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Sample records for synthase subunit dpm3

  1. Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene.

    NARCIS (Netherlands)

    Nijtmans, L.G.J.; Henderson, N.S.; Attardi, G.; Holt, L.J.

    2001-01-01

    Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome.

  2. Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate.

    Science.gov (United States)

    Burke, Charles; Croteau, Rodney

    2002-02-01

    Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C(5) units, of the respective C(10), C(15), and C(20) precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56-75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C(10) chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.

  3. Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase.

    Science.gov (United States)

    He, Jiuya; Ford, Holly C; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-03-28

    The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1 , ATP5G2 , and ATP5G3 , encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1 , ATP5G2 , and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F 1 -catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ 0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.

  4. Effect of chronic ethanol consumption on the subunit structure of the mitochondrial ATP synthase

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, W.B.; Spach, P.I.; Cunningham, C.C. (Wake Forest Univ., Winston-Salem, NC (United States))

    1991-03-11

    The relative concentrations of several subunits of the mitochondrial F{sub 0}F{sub 1} ATP synthase were determined in hepatic mitochondria and submitochrondrial particles (SMP) isolated from ethanol-fed and control rats. Animals were maintained on an ethanol-containing liquid diet for 31 days. The polypeptides were analyzed by densitometry measurements of SDS-polyacrylamide gel electrophoresis patterns. Subunit 8 was decreased 31% in intact mitochondria, whereas subunit 6 could not be measured. In contrast, there were no significant ethanol-related depressions in subunits {alpha}, {beta} and OSCP of the F{sub 0}F{sub 1} or the adenine nucleotide carrier (AdNC) in intact mitochondria. In ethanol SMP subunits 6 and 8 were decreased 41 and 34%, respectively; subunits {alpha}, {beta}, {gamma} and OSCP were also present in lowered amounts due to the loose attachment of F{sub 1} to F{sub 0}. The remainder of the F{sub 1} was observed in the soluble fraction resulting from preparation of SMP. The AdNC was present in normal amounts in ethanol SMP. These results demonstrate that ethanol consumption causes a decrease in the content of mitochondrial synthesized subunits 6 and 8 whereas no effect is exerted on the concentrations of nuclear gene products of the ATP synthase complex. Likewise, the adenine nucleotide transporter, also a nuclear gene product, is unaffected by ethanol consumption.

  5. Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase.

    Science.gov (United States)

    He, Jiuya; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-08-22

    The opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase and the oligomycin sensitivity conferral protein (OSCP), a component of the enzyme's peripheral stalk, provides the site at which cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme's catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the corresponding genes, ATP5F1 and ATP5O , had been disrupted. Both cell lines preserve the characteristic properties of the PTP; therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8, and subunit c have been eliminated previously from possible participation in the PTP; thus, the only subunits of ATP synthase that could participate in pore formation are e, f, g, diabetes-associated protein in insulin-sensitive tissues (DAPIT), and the 6.8-kDa proteolipid.

  6. Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

    International Nuclear Information System (INIS)

    Nakanishi-Matsui, Mayumi; Kashiwagi, Sachiko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu

    2010-01-01

    The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F 1 with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F 1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.

  7. Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases.

    Science.gov (United States)

    Xu, Yuquan; Zhou, Tong; Zhang, Shuwei; Espinosa-Artiles, Patricia; Wang, Luoyi; Zhang, Wei; Lin, Min; Gunatilaka, A A Leslie; Zhan, Jixun; Molnár, István

    2014-08-26

    Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into "build-couple-pair" combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.

  8. Subunit b-Dimer of the Escherichia coli ATP Synthase Can Form Left-Handed Coiled-Coils

    Science.gov (United States)

    Wise, John G.; Vogel, Pia D.

    2008-01-01

    One remaining challenge to our understanding of the ATP synthase concerns the dimeric coiled-coil stator subunit b of bacterial synthases. The subunit b-dimer has been implicated in important protein interactions that appear necessary for energy conservation and that may be instrumental in energy conservation during rotary catalysis by the synthase. Understanding the stator structure and its interactions with the rest of the enzyme is crucial to the understanding of the overall catalytic mechanism. Controversy exists on whether subunit b adopts a classic left-handed or a presumed right-handed dimeric coiled-coil and whether or not staggered pairing between nonhomologous residues in the homodimer is required for intersubunit packing. In this study we generated molecular models of the Escherichia coli subunit b-dimer that were based on the well-established heptad-repeat packing exhibited by left-handed, dimeric coiled-coils by employing simulated annealing protocols with structural restraints collected from known structures. In addition, we attempted to create hypothetical right-handed coiled-coil models and left- and right-handed models with staggered packing in the coiled-coil domains. Our analyses suggest that the available structural and biochemical evidence for subunit b can be accommodated by classic left-handed, dimeric coiled-coil quaternary structures. PMID:18326648

  9. Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit

    International Nuclear Information System (INIS)

    Roy, Ankoor; Hutcheon, Marcus L.; Duncan, Thomas M.; Cingolani, Gino

    2012-01-01

    A phosphomimetic mutation in subunit ∊ dramatically increases reproducibility for crystallization of Escherichia coli ATP synthase catalytic complex (F 1 ) (subunit composition α 3 β 3 γ∊). Diffraction data were collected to ∼3.15 Å resolution using synchrotron radiation. The bacterial ATP synthase (F O F 1 ) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli F O F 1 represents nature’s smallest rotary motor, composed of a membrane-embedded proton transporter (F O ) and a peripheral catalytic complex (F 1 ). The ATPase activity of isolated F 1 is fully expressed by the α 3 β 3 γ ‘core’, whereas single δ and ∊ subunits are required for structural and functional coupling of E. coli F 1 to F O . In contrast to mitochondrial F 1 -ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F 1 -ATPase catalytic core. Destabilizing the compact conformation of ∊’s C-terminal domain with a phosphomimetic mutation (∊S65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F 1 that diffract to ∼3.15 Å resolution

  10. Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET

    Science.gov (United States)

    Seyfert, K.; Oosaka, T.; Yaginuma, H.; Ernst, S.; Noji, H.; Iino, R.; Börsch, M.

    2011-03-01

    FoF1-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference ΔpH plus an electric potential ΔΨ across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within FoF1-ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum ΔpH ~ 4 and an unknown ΔΨ. In contrast, in living bacteria the maximum ΔpH across the plasma membrane is likely 0.75, and ΔΨ has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single FoF1-ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.

  11. Knockdown of F1 epsilon subunit decreases mitochondrial content of ATP synthase and leads to accumulation of subunit c

    Czech Academy of Sciences Publication Activity Database

    Havlíčková, Vendula; Kaplanová, Vilma; Nůsková, Hana; Drahota, Zdeněk; Houštěk, Josef

    2010-01-01

    Roč. 1797, 6-7 (2010), s. 1124-1129 ISSN 0005-2728. [European Bioenergetics Conference /16./. Warsaw, 17.07.2010-22.07.2010] R&D Projects: GA MŠk(CZ) 1M0520; GA MZd(CZ) NS9759 Grant - others:Univerzita Karlova(CZ) 97807 Institutional research plan: CEZ:AV0Z50110509 Keywords : ATP synthase * biogenesis * mitochondria Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.132, year: 2010

  12. Escherichia coli F1Fo-ATP synthase with a b/δ fusion protein allows analysis of the function of the individual b subunits.

    Science.gov (United States)

    Gajadeera, Chathurada S; Weber, Joachim

    2013-09-13

    The "stator stalk" of F1Fo-ATP synthase is essential for rotational catalysis as it connects the nonrotating portions of the enzyme. In Escherichia coli, the stator stalk consists of two (identical) b subunits and the δ subunit. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b' type. In the present study, it is shown that it is possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. This construct allowed the analysis of the roles of the individual b subunits. The full-length b subunit (which in this case is covalently linked to δ in the fusion protein) is responsible for connecting the stalk to the catalytic F1 subcomplex. It is not required for interaction with the membrane-embedded Fo subcomplex, as its transmembrane helix can be removed. Attachment to Fo is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b' type.

  13. Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

    KAUST Repository

    Beke-Somfai, T.

    2013-01-23

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

  14. 3D-localization of the a-subunit in F 0F I-ATP synthase by time resolved single-molecule FRET

    Science.gov (United States)

    Düser, Monika G.; Zarrabi, Nawid; Bi, Yumin; Zimmermann, Boris; Dunn, Stanley D.; Börsch, Michael

    2006-02-01

    F °F I-ATP synthases catalyze the ATP formation from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. Internal rotation of subunits couples the chemical reaction at the F I part to the proton translocation through the F ° part. In these enzymes, the membrane-embedded a-subunit is part of the non-rotating 'stator' subunits and provides the proton channel of the F ° motor. At present, the relative position of the a-subunit is not known. We examined the rotary movements of the ɛ-subunit with respect to the non-rotating a-subunit by time resolved singlemolecule fluorescence resonance energy transfer (FRET) using a novel pulsed laser diode. Rotation of the ɛ-subunit during ATP hydrolysis was divided into three major steps. The stopping positions of ɛ resulted in three distinct FRET efficiency levels and FRET donor lifetimes. From these FRET efficiencies the position of the FRET donor at the asubunit was calculated. Different populations of the three resting positions of ɛ, which were observed previously, enabled us to scrutinize the models for the position of the a-subunit in the F ° part.

  15. Regulation of Aerobic Energy Metabolism in Podospora anserina by Two Paralogous Genes Encoding Structurally Different c-Subunits of ATP Synthase.

    Directory of Open Access Journals (Sweden)

    Carole H Sellem

    2016-07-01

    Full Text Available Most of the ATP in living cells is produced by an F-type ATP synthase. This enzyme uses the energy of a transmembrane electrochemical proton gradient to synthesize ATP from ADP and inorganic phosphate. Proton movements across the membrane domain (FO of the ATP synthase drive the rotation of a ring of 8-15 c-subunits, which induces conformational changes in the catalytic part (F1 of the enzyme that ultimately promote ATP synthesis. Two paralogous nuclear genes, called Atp9-5 and Atp9-7, encode structurally different c-subunits in the filamentous fungus Podospora anserina. We have in this study identified differences in the expression pattern for the two genes that correlate with the mitotic activity of cells in vegetative mycelia: Atp9-7 is transcriptionally active in non-proliferating (stationary cells while Atp9-5 is expressed in the cells at the extremity (apex of filaments that divide and are responsible for mycelium growth. When active, the Atp9-5 gene sustains a much higher rate of c-subunit synthesis than Atp9-7. We further show that the ATP9-7 and ATP9-5 proteins have antagonist effects on the longevity of P. anserina. Finally, we provide evidence that the ATP9-5 protein sustains a higher rate of mitochondrial ATP synthesis and yield in ATP molecules per electron transferred to oxygen than the c-subunit encoded by Atp9-7. These findings reveal that the c-subunit genes play a key role in the modulation of ATP synthase production and activity along the life cycle of P. anserina. Such a degree of sophistication for regulating aerobic energy metabolism has not been described before.

  16. Influences of energization and nucleotide binding on the reaction of Lucifer Yellow vinyl sulfone with the alpha subunits of the chloroplast ATP synthase.

    Science.gov (United States)

    Cunningham, K M; McCarty, R E

    2000-04-18

    The catalytic portion of the chloroplast ATP synthase (CF(1)) consists of five different polypeptides in the stoichiometry alpha(3)beta(3)gammadeltaepsilon and is structurally asymmetric. Asymmetry is readily apparent in the properties of the six nucleotide binding sites and the single-copy, smaller subunits. Asymmetry is also detected in the alpha subunits by the rapid and covalent binding of Lucifer Yellow vinyl sulfone (LY) to one of the three chemically identical alpha subunits. The binding of LY to a single alpha subunit has allowed the investigation of whether asymmetry in the alpha subunits is a permanent feature of CF(1). The development of an electrochemical proton gradient across illuminated thylakoid membranes and the preincubation of CF(1) in solution with Mg(2+)-ATP were found to alter the LY distribution such that multiple alpha subunits were labeled with LY. Illumination of thylakoid membranes doubled the extent of LY labeling, and fluorescence resonance energy transfer measurements indicated that LY was bound to more than one alpha subunit. Since the change in LY distribution was inhibited by proton ionophores (uncouplers), alteration of alpha conformation by illumination is a result of the generation of a proton gradient. Preincubation of CF(1) in solution with Mg(2+)-ATP had no effect on the extent of LY labeling but resulted in multiple alpha subunits binding LY as determined by fluorescence resonance energy transfer measurements. Adenine nucleotides at substrate level concentrations inhibit the reaction of LY with the alpha subunits. No increase in LY labeling was observed when thylakoids were illuminated under conditions in which CF(1) was catalytically active.

  17. Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan adenosine triphosphate (ATP) synthases.

    Science.gov (United States)

    Walpole, Thomas B; Palmer, David N; Jiang, Huibing; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2015-04-01

    The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F1-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9-15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria. © 2015 by The American

  18. UncI protein can mediate ring-assembly of c-subunits of FoF1-ATP synthase in vitro

    International Nuclear Information System (INIS)

    Ozaki, Yoko; Suzuki, Toshiharu; Kuruma, Yutetsu; Ueda, Takuya; Yoshida, Masasuke

    2008-01-01

    In F o F 1 -ATP synthase, multimeric c-subunits are assembled to a ring (c-ring) in the membranes that rotates as protons flow across F o . We recently reported that assembly of c-ring of Propionigenium modestum in the membranes of Escherichia coli cells required P. modestum UncI, a product of the conserved uncI gene in the F o F 1 operon. However, cooperation with endogenous factors in E. coli remained unclear. Here, P. modestum c-subunit was synthesized in vitro in the presence of liposomes. When c-subunit alone was synthesized, it did not form c-ring. However, when c-subunit and P. modestum UncI were synthesized together, c-ring was formed. Fusion of the two kinds of liposomes, one containing only unassembled c-subunit and the other only UncI, resulted in gradual formation of c-ring. Thus, UncI alone can mediate in vitro post-translational c-ring assembly

  19. Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Francis, Brian R; White, Karen H; Thorsness, Peter E

    2007-04-01

    ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

  20. Cloning and characterization of homeologous cellulose synthase catalytic subunit 2 genes from allotetraploid cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Kim, Hee Jin; Triplett, Barbara A; Zhang, Hong-Bin; Lee, Mi-Kyung; Hinchliffe, Doug J; Li, Ping; Fang, David D

    2012-02-25

    Cellulose synthase catalytic subunits (CesAs) are the catalytic sites within a multisubunit complex for cellulose biosynthesis in plants. CesAs have been extensively studied in diploid plants, but are not well characterized in polyploid plants. Gossypium hirsutum is an allotetraploid cotton species producing over 90% of the world's cotton fibers. Although G. hirsutum CesAs (GhCesAs) are responsible for cellulose production in cotton fiber, very limited numbers of GhCesA genes have been identified. Here, we report isolating and characterizing a pair of homeologous CesA2 genes and their full-length cDNAs from allotetraploid cotton. The GhCesA2-A(T) gene from the A-subgenome and GhCesA2-D(T) gene from the D-subgenome were screened from a G. hirsutum BAC library. These genes shared 92% sequence similarity throughout the entire sequence. The coding sequences were nearly identical, and the deduced amino acid sequences from GhCesA2-A(T) (1,039 amino acids) and GhCesA2-D(T) (1,040 amino acids) were identical except four amino acids, whereas the noncoding sequences showed divergence. Sequence analyses showed that all exons of GhCesA2-A(T) contained consensus splice donor dinucleotides, but one exon in GhCesA2-D(T) contained nonconsensus splice donor dinucleotides. Although the nonconsensus splice donor dinucleotides were previously suggested to be involved in alternative splice or pseudogenization, our results showed that a majority of GhCesA2-A(T) and GhCesA2-D(T) transcripts consisted of functional and full-length transcripts with little evidence for alternative mRNA isoforms in developing cotton fibers. Expression analyses showed that GhCesA2-A(T) and GhCesA2-D(T) shared common temporal and spatial expression patterns, and they were highly and preferentially expressed during the cellulose biosynthesis stage in developing cotton fibers. The observations of higher expression levels of both GhCesA2-A(T) and GhCesA2-D(T) in developing fibers of one near-isogenic line (NIL

  1. Cloning and sequencing of a cDNA for the delta-subunit of photosynthetic ATP-synthase (EC 3.6.1.34) from pea (Pisum sativum).

    Science.gov (United States)

    Hoesche, J A; Berzborn, R J

    1992-12-29

    lambda gt10 cDNA clones for the nuclear encoded subunit delta of chloroplast ATP-synthase from Pisum sativum have been isolated. The 5' end was completed by PCR. The sequenced cDNA codes for the import precursor. N-Terminal sequencing of the mature protein isolated from chloroplasts revealed that the processing sites of the transit peptide from Pisum sativum and Spinacea oleracea are similar. The overall homology of the deduced amino acid sequences of the mature delta proteins from higher plants is about 40%. The conservation among hydrophilic residues is higher than for hydrophobic ones, indicating that the surface of delta is important for its function within the ATP-synthase.

  2. 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.

  3. The FoF1-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Zíková, Alena; Schnaufer, A.; Dalley, A. R.; Panigrahi, A. K.; Stuart, K. D.

