylation and the increase in phosphorylation. Our findings imply that P. gingivalis-induced disturbances in the acinar cell NO signaling pathways result from upregulation in iNOS-derived NO that causes cNOS S-nitrosylation that interferes with its activation through phosphorylation. We also show that ghrelin protection against P. gingivalis-induced disturbances involves cNOS activation associated with a decrease in its S-nitrosylation and the increase in phosphorylation.

  1. Biochemical and Structural Characterization of Germicidin Synthase: Analysis of a Type III Polyketide Synthase That Employs Acyl-ACP as a Starter Unit Donor

    Energy Technology Data Exchange (ETDEWEB)

    Chemler, Joseph A.; Buchholz, Tonia J.; Geders, Todd W.; Akey, David L.; Rath, Christopher M.; Chlipala, George E.; Smith, Janet L.; Sherman, David H. (Michigan)

    2012-08-10

    Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 {angstrom} germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.

  2. Anticonvulsion effect of acupuncture might be related to the decrease of neuronal and inducible nitric oxide synthases.

    Science.gov (United States)

    Yang, R; Huang, Z N; Cheng, J S

    1999-01-01

    To measure the levels of hippocampal nitric oxide synthase isoforms in penicillin induced epilepsy and to test the effect of electroacupuncture (EA) on changes of these levels during epilepsy, we injected penicillin into rat hippocampus to make an epilepsy model and performed electroacupuncture treatment on "Feng Fu" (DU 16) and "Jin Suo" (DU 8) points in Wistar rats. Nitric oxide synthase (NOS) mRNA levels of rat hippocampus were determined by reverse transcription-polymerase chain reaction (RT-PCR). The neuronal nitric oxide synthase (nNOS) mRNA markedly increased (pepilepsy, whereas no significant change in epithelial nitric oxide synthase (eNOS) mRNA was observed. EA inhibited the epilepsy and decreased nNOS (pepilepsy caused an increase in nNOS and iNOS, and the EA anticonvulsant effect might be related to the decrease of these nitric oxide synthases.

  3. Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

    Directory of Open Access Journals (Sweden)

    Mirian Perez Maluf

    2009-01-01

    Full Text Available In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

  4. Interest rate derivatives

    DEFF Research Database (Denmark)

    Svenstrup, Mikkel

    This Ph.D. thesis consists of four self-contained essays on valuation of interest rate derivatives. In particular derivatives related to management of interest rate risk care are considered.......This Ph.D. thesis consists of four self-contained essays on valuation of interest rate derivatives. In particular derivatives related to management of interest rate risk care are considered....

  5. Generalized Riemann derivative

    Directory of Open Access Journals (Sweden)

    Sorin Radulescu

    2013-03-01

    Full Text Available Initiated by Marshall Ash in 1966, the study of generalized Riemann derivative draw significant attention of the mathematical community and numerous studies where carried out since then. One of the major areas that benefits from these developments is the numerical analysis, as the use of generalized Riemann derivatives leads to solving a wider class of problems that are not solvable with the classical tools. This article studies the generalized Riemann derivative and its properties and establishes relationships between Riemann generalized derivative and the classical one. The existence of classical derivative implies the existence of the Riemann generalized derivative, and we study conditions necessary for the generalized Riemann derivative to imply the existence of the classical derivative. Furthermore, we provide conditions on the generalized Riemann derivative that are sufficient for the existence of the classical derivative.

  6. Interaction between Connexin 43 and nitric oxide synthase in mice heart mitochondria

    Science.gov (United States)

    Kirca, Mücella; Kleinbongard, Petra; Soetkamp, Daniel; Heger, Jacqueline; Csonka, Csaba; Ferdinandy, Péter; Schulz, Rainer