    2009-01-01

    Roč. 5, č. 5 (2009), s. 1-15 E-ISSN 1932-6203 Keywords : mitochondrion * respiratory chain * / ATP synthase * TAP-tag * RNAi Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.351, year: 2009

  4. Mitochondrial ATP synthase deficiency due to a mutation in the ATP5E gene for the F1 e subunit

    Czech Academy of Sciences Publication Activity Database

    Mayr, J. A.; Havlíčková, Vendula; Zimmermann, F.; Magler, I.; Kaplanová, Vilma; Ješina, Pavel; Pecinová, Alena; Nůsková, Hana; Koch, J.; Sperl, W.; Houštěk, Josef

    2010-01-01

    Roč. 19, č. 17 (2010), s. 3430-3439 ISSN 0964-6906 R&D Projects: GA MZd(CZ) NS9759; GA MŠk(CZ) 1M0520 Grant - others:Univerzita Karlova(CZ) 97807 Institutional research plan: CEZ:AV0Z50110509 Keywords : ATP-synthase * ATP5E * disease Subject RIV: EB - Gene tics ; Molecular Biology Impact factor: 8.058, year: 2010

  5. Aspergillus fumigatus Trehalose-Regulatory Subunit Homolog Moonlights To Mediate Cell Wall Homeostasis through Modulation of Chitin Synthase Activity

    Directory of Open Access Journals (Sweden)

    Arsa Thammahong

    2017-04-01

    Full Text Available Trehalose biosynthesis is found in fungi but not humans. Proteins involved in trehalose biosynthesis are essential for fungal pathogen virulence in humans and plants through multiple mechanisms. Loss of canonical trehalose biosynthesis genes in the human pathogen Aspergillus fumigatus significantly alters cell wall structure and integrity, though the mechanistic link between these virulence-associated pathways remains enigmatic. Here we characterize genes, called tslA and tslB, which encode proteins that contain domains similar to those corresponding to trehalose-6-phosphate phosphatase but lack critical catalytic residues for phosphatase activity. Loss of tslA reduces trehalose content in both conidia and mycelia, impairs cell wall integrity, and significantly alters cell wall structure. To gain mechanistic insights into the role that TslA plays in cell wall homeostasis, immunoprecipitation assays coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS were used to reveal a direct interaction between TslA and CsmA, a type V chitin synthase enzyme. TslA regulates not only chitin synthase activity but also CsmA sub-cellular localization. Loss of TslA impacts the immunopathogenesis of murine invasive pulmonary aspergillosis through altering cytokine production and immune cell recruitment. In conclusion, our data provide a novel model whereby proteins in the trehalose pathway play a direct role in fungal cell wall homeostasis and consequently impact fungus-host interactions.

  6. pKa of the essential Glu54 and backbone conformation for subunit c from the H+-coupled F1F0 ATP synthase from an alkaliphilic Bacillus.

    Science.gov (United States)

    Rivera-Torres, Iván O; Krueger-Koplin, Ray D; Hicks, David B; Cahill, Sean M; Krulwich, Terry A; Girvin, Mark E

    2004-09-24

    The conformation of the ATP synthase c-subunit and the pKa of its essential E54 residue were characterized in alkaliphilic Bacillus pseudofirmus OF4. The c-subunit folds as a helix-loop-helix, with inter-helical contacts demonstrated by paramagnetic relaxation effects. The E54 pKa of 7.7 is significantly higher than in non-alkaliphiles, which likely prevents proton loss from the c-rotor at high pH. The E54 pKa was unchanged in a mutant, cP51A, that has a severe ATP synthesis defect at high pH only. cP51 must have some structural role that accounts for the mutant defect, such as different subunit-subunit interactions at high pH.

  7. GFT projection NMR based resonance assignment of membrane proteins: application to subunit C of E. coli F(1)F (0) ATP synthase in LPPG micelles.

    Science.gov (United States)

    Zhang, Qi; Atreya, Hanudatta S; Kamen, Douglas E; Girvin, Mark E; Szyperski, Thomas

    2008-03-01

    G-matrix FT projection NMR spectroscopy was employed for resonance assignment of the 79-residue subunit c of the Escherichia coli F(1)F(0) ATP synthase embedded in micelles formed by lyso palmitoyl phosphatidyl glycerol (LPPG). Five GFT NMR experiments, that is, (3,2)D HNNCO, L-(4,3)D HNNC (alphabeta) C (alpha), L-(4,3)D HNN(CO)C (alphabeta) C (alpha), (4,2)D HACA(CO)NHN and (4,3)D HCCH, were acquired along with simultaneous 3D (15)N, (13)C(aliphatic), (13)C(aromatic)-resolved [(1)H,(1)H]-NOESY with a total measurement time of approximately 43 h. Data analysis resulted in sequence specific assignments for all routinely measured backbone and (13)C(beta) shifts, and for 97% of the side chain shifts. Moreover, the use of two G(2)FT NMR experiments, that is, (5,3)D HN{N,CO}{C (alphabeta) C (alpha)} and (5,3)D {C (alphabeta) C (alpha)}{CON}HN, was explored to break the very high chemical shift degeneracy typically encountered for membrane proteins. It is shown that the 4D and 5D spectral information obtained rapidly from GFT and G(2)FT NMR experiments enables one to efficiently obtain (nearly) complete resonance assignments of membrane proteins.

  8. Structure and Subunit Arrangement of the A-type ATP Synthase Complex from the Archaeon Methanococcus jannaschii Visualized by Electron Microscopy

    NARCIS (Netherlands)

    Coskun, Ünal; Chaban, Yuriy L.; Lingl, Astrid; Müller, Volker; Keegstra, Wilko; Boekema, Egbert J.; Grüber, Gerhard; Gruber, 27460

    2004-01-01

    In Archaea, bacteria, and eukarya, ATP provides metabolic energy for energy-dependent processes. It is synthesized by enzymes known as A-type or F-type ATP synthase, which are the smallest rotatory engines in nature. Here, we report the first projected structure of an intact A1A0 ATP synthase from

  9. Sequence of the gamma-subunit of Spirulina platensis : a new principle of thiol modulation of F0F1 ATP synthase?

    NARCIS (Netherlands)

    Steinemann, D.; Lill, H

    1995-01-01

    The gene encoding the gamma subunit of Spirulina platensis F0F1, the relative of the chloroplast F1 subunit responsible for thiol activation, has been cloned and sequenced. As in other cyanobacteria, a specific couple of cysteines like those involved in thiol modulation of the chloroplast enzyme was

  10. Mutations in a helix-1 motif of the ATP synthase c-subunit of Bacillus pseudofirmus OF4 cause functional deficits and changes in the c-ring stability and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Liu, Jun; Fackelmayer, Oliver J; Hicks, David B; Preiss, Laura; Meier, Thomas; Sobie, Eric A; Krulwich, Terry A

    2011-06-21

    The ATP synthase of the alkaliphile Bacillus pseudofirmus OF4 has a tridecameric c-subunit rotor ring. Each c-subunit has an AxAxAxA motif near the center of the inner helix, where neutralophilic bacteria generally have a GxGxGxG motif. Here, we studied the impact of four single and six multiple Ala-to-Gly chromosomal mutations in the A16xAxAxA22 motif on the capacity for nonfermentative growth and, for most of the mutants, on ATP synthesis by ADP- and P(i)-loaded membrane vesicles at pH 7.5 and 10.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of the holo-ATP synthases were used to probe stability of the mutant c-rotors and mobility properties of the c-rotors as well as the monomeric c-subunits that are released from them by trichloroacetic acid treatment. Mutants containing an Ala16-to-Gly mutation exhibited the most severe functional defects. Via SDS-PAGE, most of the mutant c-monomers exhibited increased mobility relative to the wild-type (WT) c-subunit, but among the intact c-rings, only Ala16-to-Gly mutants exhibited significantly increased mobility relative to that of the WT c-ring. The hypothesis that these c-rings have a decreased c-subunit stoichiometry is still untested, but the functional impact of an Ala16-to-Gly mutation clearly depended upon additional Ala-to-Gly mutation(s) and their positions. The A16/20G double mutant exhibited a larger functional deficit than both the A16G and A16/18G mutants. Most of the mutant c-rings showed in vitro instability relative to that of the WT c-ring. However, the functional deficits of mutants did not correlate well with the extent of c-ring stability loss, so this property is unlikely to be a major factor in vivo.

  11. Alteration of Polyketide Stereochemistry from anti to syn by a Ketoreductase Domain Exchange in a Type I Modular Polyketide Synthase Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Eng, Clara H.; Yuzawa, Satoshi; Wang, George; Baidoo, Edward E. K.; Katz, Leonard; Keasling, Jay D.

    2016-03-29

    Polyketide natural products have broad applications in medicine. Exploiting the modular nature of polyketide synthases to alter stereospecificity is an attractive strategy for obtaining natural product analogues with altered pharmaceutical properties. The purpose of research is to demonstrate that by retaining a dimerization element present in LipPks1+TE, we are able to use a ketoreductase domain exchange to alter α-methyl group stereochemistry with unprecedented retention of activity and simultaneously achieve a novel alteration of polyketide product stereochemistry from anti to syn. The substrate promiscuity of LipPks1+TE further provided a unique opportunity to investigate the substrate dependence of ketoreductase activity in a polyketide synthase module context.

  12. Disease: H00118 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available synthase subunit DPM3 bridges the congenital disorders of glycosylation with the dystroglycanopathies. ... JO..., Hofsteenge J, Maeda Y, van den Heuvel L, Lammens M, Lehle L, Wevers RA ... TITLE ... Deficiency of Dol-P-Man

  13. A multilevel prediction of physiological response to challenge: Interactions among child maltreatment, neighborhood crime, endothelial nitric oxide synthase gene (eNOS), and GABA(A) receptor subunit alpha-6 gene (GABRA6).

    Science.gov (United States)

    Lynch, Michael; Manly, Jody Todd; Cicchetti, Dante

    2015-11-01

    Physiological response to stress has been linked to a variety of healthy and pathological conditions. The current study conducted a multilevel examination of interactions among environmental toxins (i.e., neighborhood crime and child maltreatment) and specific genetic polymorphisms of the endothelial nitric oxide synthase gene (eNOS) and GABA(A) receptor subunit alpha-6 gene (GABRA6). One hundred eighty-six children were recruited at age 4. The presence or absence of child maltreatment as well as the amount of crime that occurred in their neighborhood during the previous year were determined at that time. At age 9, the children were brought to the lab, where their physiological response to a cognitive challenge (i.e., change in the amplitude of the respiratory sinus arrhythmia) was assessed and DNA samples were collected for subsequent genotyping. The results confirmed that complex Gene × Gene, Environment × Environment, and Gene × Environment interactions were associated with different patterns of respiratory sinus arrhythmia reactivity. The implications for future research and evidence-based intervention are discussed.

  14. GFT projection NMR based resonance assignment of membrane proteins: application to subunit c of E. coli F{sub 1}F{sub 0} ATP synthase in LPPG micelles

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Qi [State University of New York at Buffalo, Department of Chemistry (United States); Atreya, Hanudatta S. [NMR Research Centre, Indian Institute of Science (India); Kamen, Douglas E.; Girvin, Mark E. [Albert Einstein College of Medicine, Biochemistry Department (United States); Szyperski, Thomas [State University of New York at Buffalo, Department of Chemistry (United States)], E-mail: szypersk@chem.buffalo.edu

    2008-03-15

    G-matrix FT projection NMR spectroscopy was employed for resonance assignment of the 79-residue subunit c of the Escherichia coli F{sub 1}F{sub 0} ATP synthase embedded in micelles formed by lyso palmitoyl phosphatidyl glycerol (LPPG). Five GFT NMR experiments, that is, (3,2)D HNNCO, L-(4,3)D HNNC{sup {alpha}}{sup {beta}}C{sup {alpha}}, L-(4,3)D HNN(CO)C{sup {alpha}}{sup {beta}}C{sup {alpha}}, (4,2)D HACA(CO)NHN and (4,3)D HCCH, were acquired along with simultaneous 3D {sup 15}N, {sup 13}C{sup aliphatic}, {sup 13}C{sup aromatic}-resolved [{sup 1}H,{sup 1}H]-NOESY with a total measurement time of {approx}43 h. Data analysis resulted in sequence specific assignments for all routinely measured backbone and {sup 13}C{sup {beta}} shifts, and for 97% of the side chain shifts. Moreover, the use of two G{sup 2}FT NMR experiments, that is, (5,3)D HN{l_brace}N,CO{r_brace}{l_brace}C{sup {alpha}}{sup {beta}}C{sup {alpha}}{r_brace} and (5,3)D {l_brace}C{sup {alpha}}{sup {beta}}C{sup {alpha}}{r_brace}{l_brace}CON{r_brace}HN, was explored to break the very high chemical shift degeneracy typically encountered for membrane proteins. It is shown that the 4D and 5D spectral information obtained rapidly from GFT and G{sup 2}FT NMR experiments enables one to efficiently obtain (nearly) complete resonance assignments of membrane proteins.

  15. 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...

  16. Diminished synthesis of subunit a (ATP6) and altered function of ATP synthase and cytochrome c oxidase due to the mtDNA 2 bp microdeletion of TA at positions 9205 and 9206

    Czech Academy of Sciences Publication Activity Database

    Ješina, Pavel; Tesařová, M.; Fornůsková, D.; Vojtíšková, Alena; Pecina, Petr; Kaplanová, Vilma; Hansíková, H.; Zeman, J.; Houštěk, Josef

    2004-01-01

    Roč. 383, č. 3 (2004), s. 561-571 ISSN 0264-6021 R&D Projects: GA ČR(CZ) GA303/03/0749; GA MZd(CZ) NR7790; GA MZd(CZ) NR8065 Grant - others:GA UK(CZ) 14/2004 Institutional research plan: CEZ:AV0Z5011922 Keywords : ATP6 * ATP synthase * mitochondrial disease Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.278, year: 2004

  17. 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.

  18. 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, ...

  19. Alteration of structure and function of ATP synthase and cytochrome c oxidase by lack of F-o-a and Cox3 subunits caused by mitochondrial DNA 9205delTA mutation

    Czech Academy of Sciences Publication Activity Database

    Hejzlarová, Kateřina; Kaplanová, Vilma; Nůsková, Hana; Kovářová, Nikola; Ješina, Pavel; Drahota, Zdeněk; Mráček, Tomáš; Seneca, S.; Houštěk, Josef

    2015-01-01

    Roč. 466, č. 3 (2015), s. 601-611 ISSN 0264-6021 R&D Projects: GA ČR(CZ) GAP303/11/0970; GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LL1204; GA ČR(CZ) GAP303/12/1363 Institutional support: RVO:67985823 Keywords : ATP synthase * cytochrome c oxidase * mitochondrial diseases * mtDNA MT-ATP6 mutation * oxidative phosphorylation * threshold effect Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.562, year: 2015

  20. Toxocara canis and Toxocara vitulorum: molecular characterization, discrimination, and phylogenetic analysis based on mitochondrial (ATP synthase subunit 6 and 12S) and nuclear ribosomal (ITS-2 and 28S) genes.

    Science.gov (United States)

    Wickramasinghe, Susiji; Yatawara, Lalani; Rajapakse, R P V J; Agatsuma, Takeshi

    2009-06-01

    Toxocara canis and Toxocara vitulorum are two important parasites of dogs and buffaloes with public health concern. The objectives of the present study are to identify molecular markers to discriminate these closely related parasites and to determine their phylogenetic position and genetic diversity within the genus Toxocara. Thus, two mitochondrial genes (complete ATPase 6 and partial small subunit ribosomal RNA (12S rDNA)), two nuclear ribosomal genes (second internal transcribed spacer region (ITS-2)), and part of the large subunit 28S region were analyzed. Nucleotide sequence (597 bp) and predicted amino acid sequences of the complete ATPase 6 gene (199 amino acids) of both species (T. canis and T. vitulorum) are similar in size with the Toxocara cati and Toxocara malaysiensis. There was 88% nucleotide similarity between T. canis and T. vitulorum and many transversions present in the 12S gene. Analyses of the ITS-2 and 28S regions revealed that the 28S region was more conserved (95% nucleotide similarity between T. canis and T. vitulorum) than the ITS-2 region (85%). This study has provided useful molecular markers for the molecular epidemiological investigation of Toxocara species. Further, phylogenetic analyses of the ITS-2 and 28S genes have indicated that the members of the genus Toxocara form a distinct group with reference to their definitive hosts.

  1. Cryo-EM structure of the yeast ATP synthase.

    Science.gov (United States)

    Lau, Wilson C Y; Baker, Lindsay A; Rubinstein, John L

    2008-10-24

    We have used electron cryomicroscopy of single particles to determine the structure of the ATP synthase from Saccharomyces cerevisiae. The resulting map at 24 A resolution can accommodate atomic models of the F(1)-c(10) subcomplex, the peripheral stalk subcomplex, and the N-terminal domain of the oligomycin sensitivity conferral protein. The map is similar to an earlier electron cryomicroscopy structure of bovine mitochondrial ATP synthase but with important differences. It resolves the internal structure of the membrane region of the complex, especially the membrane embedded subunits b, c, and a. Comparison of the yeast ATP synthase map, which lacks density from the dimer-specific subunits e and g, with a map of the bovine enzyme that included e and g indicates where these subunits are located in the intact complex. This new map has allowed construction of a model of subunit arrangement in the F(O) motor of ATP synthase that dictates how dimerization of the complex via subunits e and g might occur.

  2. Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila.

    Directory of Open Access Journals (Sweden)

    Praveen Balabaskaran Nina

    2010-07-01

    Full Text Available 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 highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a

  3. KORRIGAN1 Interacts Specifically with Integral Components of the Cellulose Synthase Machinery

    NARCIS (Netherlands)

    Mansoori Zangir, N.; Timmers, J.F.P.; Desprez, T.; Lessa Alvim Kamei, C.; Dees, D.C.T.; Vincken, J.P.; Visser, R.G.F.; Höfte, H.; Vernhettes, S.; Trindade, L.M.

    2014-01-01

    Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases.

  4. Atypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis

    NARCIS (Netherlands)

    Yadav, K.N. Satish; Miranda-Astudillo, Héctor V; Colina-Tenorio, Lilia; Bouillenne, Fabrice; Degand, Hervé; Morsomme, Pierre; González-Halphen, Diego; Boekema, Egbert J; Cardol, Pierre

    Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex

  5. The modulator protein dissociates the catalytic subunit of hepatic protein phosphatase G from glycogen.

    OpenAIRE

    Bollen, M; Stalmans, W

    1988-01-01

    1. The phosphorylase phosphatase and glycogen-synthase phosphatase activities associated with the glycogen particles from rat liver were progressively inhibited by incubation with modulator protein. However, the phosphorylase phosphatase activity of the catalytic subunit was entirely recovered after destruction of the modulator and the regulatory subunit(s) by trypsin. 2. Inhibition of protein phosphatase G by modulator was associated with a translocation of the phosphorylase phosphatase acti...

  6. Sucrose Phosphate Synthase and Sucrose Accumulation at Low Temperature 1

    Science.gov (United States)

    Guy, Charles L.; Huber, Joan L. A.; Huber, Steven C.

    1992-01-01

    The influence of growth temperature on the free sugar and sucrose phosphate synthase content and activity of spinach (Spinacia oleracea) leaf tissue was studied. When plants were grown at 25°C for 3 weeks and then transferred to a constant 5°C, sucrose, glucose, and fructose accumulated to high levels during a 14-d period. Predawn sugar levels increased from 14- to 20-fold over the levels present at the outset of the low-temperature treatment. Sucrose was the most abundant free sugar before, during, and after exposure to 5°C. Leaf sucrose phosphate synthase activity was significantly increased by the low-temperature treatment, whereas sucrose synthase and invertases were not. Synthesis of the sucrose phosphate synthase subunit was increased during and after low-temperature exposure and paralleled an increase in the steady-state level of the subunit. The increases in sucrose and its primary biosynthetic enzyme, sucrose phosphate synthase, are discussed in relation to adjustment of metabolism to low nonfreezing temperature and freezing stress tolerance. Images Figure 1 Figure 2 Figure 3 PMID:16652990

  7. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  8. Structure of the ATP Synthase Catalytic Complex (F1) from Escherichia coli in an Autoinhibited conformation

    Energy Technology Data Exchange (ETDEWEB)

    G Cingolani; T Duncan

    2011-12-31

    ATP synthase is a membrane-bound rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, autoinhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F{sub 1}) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit {var_epsilon} adopts a heretofore unknown, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F{sub 1} structures.

  9. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    Science.gov (United States)

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

    Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

  10. Optimization of expression and properties of the recombinant acetohydroxyacid synthase of Thermotoga maritima

    Directory of Open Access Journals (Sweden)

    Mohammad S. Eram

    2015-12-01

    Full Text Available The data provide additional support of the characterization of the biophysical and biochemical properties of the enzyme acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima (Eram et al., 2015 [1]. The genes encoding the enzyme subunits have been cloned and expressed in the mesophilic host Escherichia coli. Detailed data include information about the optimization of the expression conditions, biophysical properties of the enzyme and reconstitution of the holoenzyme from individually expressed and purified subunits.

  11. 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.

  12. 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.

  13. Polyketide synthase from Fusarium

    DEFF Research Database (Denmark)

    Kvesel, Kasper; Wimmer, Reinhard; Sørensen, Jens Laurids

    Fungi produce a wide array of secondary metabolites, with interesting bioactivities by help of a number of enzyme complexes. Polyketide synthases (PKS) are a class of multidomain enzymes, producing a class of secondary metabolites called polyketides1,2. Only few structures of PKS’s have been...

  14. Atypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis.

    Science.gov (United States)

    Yadav, K N Sathish; Miranda-Astudillo, Héctor V; Colina-Tenorio, Lilia; Bouillenne, Fabrice; Degand, Hervé; Morsomme, Pierre; González-Halphen, Diego; Boekema, Egbert J; Cardol, Pierre

    2017-04-01

    Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F 1 F O -ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical α, β, γ, δ, ε, and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Cleaning up polyketide synthases.

    Science.gov (United States)

    Kwan, Jason C; Schmidt, Eric W

    2012-03-23

    Complex biosynthetic enzymes such as polyketide synthases make mistakes. In this issue of Chemistry & Biology, Jensen et al. report that a discrete family of acyltransferases is responsible for error correction, hydrolyzing key biosynthetic intermediates from a multi-enzyme complex. This activity might find use in understanding polyketide biosynthesis, particularly in uncultivated organisms and in tailoring the synthesis of small molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. 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.

  17. 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 ...

  18. Uncovering the structures of modular polyketide synthases.

    Science.gov (United States)

    Weissman, Kira J

    2015-03-01

    The modular polyketide synthases (PKSs) are multienzyme proteins responsible for the assembly of diverse secondary metabolites of high economic and therapeutic importance. These molecular 'assembly lines' consist of repeated functional units called 'modules' organized into gigantic polypeptides. For several decades, concerted efforts have been made to understand in detail the structure and function of PKSs in order to facilitate genetic engineering of the systems towards the production of polyketide analogues for evaluation as drug leads. Despite this intense activity, it has not yet been possible to solve the crystal structure of a single module, let alone a multimodular subunit. Nonetheless, on the basis of analysis of the structures of modular fragments and the study of the related multienzyme of animal fatty acid synthase (FAS), several models of modular PKS architecture have been proposed. This year, however, the situation has changed - three modular structures have been characterized, not by X-ray crystallography, but by the complementary methods of single-particle cryo-electron microscopy and small-angle X-ray scattering. This review aims to compare the cryo-EM structures and SAXS-derived structural models, and to interpret them in the context of previously obtained data and existing architectural proposals. The consequences for genetic engineering of the systems will also be discussed, as well as unresolved questions and future directions.

  19. [Advances in isoprene synthase research].

    Science.gov (United States)

    Gou, Yan; Liu, Zhongchuan; Wang, Ganggang

    2017-11-25

    Isoprene emission can lead to significant consequence for atmospheric chemistry. In addition, isoprene is a chemical compound for various industrial applications. In the organisms, isoprene is produced by isoprene synthase that eliminates the pyrophosphate from the dimethylallyl diphosphate. As a key enzyme of isoprene formation, isoprene synthase plays an important role in the process of natural emission and artificial synthesis of isoprene. So far, isoprene synthase has been found in various plants. Isoprene synthases from different sources are of conservative structural and similar biochemical properties. In this review, the biochemical and structural characteristics of isoprene synthases from different sources were compared, the catalytic mechanism of isoprene synthase was discussed, and the perspective application of the enzyme in bioengineering was proposed.

  20. Stochastic thermodynamics of a chemical nanomachine: The channeling enzyme tryptophan synthase.

    Science.gov (United States)

    Loutchko, Dimitri; Eisbach, Maximilian; Mikhailov, Alexander S

    2017-01-14

    The enzyme tryptophan synthase is characterized by a complex pattern of allosteric interactions that regulate the catalytic activity of its two subunits and opening or closing of their ligand gates. As a single macromolecule, it implements 13 different reaction steps, with an intermediate product directly channeled from one subunit to another. Based on experimental data, a stochastic model for the operation of tryptophan synthase has been earlier constructed [D. Loutchko, D. Gonze, and A. S. Mikhailov, J. Phys. Chem. B 120, 2179 (2016)]. Here, this model is used to consider stochastic thermodynamics of such a chemical nanomachine. The Gibbs energy landscape of the internal molecular states is determined, the production of entropy and its flow within the enzyme are analyzed, and the information exchange between the subunits resulting from allosteric cross-regulations and channeling is discussed.

  1. 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....

  2. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-01-01

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

  3. Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme.

    Science.gov (United States)

    Liebergesell, M; Rahalkar, S; Steinbüchel, A

    2000-08-01

    The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (Mr 40,950) and PhaC (Mr 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed.

  4. The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases.

    Science.gov (United States)

    Beck, J; Ripka, S; Siegner, A; Schiltz, E; Schweizer, E

    1990-09-11

    6-Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit. The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase. After approximately 150-fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa. By immunological screening of a genomic P. patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced. Within this sequence the MSAS gene was identified as a 5322-bp-long open reading frame coding for a protein of 1774 amino acids and 190,731 Da molecular mass. Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5' and 3' portions of the gene. The same cDNA sequences revealed the presence of a 69-bp intron within the N-terminal part of the MSAS gene. The intron contains the canonical GT and AG dinucleotides at its 5'- and 3'-splice junctions. An internal TACTGAC sequence, resembling the TACTAAC consensus element of Saccharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate. When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them. A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases. In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2-oxoacyl reductase, 2-oxoacyl synthase and acyl carrier protein domains. Besides several dissimilar, interspersed regions probably coding for MSAS- and FAS-specific functions, the sequential order of the similar domains was

  5. Crystallization of the c[subscript 14]-rotor of the chloroplast ATP synthase reveals that it contains pigments

    Energy Technology Data Exchange (ETDEWEB)

    Varco-Merth, Benjamin; Fromme, Raimund; Wang, Meitian; Fromme, Petra (AZU)

    2008-08-27

    The ATP synthase is one of the most important enzymes on earth as it couples the transmembrane electrochemical potential of protons to the synthesis of ATP from ADP and inorganic phosphage, providing the main ATP source of almost all higher life on earth. During ATP synthesis, stepwise protonation of a conserved carboxylate on each protein subunit of an oligomeric ring of 10--15 c-subunits is commonly thought to drive rotation of the rotor moiety (c{sub 10-14}{gamma}{sup {epsilon}}) relative to stator moiety ({alpha}{sub 3}{beta}{sub 3}{delta}ab{sub 2}). Here we report the isolation and crystallization of the c{sub 14}-ring of subunit c from the spinach chloroplast enzyme diffracting as far as 2.8 {angstrom}. Though ATP synthase was not previously know to contain any pigments, the crystals of the c-subunit possessed a strong yellow color. The pigment analysis revaled that they contain 1 chlorophyll and 2 carotenoids, thereby showing for the first time that the chloroplast ATP synthase contains cofactors, leading to the question of the possible roles of the functions of the pigments in the chloroplast ATP synthase.

  6. Purification and Biochemical Properties of Phytochromobilin Synthase from Etiolated Oat Seedlings1

    Science.gov (United States)

    McDowell, Michael T.; Lagarias, J. Clark

    2001-01-01

    Plant phytochromes are dependent on the covalent attachment of the linear tetrapyrrole chromophore phytochromobilin (PΦB) for photoactivity. In planta, biliverdin IXα (BV) is reduced by the plastid-localized, ferredoxin (Fd)-dependent enzyme PΦB synthase to yield 3Z-PΦB. Here, we describe the >50,000-fold purification of PΦB synthase from etioplasts from dark-grown oat (Avena sativa L. cv Garry) seedlings using traditional column chromatography and preparative electrophoresis. Thus, PΦB synthase is a very low abundance enzyme with a robust turnover rate. We estimate the turnover rate to be >100 s−1, which is similar to that of mammalian NAD(P)H-dependent BV reductase. Oat PΦB synthase is a monomer with a subunit mass of 29 kD. However, two distinct charged forms of the enzymes were identified by native isoelectric focusing. The ability of PΦB synthase to reduce BV is dependent on reduced 2Fe-2S Fds. A Km for spinach (Spinacea oleracea) Fd was determined to be 3 to 4 μm. PΦB synthase has a high affinity for its bilin substrate, with a sub-micromolar Km for BV. PMID:11500553

  7. Purification and biochemical properties of phytochromobilin synthase from etiolated oat seedlings.

    Science.gov (United States)

    McDowell, M T; Lagarias, J C

    2001-08-01

    Plant phytochromes are dependent on the covalent attachment of the linear tetrapyrrole chromophore phytochromobilin (P Phi B) for photoactivity. In planta, biliverdin IX alpha (BV) is reduced by the plastid-localized, ferredoxin (Fd)-dependent enzyme P Phi B synthase to yield 3Z-P Phi B. Here, we describe the >50,000-fold purification of P Phi B synthase from etioplasts from dark-grown oat (Avena sativa L. cv Garry) seedlings using traditional column chromatography and preparative electrophoresis. Thus, P Phi B synthase is a very low abundance enzyme with a robust turnover rate. We estimate the turnover rate to be >100 s(-1), which is similar to that of mammalian NAD(P)H-dependent BV reductase. Oat P Phi B synthase is a monomer with a subunit mass of 29 kD. However, two distinct charged forms of the enzymes were identified by native isoelectric focusing. The ability of P Phi B synthase to reduce BV is dependent on reduced 2Fe-2S Fds. A K(m) for spinach (Spinacea oleracea) Fd was determined to be 3 to 4 microM. P Phi B synthase has a high affinity for its bilin substrate, with a sub-micromolar K(m) for BV.

  8. 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.

  9. Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Kelley, M.J.; Carman, G.M.

    1987-01-01

    The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase was purified 2300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism

  10. 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...

  11. STRUCTURAL ENZYMOLOGY OF POLYKETIDE SYNTHASES

    OpenAIRE

    Tsai, Shiou-Chuan (Sheryl); Ames, Brian Douglas

    2009-01-01

    This chapter describes structural and associated enzymological studies of polyketide synthases, including isolated single domains and multidomain fragments. The sequence–structure–function relationship of polyketide biosynthesis, compared with homologous fatty acid synthesis, is discussed in detail. Structural enzymology sheds light on sequence and structural motifs that are important for the precise timing, substrate recognition, enzyme catalysis, and protein–protein interactions leading to ...

  12. Accommodating Discontinuities in Dimeric Left-Handed Coiled Coils in ATP Synthase External Stalks

    Science.gov (United States)

    Wise, John G.; Vogel, Pia D.

    2009-01-01

    ATP synthases from coupling membranes are complex rotary motors that convert the energy of proton gradients across coupling membranes into the chemical potential of the β-γ anhydride bond of ATP. Proton movement within the ring of c subunits localized in the F0-sector drives γ and ɛ rotation within the F1α3β3 catalytic core where substrates are bound and products are released. An external stalk composed of homodimeric subunits b2 in Escherichia coli or heterodimeric bb′ in photosynthetic synthases connects F0 subunit a with F1 subunits δ and most likely α. The external stalk resists rotation, and is of interest both functionally and structurally. Hypotheses that the external stalk contributes to the overall efficiency of the reaction through elastic coupling of rotational substeps, and that stalks form staggered, right-handed coiled coils, are investigated here. We report on different structures that accommodate heptad discontinuities with either local or global underwinding. Analyses of the knob-and-hole packing of the E. coli b2 and Synechocystis bb′ stalks strongly support the possibility that these proteins can adopt conventional left-handed coiled coils. PMID:19348765

  13. A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP Synthase

    Science.gov (United States)

    Stan-Lotter, Helga; Hochstein, Lawrence I.

    1989-01-01

    A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F sub 1 moiety from the Escherichia coli ATP Synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa, and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20 to 22 Mol percent). Peptide mapping of sodium dodecylsulfate-denatured subunits I and II showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F sub 1 ATPase (EC 3.6.1.34) from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, Halobacteria in general, possess an ATPase which is unlike the ubiquitous F sub o F sub 1 - ATP Synthase.

  14. The hybrid four-CBS-domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1.

    Science.gov (United States)

    Ramon, Matthew; Ruelens, Philip; Li, Yi; Sheen, Jen; Geuten, Koen; Rolland, Filip

    2013-07-01

    The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy-sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ-like proteins, plants also encode a hybrid βγ protein that combines the Four-Cystathionine β-synthase (CBS)-domain (FCD) structure in γ subunits with a glycogen-binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in-depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast-containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre-CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  15. Isolation, characterization, and mechanistic studies of (-)-alpha-gurjunene synthase from Solidago canadensis.

    Science.gov (United States)

    Schmidt, C O; Bouwmeester, H J; Bülow, N; König, W A

    1999-04-15

    The leaves of the composite Solidago canadensis (goldenrod) were shown to contain (-)-alpha-gurjunene synthase activity. This sesquiterpene is likely to be the precursor for cyclocolorenone, a sesquiterpene ketone present in high amounts in S. canadensis leaves. (-)-alpha-Gurjunene synthase was purified to apparent homogeneity (741-fold) by anion-exchange chromatography (on several matrices), dye ligand chromatography, hydroxylapatite chromatography, and gel filtration. Chromatography on a gel filtration matrix indicated a native molecular mass of 48 kDa, and SDS-PAGE showed the enzyme to be composed of one subunit with a denatured mass of 60 kDa. Its maximum activity was observed at pH 7.8 in the presence of 10 mM Mg2+ and the KM value for the substrate farnesyl diphosphate was 5.5 microM. Over a range of purification steps (-)-alpha-gurjunene and (+)-gamma-gurjunene synthase activities copurified. In addition, the product ratio of the enzyme activity under several different assay conditions was always 91% (-)-alpha-gurjunene and 9% (+)-gamma-gurjunene. This suggests that the formation of these two structurally related products is catalyzed by one enzyme. For further confirmation, we carried out a number of mechanistic studies with (-)-alpha-gurjunene synthase, in which an enzyme preparation was incubated with deuterated substrate analogues. Based on mass spectrometry analysis of the products formed, a cyclization mechanism was postulated which makes it plausible that the synthase catalyzes the formation of both sesquiterpenes. Copyright 1999 Academic Press.

  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. Cross-linking of chloroplast F0F1-ATPase subunit epsilon to gamma without effect on activity. Epsilon and gamma are parts of the rotor

    NARCIS (Netherlands)

    Schulenberg, B; Wellmer, F; Lill, H; Junge, Wolfgang; Engelbrecht, Siegfried

    1997-01-01

    Cys residues were directed into positions 17, 28, 41 and 85 of a Cys6-->Ser mutant of subunit epsilon of spinach chloroplast F0F1 ATP synthase. Wild-type and engineered epsilon were expressed in Escherichia coli, purified in the presence of urea, refolded and reassembled with spinach chloroplast F1

  18. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I

    1991-01-01

    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies.......cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  19. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    OpenAIRE

    Karuppiah, Vijaykumar; Ranaghan, Kara E.; Leferink, Nicole G. H.; Johannissen, Linus O.; Shanmugam, Muralidharan; Ní Cheallaigh, Aisling; Bennett, Nathan J.; Kearsey, Lewis J.; Takano, Eriko; Gardiner, John M.; van der Kamp, Marc W.; Hay, Sam; Mulholland, Adrian J.; Leys, David; Scrutton, Nigel S.

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS)...