    2015-01-01

    Connexin 43 (Cx43), which is highly expressed in the heart and especially in cardiomyocytes, interferes with the expression of nitric oxide synthase (NOS) isoforms. Conversely, Cx43 gene expression is down-regulated by nitric oxide derived from the inducible NOS. Thus, a complex interplay between Cx43 and NOS expression appears to exist. As cardiac mitochondria are supposed to contain a NOS, we now investigated the expression of NOS isoforms and the nitric oxide production rate in isolated mitochondria of wild-type and Cx43-deficient (Cx43Cre-ER(T)/fl) mice hearts. Mitochondria were isolated from hearts using differential centrifugation and purified via Percoll gradient ultracentrifugation. Isolated mitochondria were stained with an antibody against the mitochondrial marker protein adenine-nucleotide-translocator (ANT) in combination with either a neuronal NOS (nNOS) or an inducible NOS (iNOS) antibody and analysed using confocal laser scanning microscopy. The nitric oxide formation was quantified in purified mitochondria using the oxyhaemoglobin assay. Co-localization of predominantly nNOS (nNOS: 93 ± 4.1%; iNOS: 24.6 ± 7.5%) with ANT was detected in isolated mitochondria of wild-type mice. In contrast, iNOS expression was increased in Cx43Cre-ER(T)/fl mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43Cre-ER(T)/fl mitochondria (0.14 ± 0.02 nmol/min./mg protein) in comparison to wild-type mitochondria (0.24 ± 0.02 nmol/min./mg). These are the first data demonstrating, that a reduced mitochondrial Cx43 content is associated with a switch of the mitochondrial NOS isoform and the respective mitochondrial rate of nitric oxide formation. PMID:25678382

  7. A 1,6-ring closure mechanism for (+)-δ-cadinene synthase?

    Science.gov (United States)

    Faraldos, Juan A; Miller, David J; González, Verónica; Yoosuf-Aly, Zulfa; Cascón, Oscar; Li, Amang; Allemann, Rudolf K

    2012-04-04

    Recombinant (+)-δ-cadinene synthase (DCS) from Gossypium arboreum catalyzes the metal-dependent cyclization of (E,E)-farnesyl diphosphate (FDP) to the cadinane sesquiterpene δ-cadinene, the parent hydrocarbon of cotton phytoalexins such as gossypol. In contrast to some other sesquiterpene cyclases, DCS carries out this transformation with >98% fidelity but, as a consequence, leaves no mechanistic traces of its mode of action. The formation of (+)-δ-cadinene has been shown to occur via the enzyme-bound intermediate (3R)-nerolidyl diphosphate (NDP), which in turn has been postulated to be converted to cis-germacradienyl cation after a 1,10-cyclization. A subsequent 1,3-hydride shift would then relocate the carbocation within the transient macrocycle to expedite a second cyclization that yields the cadinenyl cation with the correct cis stereochemistry found in (+)-δ-cadinene. An elegant 1,10-mechanistic pathway that avoids the formation of (3R)-NDP has also been suggested. In this alternative scenario, the final cadinenyl cation is proposed to be formed through the intermediacy of trans, trans-germacradienyl cation and germacrene D. In addition, an alternative 1,6-ring closure mechanism via the bisabolyl cation has previously been envisioned. We report here a detailed investigation of the catalytic mechanism of DCS using a variety of mechanistic probes including, among others, deuterated and fluorinated FDPs. Farnesyl diphosphate analogues with fluorine at C2 and C10 acted as inhibitors of DCS, but intriguingly, after prolonged overnight incubations, they yielded 2F-germacrene(s) and a 10F-humulene, respectively. The observed 1,10-, and to a lesser extent, 1,11-cyclization activity of DCS with these fluorinated substrates is consistent with the postulated macrocyclization mechanism(s) en route to (+)-δ-cadinene. On the other hand, mechanistic results from incubations of DCS with 6F-FPP, (2Z,6E)-FDP, neryl diphosphate, 6,7-dihydro-FDP, and NDP seem to be in better

  8. Synthetic Bioluminescent Coelenterazine Derivatives.

    Science.gov (United States)

    Nishihara, Ryo; Citterio, Daniel; Suzuki, Koji

    2016-01-01

    The development of coelenterazine (CTZ) derivatives resulting in superior optical characteristics is an efficient method to extend the range of its possible applications. Here, we describe the synthesis of three C-6 substituted CTZ derivatives retaining the recognition by Renilla luciferase (RLuc) and its derivatives. The novel derivatives are useful as bright blue-shifted CTZ derivatives, which can be used as an alternative to hitherto reported compound DeepBlueC™.