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

    Science.gov (United States)

    Haagsma, Anna C; Driessen, Nicole N; Hahn, Marc-Manuel; Lill, Holger; Bald, Dirk

    2010-12-01

    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 in the energy metabolism of Mycobacterium tuberculosis; however, no biochemical data are available to characterize the role of ATP synthase in slow-growing mycobacterial strains. Here, we show that inverted membrane vesicles from the slow-growing model strain Mycobacterium bovis BCG are active in ATP synthesis, but ATP synthase displays no detectable ATP hydrolysis activity and does not set up a proton-motive force (PMF) using ATP as a substrate. Treatment with methanol as well as PMF activation unmasked the ATP hydrolysis activity, indicating that the intrinsic subunit ɛ and inhibitory ADP are responsible for the suppression of hydrolytic activity. These results suggest that the enzyme is needed for the synthesis of ATP, not for the maintenance of the PMF. For the development of new antimycobacterial drugs acting on ATP synthase, screening for ATP synthesis inhibitors, but not for ATP hydrolysis blockers, can be regarded as a promising strategy. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  1. Cristae architecture is determined by an interplay of the MICOS complex and the F1FOATP synthase via Mic27 and Mic10.

    Science.gov (United States)

    Eydt, Katharina; Davies, Karen M; Behrendt, Christina; Wittig, Ilka; Reichert, Andreas S

    2017-07-20

    The inner boundary and the cristae membrane are connected by pore-like structures termed crista junctions (CJs). The MICOS complex is required for CJ formation and enriched at CJs. Here, we address the roles of the MICOS subunits Mic27 and Mic10. We observe a positive genetic interaction between Mic27 and Mic60 and deletion of Mic27 results in impaired formation of CJs and altered cristae membrane curvature. Mic27 acts in an antagonistic manner to Mic60 as it promotes oligomerization of the F 1 F O -ATP synthase and partially restores CJ formation in cells lacking Mic60. Mic10 impairs oligomerization of the F 1 F O -ATP synthase similar to Mic60. Applying complexome profiling, we observed that deletion of Mic27 destabilizes the MICOS complex but does not impair formation of a high molecular weight Mic10 subcomplex. Moreover, this Mic10 subcomplex comigrates with the dimeric F 1 F O -ATP synthase in a Mic27-independent manner. Further, we observed a chemical crosslink of Mic10 to Mic27 and of Mic10 to the F 1 F O -ATP synthase subunit e. We corroborate the physical interaction of the MICOS complex and the F 1 F O -ATP synthase. We propose a model in which part of the F 1 F O -ATP synthase is linked to the MICOS complex via Mic10 and Mic27 and by that is regulating CJ formation.

  2. Cristae architecture is determined by an interplay of the MICOS complex and the F1FO ATP synthase via Mic27 and Mic10

    Directory of Open Access Journals (Sweden)

    Katharina Eydt

    2017-07-01

    Full Text Available The inner boundary and the cristae membrane are connected by pore-like structures termed crista junctions (CJs. The MICOS complex is required for CJ formation and enriched at CJs. Here, we address the roles of the MICOS subunits Mic27 and Mic10. We observe a positive genetic interaction be-tween Mic27 and Mic60 and deletion of Mic27 results in impaired formation of CJs and altered cristae membrane curvature. Mic27 acts in an antagonistic manner to Mic60 as it promotes oligomerization of the F1FO-ATP synthase and partially restores CJ formation in cells lacking Mic60. Mic10 impairs oli-gomerization of the F1FO-ATP synthase similar to Mic60. Applying complex-ome profiling, we observed that deletion of Mic27 destabilizes the MICOS complex but does not impair formation of a high molecular weight Mic10 subcomplex. Moreover, this Mic10 subcomplex comigrates with the dimeric F1FO-ATP synthase in a Mic27-independent manner. Further, we observed a chemical crosslink of Mic10 to Mic27 and of Mic10 to the F1FO-ATP synthase subunit e. We corroborate the physical interaction of the MICOS complex and the F1FO-ATP synthase. We propose a model in which part of the F1FO-ATP synthase is linked to the MICOS complex via Mic10 and Mic27 and by that is regulating CJ formation.

  3. The expression, purification, crystallization and preliminary X-ray analysis of a subcomplex of the peripheral stalk of ATP synthase from bovine mitochondria

    International Nuclear Information System (INIS)

    Silvester, Jocelyn A.; Kane Dickson, Veronica; Runswick, Michael J.; Leslie, Andrew G. W.; Walker, John E.

    2006-01-01

    A recombinant subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been crystallized and a native data set has been collected to 2.8 Å resolution. A subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been expressed to high levels in a soluble form in Escherichia coli. The subcomplex consists of residues 79–184 of subunit b, residues 1–124 of subunit d and the entire F 6 subunit (76 residues). It has been purified and crystallized by vapour diffusion. The morphology and diffraction properties of the crystals of the subcomplex were improved by the presence of thioxane or 4-methylpyridine in the crystallization liquor. With a synchrotron-radiation source, these crystals diffracted to 2.8 Å resolution. They belong to the monoclinic space group P2 1

  4. The c-Ring of the F1FO-ATP Synthase: Facts and Perspectives.

    Science.gov (United States)

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pagliarani, Alessandra

    2016-04-01

    The F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H(+) or Na(+)) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the "molecular currency unit" of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme's potential role as a drug target.

  5. 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.

  6. The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species.

    Science.gov (United States)

    Liu, Sidong; Charlesworth, Thomas J; Bason, John V; Montgomery, Martin G; Harbour, Michael E; Fearnley, Ian M; Walker, John E

    2015-05-15

    The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.

  7. The Dictyostelium discoideum cellulose synthase: Structure/function analysis and identification of interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Richard L. Blanton

    2004-02-19

    OAK-B135 The major accomplishments of this project were: (1) the initial characterization of dcsA, the gene for the putative catalytic subunit of cellulose synthase in the cellular slime mold Dictyostelium discoideum; (2) the detection of a developmentally regulated event (unidentified, but perhaps a protein modification or association with a protein partner) that is required for cellulose synthase activity (i.e., the dcsA product is necessary, but not sufficient for cellulose synthesis); (3) the continued exploration of the developmental context of cellulose synthesis and DcsA; (4) the isolation of a GFP-DcsA-expressing strain (work in progress); and (5) the identification of Dictyostelium homologues for plant genes whose products play roles in cellulose biosynthesis. Although our progress was slow and many of our results negative, we did develop a number of promising avenues of investigation that can serve as the foundation for future projects.

  8. Subunit heterogeneity in the lima bean lectin.

    Science.gov (United States)

    Roberts, D D; Etzler, M E; Goldstein, I J

    1982-08-10

    Three forms of lectin (components I, II, and III) from lima beans (Phaseolus lunatus) have been purified on an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to). Conversion of components I and II to component III has been achieved by reduction in 10(-2) M dithiothreitol. Isoelectric focusing of lima bean lectin in the presence of 8 M urea and beta-mercaptoethanol revealed charge heterogeneity of the lectin subunits. Three major subunit classes of apparent pI 7.05, 6.65, and 6.45, designated alpha, beta, and alpha', respectively, were identified; they occur in a relative abundance of 2:5:3. Green lima beans harvested before maturity lacked the alpha' subunit (pI 6.45) which appears to accumulate during seed maturation. The three subunits are glycoproteins of identical size and immunochemical reactivity. Identical NH2-terminal sequences were found for the three subunits. Amino acid analysis and tryptic peptide mapping indicated that the observed charge heterogeneity is probably due to differences in the primary structure of the subunits. Studies of subunit composition of charge isolectins provided evidence of nonrandom subunit assembly. A model is proposed involving pairing of a pI 6.65 subunit with either a pI 7.06 or 6.45 subunit to form dimeric units. Possible roles for subunit heterogeneity and ordered subunit assembly in determining the metal and sugar binding properties of lima bean lectin are discussed.

  9. Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments

    Directory of Open Access Journals (Sweden)

    Cushman Mark

    2003-12-01

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

  10. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO2/O2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

  11. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

    Science.gov (United States)

    Schep, Daniel G.; Rubinstein, John L.

    2016-01-01

    Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases. PMID:26951669

  12. Assembly of the membrane domain of ATP synthase in human mitochondria.

    Science.gov (United States)

    He, Jiuya; Ford, Holly C; Carroll, Joe; Douglas, Corsten; Gonzales, Evvia; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2018-03-20

    The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F 1 -c 8 complex inhibited by the ATPase inhibitor protein IF 1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast F o membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

  13. 28 CFR 51.6 - Political subunits.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All political...

  14. Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

    Directory of Open Access Journals (Sweden)

    Irene Mavelli

    2012-02-01

    Full Text Available Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis or “negative” (silencing mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase β subunit and Inhibitor Factor 1 (IF1. Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on β-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

  15. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    Science.gov (United States)

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids. PMID:28966840

  16. Fungal type III polyketide synthases.

    Science.gov (United States)

    Hashimoto, Makoto; Nonaka, Takamasa; Fujii, Isao

    2014-10-01

    This article covers the literature on fungal type III polyketide synthases (PKSs) published from 2005 to 2014. Since the first discovery of fungal type III PKS genes in Aspergillus oryzae, reported in 2005, putative genes for type III PKSs have been discovered in fungal genomes. Compared with type I PKSs, type III PKSs are much less abundant in fungi. However, type III PKSs could have some critical roles in fungi. This article summarizes the studies on fungal type III PKS functional analysis, including Neurospora crassa ORAS, Aspergillus niger AnPKS, Botrytis cinerea BPKS and Aspergillus oryzae CsyA and CsyB. It is mostly in vitro analysis using their recombinant enzymes that has revealed their starter and product specificities. Of these, CsyB was found to be a new kind of type III PKS that catalyses the coupling of two β-keto fatty acyl CoAs. Homology modelling reported in this article supports the importance of the capacity of the acyl binding tunnel and active site cavity in fungal type III PKSs.

  17. 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.

  18. Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET

    Science.gov (United States)

    Ernst, Stefan; Düser, Monika G.; Zarrabi, Nawid; Börsch, Michael

    2012-03-01

    The enzyme FoF1-ATP synthase provides the 'chemical energy currency' adenosine triphosphate (ATP) for living cells. Catalysis is driven by mechanochemical coupling of subunit rotation within the enzyme with conformational changes in the three ATP binding sites. Proton translocation through the membrane-bound Fo part of ATP synthase powers a 10-step rotary motion of the ring of c subunits. This rotation is transmitted to the γ and ɛ subunits of the F1 part. Because γ and ɛ subunits rotate in 120° steps, we aim to unravel this symmetry mismatch by real time monitoring subunit rotation using single-molecule Förster resonance energy transfer (FRET). One fluorophore is attached specifically to the F1 motor, another one to the Fo motor of the liposome-reconstituted enzyme. Photophysical artifacts due to spectral fluctuations of the single fluorophores are minimized by a previously developed duty cycle-optimized alternating laser excitation scheme (DCO-ALEX). We report the detection of reversible elastic deformations between the rotor parts of Fo and F1 and estimate the maximum angular displacement during the load-free rotation using Monte Carlo simulations.

  19. Functional divergence of chloroplast Cpn60α subunits during Arabidopsis embryo development.

    Directory of Open Access Journals (Sweden)

    Xiaolong Ke

    2017-09-01

    Full Text Available Chaperonins are a class of molecular chaperones that assist in the folding and assembly of a wide range of substrates. In plants, chloroplast chaperonins are composed of two different types of subunits, Cpn60α and Cpn60β, and duplication of Cpn60α and Cpn60β genes occurs in a high proportion of plants. However, the importance of multiple Cpn60α and Cpn60β genes in plants is poorly understood. In this study, we found that loss-of-function of CPNA2 (AtCpn60α2, a gene encoding the minor Cpn60α subunit in Arabidopsis thaliana, resulted in arrested embryo development at the globular stage, whereas the other AtCpn60α gene encoding the dominant Cpn60α subunit, CPNA1 (AtCpn60α1, mainly affected embryonic cotyledon development at the torpedo stage and thereafter. Further studies demonstrated that CPNA2 can form a functional chaperonin with CPNB2 (AtCpn60β2 and CPNB3 (AtCpn60β3, while the functional partners of CPNA1 are CPNB1 (AtCpn60β1 and CPNB2. We also revealed that the functional chaperonin containing CPNA2 could assist the folding of a specific substrate, KASI (β-ketoacyl-[acyl carrier protein] synthase I, and that the KASI protein level was remarkably reduced due to loss-of-function of CPNA2. Furthermore, the reduction in the KASI protein level was shown to be the possible cause for the arrest of cpna2 embryos. Our findings indicate that the two Cpn60α subunits in Arabidopsis play different roles during embryo development through forming distinct chaperonins with specific AtCpn60β to assist the folding of particular substrates, thus providing novel insights into functional divergence of Cpn60α subunits in plants.

  20. Functional divergence of chloroplast Cpn60α subunits during Arabidopsis embryo development

    Science.gov (United States)

    Ke, Xiaolong; Zou, Wenxuan; Ren, Yafang; Wang, Zhiqin; Li, Jin; Wu, Xuan

    2017-01-01

    Chaperonins are a class of molecular chaperones that assist in the folding and assembly of a wide range of substrates. In plants, chloroplast chaperonins are composed of two different types of subunits, Cpn60α and Cpn60β, and duplication of Cpn60α and Cpn60β genes occurs in a high proportion of plants. However, the importance of multiple Cpn60α and Cpn60β genes in plants is poorly understood. In this study, we found that loss-of-function of CPNA2 (AtCpn60α2), a gene encoding the minor Cpn60α subunit in Arabidopsis thaliana, resulted in arrested embryo development at the globular stage, whereas the other AtCpn60α gene encoding the dominant Cpn60α subunit, CPNA1 (AtCpn60α1), mainly affected embryonic cotyledon development at the torpedo stage and thereafter. Further studies demonstrated that CPNA2 can form a functional chaperonin with CPNB2 (AtCpn60β2) and CPNB3 (AtCpn60β3), while the functional partners of CPNA1 are CPNB1 (AtCpn60β1) and CPNB2. We also revealed that the functional chaperonin containing CPNA2 could assist the folding of a specific substrate, KASI (β-ketoacyl-[acyl carrier protein] synthase I), and that the KASI protein level was remarkably reduced due to loss-of-function of CPNA2. Furthermore, the reduction in the KASI protein level was shown to be the possible cause for the arrest of cpna2 embryos. Our findings indicate that the two Cpn60α subunits in Arabidopsis play different roles during embryo development through forming distinct chaperonins with specific AtCpn60β to assist the folding of particular substrates, thus providing novel insights into functional divergence of Cpn60α subunits in plants. PMID:28961247

  1. Acetylcholine Receptor: Complex of Homologous Subunits

    Science.gov (United States)

    Raftery, Michael A.; Hunkapiller, Michael W.; Strader, Catherine D.; Hood, Leroy E.

    1980-06-01

    The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

  2. Expression, Purification, and Characterisation of Dehydroquinate Synthase from Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    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.

  3. Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

    Science.gov (United States)

    Borejsza-Wysocki, W.; Hrazdina, G.

    1996-03-01

    p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.

  4. Risk capital allocation with autonomous subunits

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Smilgins, Aleksandrs

    2016-01-01

    Risk capital allocation problems have been widely discussed in the academic literature. We consider a set of independent subunits collaborating in order to reduce risk: that is, when subunit portfolios are merged a diversification benefit arises and the risk of the group as a whole is smaller tha...... fairness tests related directly to the problem of risk capital allocation and show that the Lorenz set satisfies all three tests in contrast to other well-known coherent methods. Finally, we discuss how to deal with non-uniqueness of the Lorenz set.......Risk capital allocation problems have been widely discussed in the academic literature. We consider a set of independent subunits collaborating in order to reduce risk: that is, when subunit portfolios are merged a diversification benefit arises and the risk of the group as a whole is smaller than...... the sum of the risks of the individual subunits. The question is how to allocate the risk capital of the group among the subunits in a fair way. In this paper we propose to use the Lorenz set as an allocation method. We show that the Lorenz set is operational and coherent. Moreover, we propose three...

  5. The affinity purification and characterization of ATP synthase complexes from mitochondria.

    Science.gov (United States)

    Runswick, Michael J; Bason, John V; Montgomery, Martin G; Robinson, Graham C; Fearnley, Ian M; Walker, John E

    2013-02-13

    The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF₁ with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF₁ with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

  6. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

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

  7. Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching

    Science.gov (United States)

    Starke, Ilka; Johnson, Kathryn M.; Petersen, Jan; Gräber, Peter; Opipari, Anthony W.; Glick, Gary D.; Börsch, Michael

    2016-03-01

    Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme FoF1-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in FoF1-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C terminus of subunit γ. Imaging the threedimensional arrangement of mitochondria in living yeast cells was possible at superresolution using structured illumination microscopy, SIM. We measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy. Our data confirmed the binding of Cy5-labeled Bz-423 to the top of the F1 domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.

  8. Post-irradiation inactivation, protection, and repair of the sulfhydryl enzyme malate synthase

    International Nuclear Information System (INIS)

    Durchschlag, H.; Zipper, P.

    1985-01-01

    Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0,30,60 h after irradiation. To elucidate the role of OH - , O 2 , and H 2 O 2 in the X-ray inactivation of the enzyme, experiments were performed in the absence of presence of different concentrations of specific additives (formate, superoxide dismutase, catalase). These additives were added to malate synthase solutions before or after X-irradiation. Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol. Experiments yielded the following results: 1. Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity Asub(r)=3% (corresponding to a D 37 =0.6 kGy), and led to further slow inactivation in the post-irradiation phase. Repairs, initiated at different times after irradiation, restored enzymic activity considerably. The repair initiated at t=0 led to Asub(r)=21%; repairs started later on resulted in somewhat lower activities. The decay of reparability, however, was found to progress more slowly than post-irradiation inactivation itself. After completion of repair the activities of repaired samples did not decrease significantly. 2. The presence of specific additives during irradiation caused significant protective effects against primary inactivation. The protection by formate was very pronounced (e.g., Asub(r)=72% and D 37 =6 kGy for 100 mM formate). The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate. (orig.)

  9. Crystallographic and enzymatic insights into the mechanisms of Mg-ADP inhibition in the A1complex of the A1AOATP synthase.

    Science.gov (United States)

    Singh, Dhirendra; Grüber, Gerhard

    2018-01-01

    F-ATP synthases are described to have mechanisms which regulate the unnecessary depletion of ATP pool during an energy limited state of the cell. Mg-ADP inhibition is one of the regulatory features where Mg-ADP gets entrapped in the catalytic site, preventing the binding of ATP and further inhibiting ATP hydrolysis. Knowledge about the existence and regulation of the related archaeal-type A 1 A O ATP synthases (A 3 B 3 CDE 2 FG 2 ac) is limited. We demonstrate MgADP inhibition of the enzymatically active A 3 B 3 D- and A 3 B 3 DF complexes of Methanosarcina mazei Gö1 A-ATP synthase and reveal the importance of the amino acids P235 and S238 inside the P-loop (GPFGSGKTV) of the catalytic A subunit. Substituting these two residues by the respective P-loop residues alanine and cysteine (GAFGCGKTV) of the related eukaryotic V-ATPase increases significantly the ATPase activity of the enzyme variant and abolishes MgADP inhibition. The atomic structure of the P235A, S238C double mutant of subunit A of the Pyrococcus horikoshii OT3 A-ATP synthase provides details of how these critical residues affect nucleotide-binding and ATP hydrolysis in this molecular engine. The qualitative data are confirmed by quantitative results derived from fluorescence correlation spectroscopy experiments. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Turner, Glenn W; Croteau, Rodney

    2004-12-01

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

  11. The Subunit Principle in Scar Face Revision.

    Science.gov (United States)

    Elshahat, Ahmed; Lashin, Riham

    2017-06-01

    Facial scaring is considered one of the most difficult cosmetic problems for any plastic surgeon to solve. The condition is more difficult if the direction of the scar is not parallel to relaxed skin tension lines. Attempts to manage this difficult situation included revisions using geometric designs, Z plasties or W plasties to camouflage the straight line visible scaring. The use of long-lasting resorbable sutures was tried too. Recently, the use of botulinum toxin during revision improved the results. Fractional CO2 lasers, microfat grafts, and platelet-rich plasma were added to the armamentarium. The scar is least visible if placed in the junction between the facial subunits. The aim of this study is to investigate the use of the subunit principle to improve the results of scar revision. Four patients were included in this study. Tissue expansion of the intact part of the subunit allowed shifting the scar to the junction between the affected subunit and the adjacent one. Tissue expansion, delivery of the expanders, and advancement of the flaps were successful in all patients. The fact that this is a 2-stage procedure and sacrifices some of the intact skin from the affected facial subunit, makes this technique reserved to patients with ugly facial scars who are ambitious to improve their appearance.

  12. Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX

    Science.gov (United States)

    Zarrabi, N.; Ernst, S.; Düser, M. G.; Golovina-Leiker, A.; Becker, W.; Erdmann, R.; Dunn, S. D.; Börsch, M.

    2009-02-01

    FoF1-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound Fo part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the γ and ɛ subunits of the F1 sector resulting in 120° steps. In order to unravel this symmetry mismatch we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme: one attached to the F1 motor, another one to the Fo motor, and the third one to a non-rotating subunit. To reduce photophysical artifacts due to spectral fluctuations of the single fluorophores, a duty cycle-optimized alternating three-laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of Fo and F1.

  13. Elastic rotation of Escherichia coli F{sub O}F{sub 1} having ε subunit fused with cytochrome b{sub 562} or flavodoxin reductase

    Energy Technology Data Exchange (ETDEWEB)

    Oka, Hideyuki [Department of Bioscience, Nagahama Institute of Bioscience and Technology, Nagahama, Shiga 526-0829 (Japan); Hosokawa, Hiroyuki; Nakanishi-Matsui, Mayumi [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Yahaba, Iwate 028-3694 (Japan); Dunn, Stanley D. [Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1 (Canada); Futai, Masamitsu [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Yahaba, Iwate 028-3694 (Japan); Iwamoto-Kihara, Atsuko, E-mail: a_iwamoto@nagahama-i-bio.ac.jp [Department of Bioscience, Nagahama Institute of Bioscience and Technology, Nagahama, Shiga 526-0829 (Japan)

    2014-04-18

    Highlights: • Intra-molecular rotation of F{sub O}F{sub 1} ATP synthase was observed using a small bead probe. • Carboxyl-terminus of the ε subunit was fused to cytochrome b{sub 562} or flavodoxin reductase. • The F{sub O}F{sub 1} showed continual rotation with similar rate to the wild-type enzyme. • The intra-molecular rotation is flexible and elastic. - Abstract: Intra-molecular rotation of F{sub O}F{sub 1} ATP synthase enables cooperative synthesis and hydrolysis of ATP. In this study, using a small gold bead probe, we observed fast rotation close to the real rate that would be exhibited without probes. Using this experimental system, we tested the rotation of F{sub O}F{sub 1} with the ε subunit connected to a globular protein [cytochrome b{sub 562} (ε-Cyt) or flavodoxin reductase (ε-FlavR)], which is apparently larger than the space between the central and the peripheral stalks. The enzymes containing ε-Cyt and ε-FlavR showed continual rotations with average rates of 185 and 148 rps, respectively, similar to the wild type (172 rps). However, the enzymes with ε-Cyt or ε-FlavR showed a reduced proton transport. These results indicate that the intra-molecular rotation is elastic but proton transport requires more strict subunit/subunit interaction.

  14. 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...

  15. Evolutionary and mechanistic insights from the reconstruction of (+)-humulene synthases from a modern (+)-Germacrene A Synthase

    OpenAIRE

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

    2014-01-01

    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

  16. 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 ...

  17. 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 ...

  18. 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.

  19. 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 ...

  20. Cloning and expression of pineapple sucrosephosphate synthase ...

    African Journals Online (AJOL)

    A 1132-base pairs (bp) polymerase-chain-reaction product of sucrose-phosphate synthase (SPS) (EC 2.3.1.14) from pineapple (Ananas comosus cv. Comte de paris) fruit was cloned and nominated as Ac- SPS1. The sequence encodes a putative 377 amino acids protein containing two serine conserved features that had ...

  1. Engineering Isoprene Synthase Expression and Activity in Cyanobacteria.

    Science.gov (United States)

    Chaves, Julie E; Rueda-Romero, Paloma; Kirst, Henning; Melis, Anastasios

    2017-12-15

    Efforts to heterologously produce quantities of isoprene hydrocarbons (C 5 H 8 ) renewably from CO 2 and H 2 O through the photosynthesis of cyanobacteria face barriers, including low levels of recombinant enzyme accumulation compounded by their slow innate catalytic activity. The present work sought to alleviate the "expression level" barrier upon placing the isoprene synthase (IspS) enzyme in different fusion configurations with the cpcB protein, the highly expressed β-subunit of phycocyanin. Different cpcB*IspS fusion constructs were made, distinguished by the absence or presence of linker amino acids between the two proteins. Composition of linker amino acids was variable with lengths of 7, 10, 16, and 65 amino acids designed to test for optimal activity of the IspS through spatial positioning between the cpcB and IspS. Results showed that fusion constructs with the highly expressed cpcB gene, as the leader sequence, improved transgene expression in the range of 61 to 275-fold over what was measured with the unfused IspS control. However, the specific activity of the IspS enzyme was attenuated in all fusion transformants, possibly because of allosteric effects exerted by the leader cpcB fusion protein. This inhibition varied depending on the nature of the linker amino acids between the cpcB and IspS proteins. In terms of isoprene production, the results further showed a trade-off between specific activity and transgenic enzyme accumulation. For example, the cpcB*L7*IspS strain showed only about 10% the isoprene synthase specific-activity of the unfused cpcB-IspS control, but it accumulated 254-fold more IspS enzyme. The latter more than countered the slower specific activity and made the cpcB*L7*IspS transformant the best isoprene producing strain in this work. Isoprene to biomass yield ratios improved from 0.2 mg g -1 in the unfused cpcB-IspS control to 5.4 mg g -1 in the cpcB*L7*IspS strain, a 27-fold improvement.

  2. ORF Sequence: Ca19AnnotatedDec2004aaSeq [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available Ca19AnnotatedDec2004aaSeq orf19.4600.1 >orf19.4600.1; Contig19-10212; complement(7...5938..76216); DPM3*; dolichol-phosphate-mannose synthase; MTKATETGLTIFALSAIYFALITGVIPTPAKIHDEILPYLPWWGLVTFGSYALSTLGWGIVTFKDKEHKYKELKIQIEEAKDFYKTKGIDLD*

  3. Immunochemical aspects of crotoxim and its subunits

    International Nuclear Information System (INIS)

    Nakazone, A.K.

    1979-01-01

    Crotamine and crotoxin with the subunits - phospholipase A and crotapotin - were obtained by purification from Crotalus durissus terrificus venom. Interaction studies of the subunits using crotalic antiserum, indicated that: crotoxin is formed of crotapotin and phospholipase A with the molar ratio of 1 to 1; using crotapotin 125 I the presence of a soluble complex was shown with the same antiserum. Immunological precipitation reactions demonstrated that crotapotin is antigenic: crotapotin and phospholipase A presented similar antigenic determinants; crotoxin antiserum reacted with each one of the submits; when the subunits are mixed to form synthetic crotoxin some antigenic determinants are masked in the process of interaction. Crotamine, interacted with crotapotin 1:1, without hidden antigenic determinants crotapotin antigenic site seems to be formed by, at least, one lysine. Enzimatical activity of phospholipase A apreared to be dependent on some reaction conditions when its arginine residues are blocked. Tyrosines of phospholipase A are more susceptible to labelling with 131 I than crotapotin. Gama irradiation of aqueous solutions of the subunits produced modifications in the ultraviolet spectra. A decrease of the enzymatic activity occured as a function of radiation dosis. Immunological activities of crotapotin and phospholipase A were not altered [pt

  4. Sequence of the human glycogen-associated regulatory subunit of type 1 protein phosphatase and analysis of its coding region and mRNA level in muscle from patients with NIDDM

    DEFF Research Database (Denmark)

    Chen, Y H; Hansen, L; Chen, Min

    1994-01-01

    Impaired insulin-stimulated glycogen synthesis of peripheral tissues is a characteristic feature of many patients with non-insulin-dependent diabetes mellitus (NIDDM) and their first-degree relatives with normal glucose tolerance, suggesting putative inherited defects in this metabolic pathway....... In previous studies, we have failed to reveal mutations in the coding regions of the muscle-specific glycogen synthase gene and the three genes that encode the catalytic subunits of protein phosphatase 1 (PP1) as frequent causes of insulin resistance. Because the glycogen-associated regulatory subunit...... of protein phosphatase 1 (PP1 G-subunit) plays a key role in the insulin stimulation of glycogen synthesis and the activity of PP1 is decreased in insulin-resistant subjects, we have now cloned the human G-subunit cDNA to search for abnormalities in the corresponding gene (designated PPP1R3 in the human...

  5. Cellulose synthase complex organization and cellulose microfibril structure.

    Science.gov (United States)

    Turner, Simon; Kumar, Manoj

    2018-02-13

    Cellulose consists of linear chains of β-1,4-linked glucose units, which are synthesized by the cellulose synthase complex (CSC). In plants, these chains associate in an ordered manner to form the cellulose microfibrils. Both the CSC and the local environment in which the individual chains coalesce to form the cellulose microfibril determine the structure and the unique physical properties of the microfibril. There are several recent reviews that cover many aspects of cellulose biosynthesis, which include trafficking of the complex to the plasma membrane and the relationship between the movement of the CSC and the underlying cortical microtubules (Bringmann et al. 2012 Trends Plant Sci. 17 , 666-674 (doi:10.1016/j.tplants.2012.06.003); Kumar & Turner 2015 Phytochemistry 112 , 91-99 (doi:10.1016/j.phytochem.2014.07.009); Schneider et al. 2016 Curr. Opin. Plant Biol. 34 , 9-16 (doi:10.1016/j.pbi.2016.07.007)). In this review, we will focus on recent advances in cellulose biosynthesis in plants, with an emphasis on our current understanding of the structure of individual catalytic subunits together with the local membrane environment where cellulose synthesis occurs. We will attempt to relate this information to our current knowledge of the structure of the cellulose microfibril and propose a model in which variations in the structure of the CSC have important implications for the structure of the cellulose microfibril produced.This article is part of a discussion meeting issue 'New horizons for cellulose nanotechnology'. © 2017 The Author(s).

  6. Riboprinting of Naegleria spp.: small-subunit versus large-subunit rDNA.

    Science.gov (United States)

    De Jonckheere, J F

    1994-01-01

    The nonpathogenic amoeba Naegleria lovaniensis is closely related to the human pathogen N. fowleri. Both grow at a maximal temperature of 45 degrees C and, therefore, are often found together in the environment. As they are morphologically inseparable at the light-microscope level, refined techniques are necessary to separate the two species. I have used restriction-fragment-length polymorphism analysis of the polymerase chain reaction (PCR)-amplified ribosomal RNA gene, or riboprinting, to distinguish between the different Naegleria spp. Riboprints generated from the small subunit and the large subunit separate N. fowleri from N. lovaniensis. To examine the taxonomic relationships among all Naegleria spp., analysis of the large subunit has to be performed; the small subunit contains a 1.3-kb group I intron, which interferes with tree building based upon restriction sites.

  7. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    KAUST Repository

    Beke-Somfai, Tamás

    2010-01-26

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in βTP and, thus, promote ATP. © 2009 American Chemical Society.

  8. Acetohydroxyacid synthase (AHAS) in vivo assay for screening imidazolinone-resistance in sunflower (Helianthus annuus L.).

    Science.gov (United States)

    Vega, T; Breccia, G; Gil, M; Zorzoli, R; Picardi, L; Nestares, G

    2012-12-01

    The objective of this work was to evaluate the in vivo acetohydroxyacid synthase (AHAS) activity response to imidazolinones and its possible use as a selection method for evaluating AHAS inhibitor resistance. In vivo AHAS assay and the comparison of parameters from dose-response curves have been used as a valid tool for comparing sunflower lines and hybrids differing in imidazolinone resistance. The sunflower resistant genotypes evaluated here were 100-fold and 20-fold more resistant compared with the susceptible line for imazethapyr and imazapyr, respectively. This assay also allowed discrimination of homozygous from heterozygous genotypes for I(mr1) locus that codify for the catalytic subunit of AHAS. The in vivo AHAS assay described in this study was useful for the selection of sunflower genotypes differing in herbicide resistance and could be a useful tool when breeding for imidazolinone resistance in sunflower. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  9. Engineering of plant type III polyketide synthases.

    Science.gov (United States)

    Wakimoto, Toshiyuki; Morita, Hiroyuki; Abe, Ikuro

    2012-01-01

    Members of the chalcone synthase superfamily of type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkable structural diversity and biological activities. The homodimeric type III PKSs share a common three-dimensional overall fold with a conserved Cys-His-Asn catalytic triad; notably, only a slight modification of the active site dramatically expands the catalytic repertoire of the enzymes. In addition, the enzymes exhibit extremely promiscuous substrate specificities, and accept a variety of nonphysiological substrates, making the type III PKSs an excellent platform for the further production of unnatural, novel polyketide scaffolds with promising biological activities. This chapter summarizes recent advances in the engineering of plant type III PKS enzymes in our laboratories, using approaches combining structure-based enzyme engineering and precursor-directed biosynthesis with rationally designed substrate analogs. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    Science.gov (United States)

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

  11. The Biological Role of the ζ Subunit as Unidirectional Inhibitor of the F1FO-ATPase of Paracoccus denitrificans

    Directory of Open Access Journals (Sweden)

    Francisco Mendoza-Hoffmann

    2018-01-01

    Full Text Available The biological roles of the three natural F1FO-ATPase inhibitors, ε, ζ, and IF1, on cell physiology remain controversial. The ζ subunit is a useful model for deletion studies since it mimics mitochondrial IF1, but in the F1FO-ATPase of Paracoccus denitrificans (PdF1FO, it is a monogenic and supernumerary subunit. Here, we constructed a P. denitrificans 1222 derivative (PdΔζ with a deleted ζ gene to determine its role in cell growth and bioenergetics. The results show that the lack of ζ in vivo strongly restricts respiratory P. denitrificans growth, and this is restored by complementation in trans with an exogenous ζ gene. Removal of ζ increased the coupled PdF1FO-ATPase activity without affecting the PdF1FO-ATP synthase turnover, and the latter was not affected at all by ζ reconstitution in vitro. Therefore, ζ works as a unidirectional pawl-ratchet inhibitor of the PdF1FO-ATPase nanomotor favoring the ATP synthase turnover to improve respiratory cell growth and bioenergetics.

  12. Engineering cotton (+)-delta-cadinene synthase to an altered function: germacrene D-4-ol synthase.

    Science.gov (United States)

    Yoshikuni, Yasuo; Martin, Vincent J J; Ferrin, Thomas E; Keasling, Jay D

    2006-01-01

    The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-cadinene synthase suggested that the G helix plays a very important role in (+)-delta-cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).

  13. 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.

  14. allelic variation of hmw glutenin subunits of ethiopian bread wheat ...

    African Journals Online (AJOL)

    journal

    reduced subunits of glutenin proteins bands are separated: the high molecular weight (HMW) and low molecular weight (LMW) subunits (Payne et al.,1980; Jackson et al., 1983). The HMW glutenin subunits (GS) of wheat protein are quantitatively minor, but functionally an important group of gluten proteins in the process of ...

  15. Crystal Structures of Two Isozymes of Citrate Synthase from Sulfolobus tokodaii Strain 7

    Directory of Open Access Journals (Sweden)

    Midori Murakami

    2016-01-01

    Full Text Available Thermoacidophilic archaeon Sulfolobus tokodaii strain 7 has two citrate synthase genes (ST1805-CS and ST0587-CS in the genome with 45% sequence identity. Because they exhibit similar optimal temperatures of catalytic activity and thermal inactivation profiles, we performed structural comparisons between these isozymes to elucidate adaptation mechanisms to high temperatures in thermophilic CSs. The crystal structures of ST1805-CS and ST0587-CS were determined at 2.0 Å and 2.7 Å resolutions, respectively. Structural comparison reveals that both of them are dimeric enzymes composed of two identical subunits, and these dimeric structures are quite similar to those of citrate synthases from archaea and eubacteria. ST0587-CS has, however, 55 ion pairs within whole dimer structure, while having only 36 in ST1805-CS. Although the number and distributions of ion pairs are distinct from each other, intersubunit ion pairs between two domains of each isozyme are identical especially in interterminal region. Because the location and number of ion pairs are in a trend with other CSs from thermophilic microorganisms, the factors responsible for thermal adaptation of ST-CS isozymes are characterized by ion pairs in interterminal region.