  9. The Cer-cqu gene cluster determines three key players in a β-diketone synthase polyketide pathway synthesizing aliphatics in epicuticular waxes

    DEFF Research Database (Denmark)

    Schneider, Lizette Marais; Adamski, Nikolai M.; Christensen, Caspar Elo

    2016-01-01

    five candidates, of which three were missing in apparent cer-cqu triple mutants. Sequencing more than 50 independent mutants for each gene confirmed their identification. Cer-c is a chalcone synthase-like polyketide synthase, designated diketone synthase (DKS), Cer-q is a lipase/carboxyl transferase...

  10. Mechanistic insight with HBCH2CoA as a probe to polyhydroxybutyrate (PHB) synthases.

    Science.gov (United States)

    Zhang, Wei; Shrestha, Ruben; Buckley, Rachael M; Jewell, Jamie; Bossmann, Stefan H; Stubbe, JoAnne; Li, Ping

    2014-08-15

    Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1-2 MDa. A substrate analogue HBCH2CoA, in which the S in HBCoA is replaced with a CH2 group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with Kis of 40 and 14 μM, respectively. To probe the elongation steps of the polymerization, HBCH2CoA was incubated with a synthase acylated with a [(3)H]-saturated trimer-CoA ([(3)H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [(3)H]-sTCoA ([(3)H]-sT-CH2-CoA), saturated dimer-([(3)H]-sD-CO2H), and trimer-acid ([(3)H]-sT-CO2H), distinct from the expected methylene analogue of [(3)H]-saturated tetramer-CoA ([(3)H]-sTet-CH2-CoA). Detection of [(3)H]-sT-CH2-CoA and its slow rate of formation suggest that HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation.

  11. Immunofluorescent localization of thymidylate synthase in the development of Trichinella spiralis and Caenorhabditis elegans.

    Science.gov (United States)

    Gołos, Barbara; Dąbrowska, Magdalena; Wałajtys-Rode, Elżbieta; Zieliński, Zbigniew; Wińska, Patrycja; Cieśla, Joanna; Jagielska, Elżbieta; Moczoń, Tadeusz; Rode, Wojciech

    2012-05-01

    Localization of thymidylate synthase protein in Trichinella spiralis and Caenorhabditis elegans development was followed with the use of confocal microscopy, revealing similar expression patterns in both nematode species. In T. spiralis premature muscle larvae and C. elegans dauer, L3 and L4 larvae, thymidylate synthase was detected in the nerve ring and gonad primordia, as well as T. spiralis stichosome and C. elegans pharyngeal glandular cells. In developmentally arrested T. spiralis muscle larvae, the enzyme was found localized to the gonad primordia and stichosome. High enzyme level was also observed in the embryos developing in uteri of T. spiralis female adult and C. elegans hermaphrodite forms. In the case of T. spiralis adult forms, thymidylate synthase was detected in stichosome, along esophagus wall, as well as in egg and sperm cells. While the enzyme protein present in the embryos remains in accord with its known association with proliferating systems, thymidylate synthase presence in the nerve ring, and reproductive and secretory (T. spiralis stichosomal and C. elegans pharyngeal glandular cells) systems, points to a state of cell cycle-arrest, also known to preserve the enzyme protein. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. THYMIDYLATE SYNTHASE LEVELS IN TUMOR-BIOPSIES FROM PATIENTS WITH COLORECTAL-CANCER

    NARCIS (Netherlands)

    MULDER, NH; TIMMERBOSSCHA, H; MEERSMA, GJ; VERSCHUEREN, RCJ

    1994-01-01

    Catalytic activity and FdUMP binding characteristics of thymidylate synthase (TS) were determined in 22 rumor biopsies of patients to be treated (15) or just treated (7) for colorectal cancer with 5-fluorouracil and leucovorin. In 19 samples both parameters could be determined and were found to

  13. In situ localization of chalcone synthase mRNA in pea root nodule development.

    NARCIS (Netherlands)

    Yang, W.C.; Canter Cremers, H.C.J.; Hogendijk, P.; Katinakis, P.; Wijffelman, C.A.; Franssen, H.J.; Kammen, van A.; Bisseling, T.

    1992-01-01

    In this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it

  14. Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria ananassa)

    NARCIS (Netherlands)

    Lunkenbein, S.; Coiner, H.; Vos, de C.H.; Schaart, J.G.; Boone, M.J.; Krens, F.A.; Schwab, W.; Salentijn, E.M.J.

    2006-01-01

    An octaploid (Fragaria × ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria × ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens.

  15. The absence of functional glucosylceramide synthase does not sensitize melanoma cells for anticancer drugs

    NARCIS (Netherlands)

    Veldman, RJ; Mita, A; Cuvillier, O; Garcia, [No Value; Klappe, K; Medin, JA; Campbell, JD; Carpentier, S; Kok, JW; Levade, T

    Conversion of ceramide, a putative mediator of anticancer drug-induced apoptosis, into glucosylceramide, by the action of glucosylceramide synthase (GCS), has been implicated in drug resistance. Herein, we compared GM95 mouse melanoma cells deficient in GCS activity, with cells stably transfected

  16. Cloning and sequence analysis of putative type II fatty acid synthase ...

    Indian Academy of Sciences (India)

    Prakash

    Peanut (A. hypogaea cultivar luhua-14) was grown in the farm and gynophores were labelled. The immature ..... Planta 191 102–111. Lai C Y and Cronan J E 2003 β-ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis;. J. Biol. Chem. 278 51494–51503. Lamppa G and Jacks C 1991 ...

  17. Distribution of genotypes and alleles of the aldosterone-synthase gene in patients with abdominal obesity

    Directory of Open Access Journals (Sweden)

    D. L. Brovin

    2015-01-01

    Full Text Available We observed 140 patients with abdominal obesity (AO (IDF, 2005, the residents of St. Petersburg (44.6 ± 0.6 years. Metabolic syndrome (MS (IDF, 2005 was diagnosed in 49.2% of patients with AO. The most frequent component of MS in patients with AO was arterial hypertension (AH. The distribution of genotypes and -alleles of the aldosterone-synthase gene in patients with AO and in the comparison group (56 subjects without AO, 41.0 ± 1.1 years didn't differ (p> 0.05. Levels of both systolic and diastolic blood pressure (BP were higher in carriers of -344T allele of aldosterone-synthase gene. Plasma renin activity, plasma aldosterone and glucose levels, anthropometric parameters, serum blood lipids and carbohydrate metabolism indices in obese patients with different genotypes of aldosterone-synthase gene didn't differ. -344T allele of aldosterone-synthase gene in patients with AO is associated with the increased risk of AH.

  18. Cloning and characterization of ATP synthase CF1 α gene from ...

    African Journals Online (AJOL)

    ATP synthase CF1 α subunit protein is a key enzyme for energy metabolism in plant kingdom, and plays an important role in multiple cell processes. In this study, the complete atpA gene (accession no. JN247444) was cloned from sweet potato (Ipomoea batatas L. Lam) by reverse transcriptasepolymerase chain reaction ...

  19. DNA methylation status is not impaired in treated cystathionine beta-synthase (CBS) deficient patients.

    NARCIS (Netherlands)

    Heil, S.G.; Riksen, N.P.; Boers, G.H.J.; Smulders, Y.; Blom, H.J.

    2007-01-01

    BACKGROUND: Cystathionine beta-synthase (CBS) deficiency is an inborn error of metabolism that is biochemically characterized by severe hyperhomocysteinemia and homocystinuria. In tissues of mice deficient for CBS it has been demonstrated that global DNA methylation and DNA methylation of the H19

  20. Organellar and cytosolic localization of four phosphoribosyl diphosphate synthase isozymes in spinach

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1999-01-01

    Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Δprs mutation. The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate. Two of the enzymes (isozymes 1 and 2...