  16. Kinetic and hysteretic behavior of ATP hydrolysis of the highly stable dimeric ATP synthase of Polytomella sp.

    Science.gov (United States)

    Villavicencio-Queijeiro, Alexa; Pardo, Juan Pablo; González-Halphen, Diego

    2015-06-01

    The F1FO-ATP synthase of the colorless alga Polytomella sp. exhibits a robust peripheral arm constituted by nine atypical subunits only present in chlorophycean algae. The isolated dimeric enzyme exhibits a latent ATP hydrolytic activity which can be activated by some detergents. To date, the kinetic behavior of the algal ATPase has not been studied. Here we show that while the soluble F1 sector exhibits Michaelis-Menten kinetics, the dimer exhibits a more complex behavior. The kinetic parameters (Vmax and Km) were obtained for both the F1 sector and the dimeric enzyme as isolated or activated by detergent, and this activation was also seen on the enzyme reconstituted in liposomes. Unlike other ATP synthases, the algal dimer hydrolyzes ATP on a wide range of pH and temperature. The enzyme was inhibited by oligomycin, DCCD and Mg-ADP, although oligomycin induced a peculiar inhibition pattern that can be attributed to structural differences in the algal subunit-c. The hydrolytic activity was temperature-dependent and exhibited activation energy of 4 kcal/mol. The enzyme also exhibited a hysteretic behavior with a lag phase strongly dependent on temperature but not on pH, that may be related to a possible regulatory role in vivo. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

    Energy Technology Data Exchange (ETDEWEB)

    Harford, N.; De Wilde, M.

    1987-05-19

    A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce an immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.

  18. 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...... in the sarcolemma as well as the cytoplasm of type I muscle fibres. NADPH diaphorase activity confirmed a higher level of NO synthase activity in the sarcolemma as well as the cytoplasm of type I muscle fibers. Histochemical staining for cytochrome oxidase showed a staining pattern similar to that observed for type...... 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...

  19. Functional plasticity of paralogous diterpene synthases involved in conifer defense

    OpenAIRE

    Keeling, Christopher I.; Weisshaar, Sabrina; Lin, Roy P. C.; Bohlmann, Jörg

    2008-01-01

    The diversity of terpenoid compounds produced by plants plays an important role in mediating various plant–herbivore, plant–pollinator, and plant–pathogen interactions. This diversity has resulted from gene duplication and neofunctionalization of the enzymes that synthesize and subsequently modify terpenes. Two diterpene synthases in Norway spruce (Picea abies), isopimaradiene synthase and levopimaradiene/abietadiene synthase, provide the hydrocarbon precursors for most of the diterpene resin...

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

    Science.gov (United States)

    Croteau, Rodney Bruce [Pullman, WA; Burke, Charles Cullen [Moscow, ID

    2008-06-24

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

  1. Oligomycin frames a common drug-binding site in the ATP synthase

    Energy Technology Data Exchange (ETDEWEB)

    Symersky, Jindrich; Osowski, Daniel; Walters, D. Eric; Mueller, David M. (Rosalind)

    2015-12-01

    We report the high-resolution (1.9 {angstrom}) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common 'drug-binding site.' We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

  2. Crystal structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase of riboflavin biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Calabrese, J.C.; Wawrzak, Z.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    2010-03-05

    3,4-Dihydroxy-2-butanone-4-phosphate synthase catalyzes a commitment step in the biosynthesis of riboflavin. On the enzyme, ribulose 5-phosphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate and formate in steps involving enolization, ketonization, dehydration, skeleton rearrangement, and formate elimination. The enzyme is absent in humans and an attractive target for the discovery of antimicrobials for pathogens incapable of acquiring sufficient riboflavin from their hosts. The homodimer of 23 kDa subunits requires Mg{sup 2+} for activity. The first three-dimensional structure of the enzyme was determined at 1.4 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on Escherichia coli protein crystals containing gold. The protein consists of an {alpha} + {beta} fold having a complex linkage of {beta} strands. Intersubunit contacts are mediated by numerous hydrophobic interactions and three hydrogen bond networks. A proposed active site was identified on the basis of amino acid residues that are conserved among the enzyme from 19 species. There are two well-separated active sites per dimer, each of which comprise residues from both subunits. In addition to three arginines and two threonines, which may be used for recognizing the phosphate group of the substrate, the active site consists of three glutamates, two aspartates, two histidines, and a cysteine which may provide the means for general acid and base catalysis and for coordinating the Mg{sup 2+} cofactor within the active site.

  3. Impairment of Cellulose Synthases Required for Arabidopsis Secondary Cell Wall Formation Enhances Disease Resistance[W

    Science.gov (United States)

    Hernández-Blanco, Camilo; Feng, Dong Xin; Hu, Jian; Sánchez-Vallet, Andrea; Deslandes, Laurent; Llorente, Francisco; Berrocal-Lobo, Marta; Keller, Harald; Barlet, Xavier; Sánchez-Rodríguez, Clara; Anderson, Lisa K.; Somerville, Shauna; Marco, Yves; Molina, Antonio

    2007-01-01

    Cellulose is synthesized by cellulose synthases (CESAs) contained in plasma membrane–localized complexes. In Arabidopsis thaliana, three types of CESA subunits (CESA4/IRREGULAR XYLEM5 [IRX5], CESA7/IRX3, and CESA8/IRX1) are required for secondary cell wall formation. We report that mutations in these proteins conferred enhanced resistance to the soil-borne bacterium Ralstonia solanacearum and the necrotrophic fungus Plectosphaerella cucumerina. By contrast, susceptibility to these pathogens was not altered in cell wall mutants of primary wall CESA subunits (CESA1, CESA3/ISOXABEN RESISTANT1 [IXR1], and CESA6/IXR2) or POWDERY MILDEW–RESISTANT5 (PMR5) and PMR6 genes. Double mutants indicated that irx-mediated resistance was independent of salicylic acid, ethylene, and jasmonate signaling. Comparative transcriptomic analyses identified a set of common irx upregulated genes, including a number of abscisic acid (ABA)–responsive, defense-related genes encoding antibiotic peptides and enzymes involved in the synthesis and activation of antimicrobial secondary metabolites. These data as well as the increased susceptibility of ABA mutants (abi1-1, abi2-1, and aba1-6) to R. solanacearum support a direct role of ABA in resistance to this pathogen. Our results also indicate that alteration of secondary cell wall integrity by inhibiting cellulose synthesis leads to specific activation of novel defense pathways that contribute to the generation of an antimicrobial-enriched environment hostile to pathogens. PMID:17351116

  4. Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer

    International Nuclear Information System (INIS)

    Kaur, Jatinder; Sawhney, Meenakshi; DattaGupta, Siddartha; Shukla, Nootan K; Srivastava, Anurag; Ralhan, Ranju

    2010-01-01

    We reported increased levels of Phosphatidyl Inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST) exposure. Tissue microarray (TMA) Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000). Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005) and tobacco consumption (p = 0.03, OR = 9.0). Exposure of oral cell systems to smokeless tobacco (ST) in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K) and cyclin D1 levels. Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco

  5. Divinyl ether synthase gene and protein, and uses thereof

    Science.gov (United States)

    Howe, Gregg A [East Lansing, MI; Itoh, Aya [Tsuruoka, JP

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  6. Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries.

    Science.gov (United States)

    Lücker, Joost; Bowen, Pat; Bohlmann, Jörg

    2004-10-01

    Valencene is a volatile sesquiterpene emitted from flowers of grapevine, Vitis vinifera L. A full-length cDNA from the cultivar Gewürztraminer was functionally expressed in Escherichia coli and found to encode valencene synthase (VvVal). The two major products formed by recombinant VvVal enzyme activity with farnesyl diphosphate (FPP) as substrate are (+)-valencene and (-)-7-epi-alpha-selinene. Grapevine valencene synthase is closely related to a second sesquiterpene synthase from this species, (-)-germacrene D synthase (VvGerD). VvVal and VvGerD cDNA probes revealed strong signals in Northern hybridizations with RNA isolated from grapevine flower buds. Transcript levels were lower in open pre-anthesis flowers, flowers after anthesis, or at early onset of fruit development. Similar results were obtained using a third probe, (-)-alpha-terpineol synthase, a monoterpenol synthase. Sesquiterpene synthase and monoterpene synthase transcripts were not detected in the mesocarp and exocarp during early stages of fruit development, but transcripts hybridizing with VvVal appeared during late ripening of the berries. Sesquiterpene synthase transcripts were also detected in young seeds.

  7. Deregulation of acetohydroxy-acid synthase: loss of allosteric inhibition conferred by mutations in the catalytic subunit

    Czech Academy of Sciences Publication Activity Database

    Kopecký, Jan; Kyselková, Martina; Šigutová, Lucie; Pospíšil, Stanislav; Felsberg, Jürgen; Spížek, Jaroslav; Janata, Jiří

    2008-01-01

    Roč. 53, č. 6 (2008), s. 467-471 ISSN 0015-5632 R&D Projects: GA ČR GA204/01/1001; GA ČR GA204/05/0616 Institutional research plan: CEZ:AV0Z50200510 Keywords : acetohydroxy acid * ilvb genes * sequencing Subject RIV: EE - Microbiology, Virology Impact factor: 1.172, year: 2008

  8. Genetic differences in the Chlamydia trachomatis tryptophan synthase alpha-subunit can explain variations in serovar pathogenesis

    DEFF Research Database (Denmark)

    Shaw, A C; Christiansen, G; Roepstorff, P

    2000-01-01

    The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium, characterized by a developmental cycle that alternates between the infectious, extracellular elementary bodies and intracellular, metabolically active reticulate bodies. The cellular immune effector interferon gamma...

  9. Exocyst subunit EXO70H4 has a specific role in callose synthase secretion and silica accumulation

    Czech Academy of Sciences Publication Activity Database

    Kulich, I.; Vojtíková, Z.; Sabol, P.; Ortmannová, Jitka; Neděla, Vilém; Tihlaříková, Eva; Žárský, Viktor

    2018-01-01

    Roč. 176, č. 3 (2018), s. 2040-2051 ISSN 0032-0889 Institutional support: RVO:61389030 ; RVO:68081731 Keywords : COMPLETE PENETRATION RESISTANCE * SCANNING-ELECTRON-MICROSCOPE * POWDERY MILDEW Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 6.456, year: 2016

  10. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    DEFF Research Database (Denmark)

    Schmidt, Signe Tandrup; Foged, Camilla; Korsholm, Karen Smith

    2016-01-01

    for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce......The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens...... been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly...

  11. 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...

  12. MspA nanopores from subunit dimers.

    Directory of Open Access Journals (Sweden)

    Mikhail Pavlenok

    Full Text Available Mycobacterium smegmatis porin A (MspA forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore

  13. Glycine receptor subunits expression in the developing rat retina.

    Science.gov (United States)

    Sánchez-Chávez, Gustavo; Velázquez-Flores, Miguel Ángel; Ruiz Esparza-Garrido, Ruth; Salceda, Rocío

    2017-09-01

    Glycine receptor (GlyR) consists of two α (1-4) and three β subunits. Considerable evidence indicates that the adult retina expresses the four types of α subunits; however, the proportion of these subunits in adult and immature retina is almost unknown. In this report we have studied mRNA and the protein expression of GlyR subunits in the retina during postnatal rat development by Real-Time qRT-PCR and western blot. mRNA and protein expression indicated a gradual increase of the α1, α3, α4 and β GlyR subunits during postnatal ages tested. The mRNA β subunit showed higher expression levels (∼3 fold) than those observed for the α1 and α3 subunits. Very interestingly, the α2 GlyR subunit had the highest expression in the retina, even in the adult. These results revealed the expression of GlyR at early postnatal ages, supporting its role in retina development. In addition, our results indicated that the adult retina expressed a high proportion of the α2 subunit, suggesting the expression of monomeric and/or heteromeric receptors. A variety of studies are needed to further characterize the role of the specific subunits in both adult and immature retina. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. 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...

  15. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

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

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed ...

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

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... sequencing of sucrose synthase gene fragment from sor- ghum using primers designed at their conserved exons. MATERIALS AND METHODS. Multiple sequence alignment. Sucrose synthase gene sequences of various cereals like rice, maize, and barley were accessed from NCBI Genbank database.

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

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i...

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

    Science.gov (United States)

    Turner, Glenn W.; Croteau, Rodney

    2004-01-01

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

  19. Subunit Arrangement and Function in NMDA Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Furukawa,H.; Singh, S.; Mancusso, R.; Gouaux, E.

    2005-01-01

    Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.

  20. Sodium channel β subunits: emerging targets in channelopathies.

    Science.gov (United States)

    O'Malley, Heather A; Isom, Lori L

    2015-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Although β subunits were originally termed auxiliary, we now know that they are multifunctional signaling molecules that play roles in both excitable and nonexcitable cell types and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. Although VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target.

  1. Transcriptional regulators of Na, K-ATPase subunits

    OpenAIRE

    Zhiqin eLi; Sigrid A Langhans

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

  2. 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.

  3. Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

    Science.gov (United States)

    Ober, Dietrich; Hartmann, Thomas

    1999-01-01

    Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids. PMID:10611289

  4. Sequence of the human glycogen-associated regulatory subunit of type 1 protein phosphatase and analysis of its coding region and mRNA level in muscle from patients with NIDDM

    DEFF Research Database (Denmark)

    Chen, Y H; Hansen, L; Chen, Min

    1994-01-01

    . In previous studies, we have failed to reveal mutations in the coding regions of the muscle-specific glycogen synthase gene and the three genes that encode the catalytic subunits of protein phosphatase 1 (PP1) as frequent causes of insulin resistance. Because the glycogen-associated regulatory subunit...... of protein phosphatase 1 (PP1 G-subunit) plays a key role in the insulin stimulation of glycogen synthesis and the activity of PP1 is decreased in insulin-resistant subjects, we have now cloned the human G-subunit cDNA to search for abnormalities in the corresponding gene (designated PPP1R3 in the human...... genome nomenclature) in patients with NIDDM. The human cDNA was isolated from a skeletal muscle cDNA library and was found to encode a 126-kDa protein, which shows 73% amino acid identity to the rabbit PP1 G-subunit. The human G-subunit cDNA from 30 insulin-resistant NIDDM patients was analyzed...

  5. The F1 -ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit.

    Science.gov (United States)

    Gahura, Ondřej; Šubrtová, Karolína; Váchová, Hana; Panicucci, Brian; Fearnley, Ian M; Harbour, Michael E; Walker, John E; Zíková, Alena

    2018-02-01

    The F-ATPases (also called the F 1 F o -ATPases or ATP synthases) are multi-subunit membrane-bound molecular machines that produce ATP in bacteria and in eukaryotic mitochondria and chloroplasts. The structures and enzymic mechanisms of their F 1 -catalytic domains are highly conserved in all species investigated hitherto. However, there is evidence that the F-ATPases from the group of protozoa known as Euglenozoa have novel features. Therefore, we have isolated pure and active F 1 -ATPase from the euglenozoan parasite, Trypanosoma brucei, and characterized it. All of the usual eukaryotic subunits (α, β, γ, δ, and ε) were present in the enzyme, and, in addition, two unique features were detected. First, each of the three α-subunits in the F 1 -domain has been cleaved by proteolysis in vivo at two sites eight residues apart, producing two assembled fragments. Second, the T. brucei F 1 -ATPase has an additional subunit, called p18, present in three copies per complex. Suppression of expression of p18 affected in vitro growth of both the insect and infectious mammalian forms of T. brucei. It also reduced the levels of monomeric and multimeric F-ATPase complexes and diminished the in vivo hydrolytic activity of the enzyme significantly. These observations imply that p18 plays a role in the assembly of the F 1 domain. These unique features of the F 1 -ATPase extend the list of special characteristics of the F-ATPase from T. brucei, and also, demonstrate that the architecture of the F 1 -ATPase complex is not strictly conserved in eukaryotes. © 2017 Federation of European Biochemical Societies.

  6. Novel applications of plant polyketide synthases.

    Science.gov (United States)

    Abe, Ikuro

    2012-04-01

    The structurally and mechanistically simple type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkably diverse structures and biological activities. By exploiting the enzymes, we combined precursor-directed biosynthesis with nitrogen-containing substrates and structure-based enzyme engineering and generated unnatural, novel polyketide-alkaloid scaffolds with promising biological activities. The nucleophilic nitrogen atom and the engineered enzymes thus facilitated the formation of additional CC and CN bonds during the enzymatic transformations. The methodology will contribute to the further production of chemically and structurally divergent, unnatural natural products, as well as the rational design of novel biocatalysts with unprecedented catalytic functions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Tyrosine nitration affects thymidylate synthase properties.

    Science.gov (United States)

    Dąbrowska-Maś, Elżbieta; Frączyk, Tomasz; Ruman, Tomasz; Radziszewska, Karolina; Wilk, Piotr; Cieśla, Joanna; Zieliński, Zbigniew; Jurkiewicz, Agata; Gołos, Barbara; Wińska, Patrycja; Wałajtys-Rode, Elżbieta; Leś, Andrzej; Nizioł, Joanna; Jarmuła, Adam; Stefanowicz, Piotr; Szewczuk, Zbigniew; Rode, Wojciech

    2012-01-14

    Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

  8. 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

  9. The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid subunits

    Science.gov (United States)

    Hilario, E.; Gogarten, J. P.

    1998-01-01

    Changes in the primary and quarternary structure of vacuolar and archaeal type ATPases that accompany the prokaryote-to-eukaryote transition are analyzed. The gene encoding the vacuolar-type proteolipid of the V-ATPase from Giardia lamblia is reported. Giardia has a typical vacuolar ATPase as observed from the common motifs shared between its proteolipid subunit and other eukaryotic vacuolar ATPases, suggesting that the former enzyme works as a hydrolase in this primitive eukaryote. The phylogenetic analyses of the V-ATPase catalytic subunit and the front and back halves of the proteolipid subunit placed Giardia as the deepest branch within the eukaryotes. Our phylogenetic analysis indicated that at least two independent duplication and fusion events gave rise to the larger proteolipid type found in eukaryotes and in Methanococcus. The spatial distribution of the conserved residues among the vacuolar-type proteolipids suggest a zipper-type interaction among the transmembrane helices and surrounding subunits of the V-ATPase complex. Important residues involved in the function of the F-ATP synthase proteolipid have been replaced during evolution in the V-proteolipid, but in some cases retained in the archaeal A-ATPase. Their possible implication in the evolution of V/F/A-ATPases is discussed.

  10. Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels

    Science.gov (United States)

    Muhitch, Michael J.

    1988-01-01

    Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis. PMID:16665871

  11. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    Directory of Open Access Journals (Sweden)

    Signe Tandrup Schmidt

    2016-03-01

    Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

  12. Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αβγ are linked to hibernating state in hamsters

    Directory of Open Access Journals (Sweden)

    Alò Raffaella

    2010-09-01

    Full Text Available Abstract Background The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB and hibernating (HIB hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators. Results Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p 1 ratio (over total α subunits considered in the present study in the medial preoptic area (MPOA and arcuate nucleus (Arc of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%. Regarding the other two subunits (β and γ, elevated β3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β3 and γ2 during hibernating conditions. Conclusion We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional

  13. Role of a Highly Conserved and Catalytically Important Glutamate-49 in the Enterococcus faecalis Acetolactate Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Miyoung; Lee, Sangchoon; Cho, Junehaeng; Ryu, Seong Eon; Yoon, Moonyoung [Hanyang Univ., Seoul (Korea, Republic of); Koo, Bonsung [Rural Development Administration, Suwon (Korea, Republic of)

    2013-02-15

    Acetolactate synthase (ALS) is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylation of pyruvate and then condenses the hydroxyethyl moiety with another molecule of pyruvate to give 2-acetolactate (AL). AL is a key metabolic intermediate in various metabolic pathways of microorganisms. In addition, AL can be converted to acetoin, an important physiological metabolite that is excreted by many microorganisms. There are two types of ALSs reported in the literature, anabolic aceto-hydroxyacid synthase (AHAS) and catabolic ALSs (cALS). The anabolic AHAS is primarily found in plants, fungi, and bacteria, is involved in the biosynthesis of branched-chain amino acids (BCAAs), and contains flavin adenine dinucleotide (FAD), whereas the cALS is found only in some bacteria and is involved in the butanediol fermentation pathway. Both of the enzymes are ThDP-dependent and require a divalent metal ion for catalytic activity. Despite the similarities of the reactions catalyzed, the cALS can be distinguished from anabolic AHAS by a low optimal pH of about 6.0, FAD-independent functionality, a genetic location within the butanediol operon, and lack of a regulatory subunit. It is noteworthy that the structural and functional features of AHAS have been extensively studied, in contrast to those of cALS, for which only limited information is available. To date, the only crystal structure of cALS reported is from Klebsiella pneumonia, which revealed that the overall structure of K. pneumonia ALS is similar to that of AHAS except for the FAD binding region found in AHAS.

  14. INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

    Science.gov (United States)

    Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

    2012-01-01

    SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

  15. 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.

  16. Regulation of BK channels by auxiliary γ subunits

    Directory of Open Access Journals (Sweden)

    Jiyuan eZhang

    2014-10-01

    Full Text Available The large-conductance, calcium- and voltage-activated potassium (BK channel has the largest single-channel conductance among potassium channels and can be activated by both membrane depolarization and increases in intracellular calcium concentration. BK channels consist of pore-forming, voltage- and calcium-sensing α subunits, either alone or in association with regulatory subunits. BK channels are widely expressed in various tissues and cells including both excitable and non-excitable cells and display diverse biophysical and pharmacological characteristics. This diversity can be explained in part by posttranslational modifications and alternative splicing of the α subunit, which is encoded by a single gene, KCNMA1, as well as by tissue-specific β subunit modulation. Recently, a leucine-rich repeat-containing membrane protein, LRRC26, was found to interact with BK channels and cause an unprecedented large negative shift (~-140 mV in the voltage dependence of the BK channel activation. LRRC26 allows BK channels to open even at near-physiological calcium concentration and membrane voltage in non-excitable cells. Three LRRC26-related proteins, LRRC52, LRRC55, and LRRC38, were subsequently identified as BK channel modulators. These LRRC proteins are structurally and functionally distinct from the BK channel β subunits and were designated as γ subunits. The discovery of the γ subunits adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. Unlike BK channel β subunits, which have been intensively investigated both mechanistically and physiologically, our understanding of the γ subunits is very limited at this stage. This article reviews the structure, modulatory mechanisms, physiological relevance, and potential therapeutic implications of γ subunits as they are currently understood.

  17. 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.

  18. 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.

  19. 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.

  20. Engineering fatty acid synthases for directed polyketide production.

    Science.gov (United States)

    Gajewski, Jan; Buelens, Floris; Serdjukow, Sascha; Janßen, Melanie; Cortina, Niña; Grubmüller, Helmut; Grininger, Martin

    2017-04-01

    In this study, we engineered fatty acid synthases (FAS) for the biosynthesis of short-chain fatty acids and polyketides, guided by a combined in vitro and in silico approach. Along with exploring the synthetic capability of FAS, we aim to build a foundation for efficient protein engineering, with the specific goal of harnessing evolutionarily related megadalton-scale polyketide synthases (PKS) for the tailored production of bioactive natural compounds.

  1. 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.

  2. Understanding plant cellulose synthases through a comprehensive investigation of the cellulose synthase family sequences.

    Directory of Open Access Journals (Sweden)

    Andrew eCarroll

    2011-03-01

    Full Text Available The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized subfamilies and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair cellulose synthase function, and compare these sites to those observed in the closest cellulose synthase-like (Csl families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.

  3. The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

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    Santos Diógenes S

    2008-04-01

    Full Text Available Abstract Background The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB. The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS, molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and

  4. Homocystinuria due to cystathionine beta synthase deficiency

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    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.

  5. Type III polyketide synthases in microorganisms.

    Science.gov (United States)

    Katsuyama, Yohei; Ohnishi, Yasuo

    2012-01-01

    Type III polyketide synthases (PKSs) are simple homodimers of ketosynthases which catalyze the condensation of one to several molecules of extender substrate onto a starter substrate through iterative decarboxylative Claisen condensation reactions. Type III PKSs have been found in bacteria and fungi, as well as plants. Microbial type III PKSs, which are involved in the biosynthesis of some lipidic compounds and various secondary metabolites, have several interesting characteristics that are not shared by plant type III PKSs. Further, many compounds produced by microbial type III PKSs have significant biological functions and/or important pharmaceutical activities. Thus, studies on this class of enzymes will expand our knowledge of the biosynthetic machineries that generate natural products and generate new findings about microbial physiology. The recent development of next-generation DNA sequencing has allowed for an increase in the number of microbial genomes sequenced and the discovery of many microbial type III PKS genes. Here, we describe basic methods to study microbial type III PKSs whose genes are easy to clone. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

    Science.gov (United States)

    Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

    2014-07-10

    There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism.

  7. Nitric oxide synthase in ferret brain: localization and characterization.

    Science.gov (United States)

    Matsumoto, T.; Mitchell, J. A.; Schmidt, H. H.; Kohlhaas, K. L.; Warner, T. D.; Förstermann, U.; Murad, F.

    1992-01-01

    1. In the present study, we have investigated the distribution of nitric oxide synthase in the ferret brain. Nitric oxide synthase was determined biochemically and immunochemically. 2. In the rat brain, the highest nitric oxide synthase activity has been detected in the cerebellum. However, in the ferret brain, the highest activity was found in the striatum and the lowest in the cerebellum and cerebral cortex. The enzymatic activity was localized predominantly in the cytosolic fractions, it was dependent on NADPH and Ca2+, and inhibited by NG-nitro-L-arginine or NG-methyl-L-arginine. 3. Western blot analysis revealed that all regions of the ferret brain contained a 160 kD protein crossreacting with an antibody to nitric oxide synthase purified from the rat cerebellum, and the levels of relative intensity of staining by the antibody correlated with the distribution of nitric oxide synthase activity. 4. These results indicate that the ferret brain contains a nitric oxide synthase similar to the rat brain, but the distribution of enzymatic activity in the ferret brain differs markedly from the rat brain. Images Figure 1 PMID:1282076

  8. Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

    Science.gov (United States)

    Dienerowitz, Maria; Ilchenko, Mykhailo; Su, Bertram; Deckers-Hebestreit, Gabriele; Mayer, Günter; Henkel, Thomas; Heitkamp, Thomas; Börsch, Michael

    2016-02-01

    Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap -- a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.

  9. Selective decrease of components of the creatine kinase system and ATP synthase complex in chronic Chagas disease cardiomyopathy.

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    Priscila Camillo Teixeira

    2011-06-01

    Full Text Available BACKGROUND: Chronic Chagas disease cardiomyopathy (CCC is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC and ischemic (IC cardiomyopathies. METHODOLOGY/PRINCIPAL FINDINGS: Myocardium homogenates from CCC (N=5, IC (N=5 and IDC (N=5 patients, as well as from heart donors (N=5 were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit and muscular creatine kinase (CKM and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. CONCLUSIONS/SIGNIFICANCE: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

  10. The major antigenic membrane protein of "Candidatus Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors.

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    Luciana Galetto

    Full Text Available Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP strain, and three others as non-vectors. Interactions between a labelled (recombinant CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

  11. Inhibition of 26S protease regulatory subunit 7 (MSS1 suppresses neuroinflammation.

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    Wei Bi

    Full Text Available Recently, researchers have focused on immunosuppression induced by rifampicin. Our previous investigation found that rifampicin was neuroprotective by inhibiting the production of pro-inflammatory mediators, thereby suppressing microglial activation. In this study, using 2-dimensional gel electrophoresis (2-DE and mass spectrometry (MS, we discovered that 26S protease regulatory subunit 7 (MSS1 was decreased in rifampicin-treated microglia. Western blot analysis verified the downregulation of MSS1 expression by rifampicin. As it is indicated that the modulation of the ubiquitin-26S proteasome system (UPS with proteasome inhibitors is efficacious for the treatment of neuro-inflammatory disorders, we next hypothesized that silencing MSS1 gene expression might inhibit microglial inflammation. Using RNA interference (RNAi, we showed significant reduction of IkBα degradation and NF-kB activation. The production of lipopolysaccharides-induced pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS, nitric oxide, cyclooxygenase-2, and prostaglandin E(2 were also reduced by MSS1 gene knockdown. Taken together, our findings suggested that rifampicin inhibited microglial inflammation by suppressing MSS1 protein production. Silencing MSS1 gene expression decreased neuroinflammation. We concluded that MSS1 inhibition, in addition to anti-inflammatory rifampicin, might represent a novel mechanism for the treatment of neuroinflammatory disorders.

  12. Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

    Science.gov (United States)

    Monteiro, Karina M.; Cardoso, Mateus B.; Follmer, Cristian; da Silveira, Nádya P.; Vargas, Daiani M.; Kitajima, Elliot W.; Zaha, Arnaldo; Ferreira, Henrique B.

    2012-01-01

    Background Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the

  13. Converting S-limonene synthase to pinene or phellandrene synthases reveals the plasticity of the active site.

    Science.gov (United States)

    Xu, Jinkun; Ai, Ying; Wang, Jianhui; Xu, Jingwei; Zhang, Yongkang; Yang, Dong

    2017-05-01

    S-limonene synthase is a model monoterpene synthase that cyclizes geranyl pyrophosphate (GPP) to form S-limonene. It is a relatively specific enzyme as the majority of its products are composed of limonene. In this study, we converted it to pinene or phellandrene synthases after introducing N345A/L423A/S454A or N345I mutations. Further studies on N345 suggest the polarity of this residue plays a critical role in limonene production by stabilizing the terpinyl cation intermediate. If it is mutated to a non-polar residue, further cyclization or hydride shifts occurs so the carbocation migrates towards the pyrophosphate, leading to the production of pinene or phellandrene. On the other hand, mutant enzymes that still possess a polar residue at this position produce limonene as the major product. N345 is not the only polar residue that may stabilize the terpinyl cation because it is not strictly conserved among limonene synthases across species and there are also several other polar residues in this area. These residues could form a "polar pocket" that may collectively play this stabilizing role. Our study provides important insights into the catalytic mechanism of limonene synthases. Furthermore, it also has wider implications on the evolution of terpene synthases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Yeast dihydroxybutanone phosphate synthase, an enzyme of the riboflavin biosynthetic pathway, has a second unrelated function in expression of mitochondrial respiration.

    Science.gov (United States)

    Jin, Can; Barrientos, Antoni; Tzagoloff, Alexander

    2003-04-25

    aE280/U1 is a pet mutant of Saccharomyces cerevisiae partially deficient in cytochromes a, a3, and cytochrome b. The ability of this mutant to respire is restored by RIB3, a gene previously shown to code for 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBP synthase), an enzyme of the riboflavin biosynthetic pathway. The sequences of RIB3 from wild type and aE280/U1 indicated a single base change resulting in an A137T substitution. The alanine 137 is a conserved residue located in a cavity on the surface of the protein distant from the active site and from the subunit interaction domain involved in homodimer formation. The respiratory defect elicited by this mutation cannot be explained by a flavin insufficiency based on the following evidence: 1) growth of the aE280/U1 on respiratory substrates is not rescued by exogenous riboflavin; 2) the levels of flavin nucleotides are not significantly different in the mutant and wild type. We proposed that in addition to its known function in riboflavin synthesis, RIB3 also functions in expression of mitochondrial respiration. Restoration by riboflavin of growth of a rib3 deletion mutant on glucose but not glycerol/ethanol also supported this conclusion. An antibody against the N-terminal half of DHBP synthase was used to study its subcellular distribution. Most of the protein was localized in the cytosolic fraction, but a small fraction was detected in the mitochondrial intermembrane space.

  15. 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.

  16. 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.

  17. Cellular localization and detergent dependent oligomerization of rice allene oxide synthase-1.

    Science.gov (United States)

    Yoeun, Sereyvath; Kim, Jeong-Il; Han, Oksoo

    2015-01-01

    Allene oxide synthase-1 from Oryza sativa (OsAOS1) localizes to the chloroplast, but lacks a putative chloroplast targeting sequence typically found in dicot AOS. Here, kinetic parameters and the oligomerization state/subunit composition of OsAOS1 were characterized in vitro in the absence or presence of detergent micelles. The catalytic efficiency (k(cat)/K(m)) of OsAOS1 reached a maximum near the critical micelle concentration for polyoxyethylene 10 tridecyl ether. Native gel analysis showed that OsAOS1 exists as a multimer in the absence of detergent micelles. The multimeric form of OsAOS1 was stably cross-linked in the absence of detergents, while only monomeric OsAOS1 was detected in the presence of detergent micelles. Gel filtration analysis indicated that the oligomeric state of OsAOS1 depends strongly on the detergents and that the monomer becomes the predominant form in the presence of detergent micelles. These data suggest that the detergent-dependent oligomeric state of OsAOS1 is an important factor for the regulation of its catalytic efficiency.

  18. Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota.

    Science.gov (United States)

    Cella, R; Carbonera, D; Orsi, R; Ferri, G; Iadarola, P

    1991-06-01

    The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated Mr of 124,000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.

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

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    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.

  20. Metagenomic Survey of Potential Symbiotic Bacteria and Polyketide Synthase Genes in an Indonesian Marine Sponge

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    Nia M. Kurnia

    2017-01-01

    Full Text Available There has been emerging evidence that the bacteria associated with marine sponges are the key producers of many complex bioactive compounds. The as-yet uncultured candidate bacterial genus “Candidatus Entotheonella” of the marine sponge Theonella swinhoei from Japan have recently been recognized as the source of numerous pharmacologically relevant polyketides and modified peptides, as previously reported by the Piel group (Wilson et al. 2014. This work reported the presence of “Candidatus Entotheonella sp.” in the highly complex microbiome of an Indonesian marine sponge from Kapoposang Island, South Sulawesi. We further identified the Kapoposang sponge specimen used in this work as Rhabdastrella sp. based on the integrated morphological, histological, and cytochrome oxidase subunit I (COI gene analyses. To detect the polyketide biosynthetic machinery called type I polyketide synthase (PKS in this Indonesian Rhabdastrella sp., we amplified and cloned the ketosynthase-encoding DNA regions of approximately 700 bp from the uncultured sponge's microbiome. Further sequencing and analysis of several randomly chosen clones indicated that all of them are mostly likely involved in the biosynthesis of methyl-branched fatty acids. However, employing a PKS-targeting primer designed in this work led to the isolation of four positive clones. BlastX search and subsequent phylogenetic analysis showed that one of the positive clones, designed as RGK32, displayed high homology with ketosynthase domains of many type I PKS systems and may belong to the subclass cis-AT PKS group.

  1. Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

    Energy Technology Data Exchange (ETDEWEB)

    Min, Li; Jin, Zhongmin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang (GUW); (Maryland); (GWU); (Georgia)

    2010-01-07

    N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in L-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by L-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with L-arginine bound and in the active R-state complexed with CoA and L-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of L-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by {approx}10 {angstrom} and decreases its height by {approx}20{angstrom}. AAK dimers move 5{angstrom} outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by {approx}4{sup o}. The NAT domains rotate {approx}109{sup o} relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the L-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.

  2. A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

    Science.gov (United States)

    Du, Z Q; Wang, J Y

    2015-10-27

    Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

  3. Glycogen synthase kinase-3β regulates leucine-309 demethylation of protein phosphatase-2A via PPMT1 and PME-1.

    Science.gov (United States)

    Yao, Xiu-Qing; Li, Xia-Chun; Zhang, Xiao-Xue; Yin, Yang-Yang; Liu, Bin; Luo, Dan-Ju; Wang, Qun; Wang, Jian-Zhi; Liu, Gong-Ping

    2012-07-30

    Protein phosphatase-2A (PP2A) activity is significantly suppressed in Alzheimer's disease. We have reported that glycogen synthase kinase-3β (GSK-3β) inhibits PP2A via upregulating the phosphorylation of PP2A catalytic subunit (PP2A(C)). Here we studied the effects of GSK-3β on the inhibitory demethylation of PP2A at leucine-309 (dmL309-PP2A(C)). We found that GSK-3β regulates dmL309-PP2A(C) level by regulating PME-1 and PPMT1. Knockdown of PME-1 or PPMT1 eliminated the effects of GSK-3β on PP2A(C). GSK-3 could negatively regulate PP2A regulatory subunit protein level. We conclude that GSK-3β can inhibit PP2A by increasing the inhibitory L309-demethylation involving upregulation of PME-1 and inhibition of PPMT1. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

  5. Plasticity and evolution of (+)-3-carene synthase and (-)-sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance.

    Science.gov (United States)

    Roach, Christopher R; Hall, Dawn E; Zerbe, Philipp; Bohlmann, Jörg

    2014-08-22

    The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (-)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (-)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (-)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. 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.

  7. 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.

  8. Bornyl-diphosphate synthase from Lavandula angustifolia: A major monoterpene synthase involved in essential oil quality.

    Science.gov (United States)

    Despinasse, Yolande; Fiorucci, Sébastien; Antonczak, Serge; Moja, Sandrine; Bony, Aurélie; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis; Jullien, Frédéric

    2017-05-01

    Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221. Copyright © 2017. Published by Elsevier Ltd.

  9. Understanding Plant Cellulose Synthases through a Comprehensive Investigation of the Cellulose Synthase Family Sequences.

    Science.gov (United States)

    Carroll, Andrew; Specht, Chelsea D

    2011-01-01

    The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA) family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized clades and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair CesA function, and compare these sites to those observed in the closest cellulose synthase-like (Csl) families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.

  10. Mitochondrial permeability transition pore induction is linked to formation of the complex of ATPase C-subunit, polyhydroxybutyrate and inorganic polyphosphate.

    Science.gov (United States)

    Elustondo, P A; Nichols, M; Negoda, A; Thirumaran, A; Zakharian, E; Robertson, G S; Pavlov, E V

    Mitochondrial permeability transition pore (mPTP) opening allows free movement of ions and small molecules leading to mitochondrial membrane depolarization and ATP depletion that triggers cell death. A multi-protein complex of the mitochondrial ATP synthase has an essential role in mPTP. However, the molecular identity of the central 'pore' part of mPTP complex is not known. A highly purified fraction of mammalian mitochondria containing C-subunit of ATPase (C-subunit), calcium, inorganic polyphosphate (polyP) and polyhydroxybutyrate (PHB) forms ion channels with properties that resemble the native mPTP. We demonstrate here that amount of this channel-forming complex dramatically increases in intact mitochondria during mPTP activation. This increase is inhibited by both Cyclosporine A, an inhibitor of mPTP and Ruthenium Red, an inhibitor of the Mitochondrial Calcium Uniporter. Similar increases in the amount of complex formation occurs in areas of mouse brain damaged by ischemia-reperfusion injury. These findings suggest that calcium-induced mPTP is associated with de novo assembly of a channel comprising C-subunit, polyP and PHB.

  11. Enzymatic Assays to Investigate Acyl-Homoserine Lactone Autoinducer Synthases.

    Science.gov (United States)

    Shin, Daniel; Nagarajan, Rajesh

    2018-01-01

    Bacteria use chemical molecules called autoinducers as votes to poll their numerical strength in a colony. This polling mechanism, commonly referred to as quorum sensing, enables bacteria to build a social network and provide a collective response for fighting off common threats. In Gram-negative bacteria, AHL synthases synthesize acyl-homoserine lactone (AHL) autoinducers to turn on the expression of several virulent genes including biofilm formation, protease secretion, and toxin production. Therefore, inhibiting AHL signal synthase would limit quorum sensing and virulence. In this chapter, we describe four enzymatic methods that could be adopted to investigate a broad array of AHL synthases. The enzymatic assays described here should accelerate our mechanistic understanding of quorum-sensing signal synthesis that could pave the way for discovery of potent antivirulence compounds.

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

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    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......, the molecular mechanisms responsible for this defect remain unknown. Recently, the use of phospho-specific antibodies in human diabetic muscle has revealed hyperphosphorylation of glycogen synthase at sites not regulated by the classical insulin signaling pathway. In addition, novel approaches such as gene...

  13. Identification and characterization of a chondroitin synthase from Avibacterium paragallinarum.

    Science.gov (United States)

    Wang, Ting-Ting; Zhu, Chen-Ye; Zheng, Shuang; Meng, Cai-Cai; Wang, Tian-Tian; Meng, Dan-Hua; Li, Yi-Jun; Zhu, Hao-Miao; Wang, Feng-Shan; Sheng, Ju-Zheng

    2018-04-03

    Avibacterium paragallinarum is a Gram-negative bacterium that causes infectious coryza in chicken. It was reported that the capsule polysaccharides extracted from Av. paragallinarum genotype A contained chondroitin. Chondroitin synthase of Av. paragallinarum (ApCS) encoded by one gene within the presumed capsule biosynthesis gene cluster exhibited considerable homology to identified bacterial chondroitin synthases. Herein, we report the identification and characterization of ApCS. This enzyme indeed displays chondroitin synthase activity involved in the biosynthesis of the capsule. ApCS is a bifunctional protein catalyzing the elongation of the chondroitin chain by alternatively transferring the glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) residues from their nucleotide forms to the non-reducing ends of the saccharide chains. GlcA with a para-nitrophenyl group (pNP) could serve as the acceptor for ApCS; this enzyme shows a stringent donor tolerance when the acceptor is as small as this monosaccharide. Then, UDP-GalNAc and GlcA-pNP were injected sequentially through the chip-immobilized chondroitin synthases, and the surface plasmon resonance data demonstrated that the up-regulated extent caused by the binding of the donor is one possibly essential factor in successful polymerization reaction. This conclusion will, therefore, enhance the understanding of the mode of action of glycosyltransferase. Surprisingly, high activity at near-zero temperature as well as weak temperature dependence of this novel bacterial chondroitin synthase indicate that ApCS was a cold-active enzyme. From all accounts, ApCS becomes the fourth known bacterial chondroitin synthase, and the potential applications in artificial chondroitin sulfate and glycosaminoglycan synthetic approaches make it an attractive glycosyltransferase for further investigation.

  14. Synthesis and Physical Properties of Polyhydroxyalkanoate Polymers with Different Monomer Compositions by Recombinant Pseudomonas putida LS46 Expressing a Novel PHA SYNTHASE (PhaC116 Enzyme

    Directory of Open Access Journals (Sweden)

    Parveen K. Sharma

    2017-03-01

    Full Text Available A recombinant of Pseudomonas putida LS461 (deletion of the phaC1phaZphaC2 genes was constructed by introducing cosmid JC123 carrying a novel phaC116 gene from a metagenomic clone. The resulting strain, P. putida LS46123, was able to synthesize polyhydroxyalkanoate (PHA polymers with novel monomer compositions when cultured on glucose or free fatty acids, and accumulated PHAs from 9.24% to 27.09% of cell dry weight. The PHAs synthesized by P. putida LS46123 contained up to 50 mol % short chain length subunits (3-hydroxybutyrate and 3-hydroxyvalerate, with the remaining monomers consisting of various medium chain length subunits. The PhaC116 protein expressed by P. putida LS46123 had an amino acid sequence similarity of 45% with the PhaC1 protein of the parent strain, P. putida LS46. Predicted 3D structures of the PhaC116 proteins from P. putida LS46123 and P. putida LS46 revealed several differences in the numbers and locations of protein secondary structures. The physical and thermal properties of the novel polymers synthesized by P. putida LS46123 cultured with glucose or free fatty acids differed significantly from those produced by P. putida LS46 grown on the same substrates. PHA polymers with different subunit compositions, and hence different physical and thermal properties, can be tailor-made using novel PHA synthase for specific applications.

  15. Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

    Directory of Open Access Journals (Sweden)

    Oliveira M.C.

    1999-01-01

    Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

  16. Modes of Operation of the BKCa Channel β2 Subunit

    Science.gov (United States)

    Savalli, Nicoletta; Kondratiev, Andrei; de Quintana, Sarah Buxton; Toro, Ligia; Olcese, Riccardo

    2007-01-01

    The β2 subunit of the large conductance Ca2+- and voltage-activated K+ channel (BKCa) modulates a number of channel functions, such as the apparent Ca2+/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the β2 subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BKCa channels (hSlo), we have investigated the mechanisms of operation of the β2 subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with β2 subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the β2 subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BKCa channels coexpressed with the β2 subunit. The experimental observations are consistent with the view that the β2 subunit of BKCa channels facilitates channel activation by changing the voltage sensor equilibrium and that the β2-induced inactivation process does not follow a typical N-type mechanism. PMID:17591990

  17. Modes of operation of the BKCa channel beta2 subunit.

    Science.gov (United States)

    Savalli, Nicoletta; Kondratiev, Andrei; de Quintana, Sarah Buxton; Toro, Ligia; Olcese, Riccardo

    2007-07-01

    The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.

  18. NOX Activation by Subunit Interaction and Underlying Mechanisms in Disease.

    Science.gov (United States)

    Rastogi, Radhika; Geng, Xiaokun; Li, Fengwu; Ding, Yuchuan

    2016-01-01

    Nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX) is an enzyme complex with the sole function of producing superoxide anion and reactive oxygen species (ROS) at the expense of NADPH. Vital to the immune system as well as cellular signaling, NOX is also involved in the pathologies of a wide variety of disease states. Particularly, it is an integral player in many neurological diseases, including stroke, TBI, and neurodegenerative diseases. Pathologically, NOX produces an excessive amount of ROS that exceed the body's antioxidant ability to neutralize them, leading to oxidative stress and aberrant signaling. This prevalence makes it an attractive therapeutic target and as such, NOX inhibitors have been studied and developed to counter NOX's deleterious effects. However, recent studies of NOX have created a better understanding of the NOX complex. Comprised of independent cytosolic subunits, p47- phox , p67- phox , p40- phox and Rac , and membrane subunits, gp91- phox and p22- phox , the NOX complex requires a unique activation process through subunit interaction. Of these subunits, p47- phox plays the most important role in activation, binding and translocating the cytosolic subunits to the membrane and anchoring to p22- phox to organize the complex for NOX activation and function. Moreover, these interactions, particularly that between p47- phox and p22- phox , are dependent on phosphorylation initiated by upstream processes involving protein kinase C (PKC). This review will look at these interactions between subunits and with PKC. It will focus on the interaction involving p47- phox with p22- phox , key in bringing the cytosolic subunits to the membrane. Furthermore, the implication of these interactions as a target for NOX inhibitors such as apocynin will be discussed as a potential avenue for further investigation, in order to develop more specific NOX inhibitors based on the inhibition of NOX assembly and activation.

  19. Nicotinic acetylcholine receptor subunits in rhesus monkey retina.

    Science.gov (United States)

    Liu, Ji; McGlinn, Alice M; Fernandes, Alcides; Milam, Ann H; Strang, Christianne E; Andison, Margot E; Lindstrom, Jon M; Keyser, Kent T; Stone, Richard A

    2009-03-01

    The purpose of this study was to detect and establish the cellular localizations of nicotinic acetylcholine receptor (nAChR) subunits in Rhesus monkey retina. Retinas were dissected from the eyes of monkeys killed after unrelated experiments. RNA was extracted and analyzed by RT-PCR, using primers designed against human sequences of alpha3-alpha7, alpha9, and beta2-beta4 nAChR subunits. The RT-PCR products were separated by gel electrophoresis and sequenced. Frozen sections of postmortem fixed monkey eyes were immunolabeled with well-characterized and specific monoclonal antibodies against the alpha3, alpha4, alpha6, alpha7, beta2, or beta4 nAChR subunits and visualized with fluorescence labeling. Products of the predicted size for the alpha3-alpha7, alpha9, and beta2-beta4 nAChR subunits were detected by RT-PCR in Rhesus monkey retina. Homology between transcripts from monkey retina and human nucleotide sequences ranged from 93 to 99%. Immunohistochemical studies demonstrated that neurons in various cell layers of monkey retina expressed alpha3, alpha4, alpha7, or beta2 nAChR subunits and cells with the morphology of microglia were immunoreactive for the alpha6 or beta4 nAChR subunits. nAChR subunits are expressed in the monkey retina and localize to diverse retinal neurons as well as putative microglia. Besides mediating visual processing, retinal nAChRs may influence refractive development and ocular pathologies such as neovascularization.

  20. A bioinformatic and computational study of myosin phosphatase subunit diversity.

    Science.gov (United States)

    Dippold, Rachael P; Fisher, Steven A

    2014-08-01

    Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational algorithms to investigate the phylogenetic diversity of MP regulatory (PPP1R12A-C) and inhibitory (PPP1R14A-D) subunits. The comparison of exonic coding sequences and expression data confirmed or refuted the existence of isoforms and their tissue-specific expression in different model organisms. The comparison of intronic and exonic sequences identified potential expressional regulatory elements. As examples, smooth muscle MP regulatory subunit (PPP1R12A) is highly conserved through evolution. Its alternative exon E24 is present in fish through mammals with two invariant features: 1) a reading frame shift generating a premature termination codon and 2) a hexanucleotide sequence adjacent to the 3' splice site hypothesized to be a novel suppressor of exon splicing. A characteristic of the striated muscle MP regulatory subunit (PPP1R12B) locus is numerous and phylogenetically variable transcriptional start sites. In fish this locus only codes for the small (M21) subunit, suggesting the primordial function of this gene. Inhibitory subunits show little intragenic variability; their diversity is thought to have arisen by expansion and tissue-specific expression of different gene family members. We demonstrate differences in the regulatory landscape between smooth muscle enriched (PPP1R14A) and more ubiquitously expressed (PPP1R14B) family members and identify deeply conserved intronic sequence and predicted transcriptional cis-regulatory elements. This bioinformatic and computational study has uncovered a number of attributes of MP subunits that supports selection of ideal model organisms and testing of hypotheses regarding their physiological significance and regulated expression. Copyright © 2014 the American Physiological Society.

  1. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

    Directory of Open Access Journals (Sweden)

    Yan Ping Lim

    2016-06-01

    Full Text Available Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development.

  2. Inhibitors of Fatty Acid Synthase for Prostate Cancer

    Science.gov (United States)

    2010-05-31

    inhibitors into the clinic. fatty acid synthase, thioesterase, inhibitors, drug development U U U UU 44 USAMRMC Table of Contents ...targeting. Ursolic acid , a pentacyclic triterpenoid acid , as well as the tea polyphenols, epigallocatechin gallate (EGCG) and epicatechin gallate...2007,  6(7), 2120‐2126.  73.  Liu, Y., Tian, W., Ma, X., and Ding, W. Evaluation of  inhibition of  fatty  acid  synthase by  ursolic   acid : positive

  3. Virtual Screening of Novel Glucosamine-6-Phosphate Synthase Inhibitors.

    Science.gov (United States)

    Lather, Amit; Khatkar, Anurag; Sharma, Sunil

    2018-03-29

    Infections caused by microorganisms are the major cause of death today. The tremendous and improper use of antimicrobial agents leads to antimicrobial resistance. Various currently available antimicrobial drugs are inadequate to control the infections and lead to various adverse drug reactions. Efforts based on computer-aided drug design (CADD) can excavate a large number of databases to generate new, potent hits and minimize the requirement of time as well as money for the discovery of newer antimicrobials. Pharmaceutical sciences also have made development with advances in drug designing concepts. The current research article focuses on the study of various G-6-P synthase inhibitors from literature cited molecular database. Docking analysis was conducted and ADMET data of various molecules was evaluated by Schrodinger Glide and PreADMET software, respectively. Here, the results presented efficacy of various inhibitors towards enzyme G-6-P synthase. Docking scores, binding energy and ADMET data of various molecules showed good inhibitory potential toward G-6-P synthase as compared to standard antibiotics. This novel antimicrobial drug target G-6-P synthase has not so extensively been explored for its application in antimicrobial therapy, so the work done so far proved highly essential. This article has helped the drug researchers and scientists to intensively explore about this wonderful antimicrobial drug target. The Schrodinger, Inc. (New York, USA) software was utilized to carry out the computational calculations and docking studies. The hardware configuration was Intel® core (TM) i5-4210U CPU @ 2.40GHz, RAM memory 4.0 GB under 64-bit window operating system. The ADMET data was calculated by using the PreADMET tool (PreADMET ver. 2.0). All the computational work was completed in the Laboratory for Enzyme Inhibition Studies, Department of Pharmaceutical Sciences, M.D. University, Rohtak, INDIA. Molecular docking studies were carried out to identify the binding

  4. Probing the functional subunits of the tonoplast H+-ATPase

    International Nuclear Information System (INIS)

    Randall, S.K.; Lai, S.; Sze, H.

    1986-01-01

    The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind 14 C]-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and [ 14 C]N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial F 1 -ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H + pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site

  5. Separation and characterization of alpha-chain subunits from tilapia (Tilapia zillii) skin gelatin using ultrafiltration.

    Science.gov (United States)

    Chen, Shulin; Tang, Lanlan; Su, Wenjin; Weng, Wuyin; Osako, Kazufumi; Tanaka, Munehiko

    2015-12-01

    Alpha-chain subunits were separated from tilapia skin gelatin using ultrafiltration, and the physicochemical properties of obtained subunits were investigated. As a result, α1-subunit and α2-subunit could be successfully separated by 100 kDa MWCO regenerated cellulose membranes and 150 kDa MWCO polyethersulfone membranes, respectively. Glycine was the most dominant amino acid in both α1-subunit and α2-subunit. However, the tyrosine content was higher in α2-subunit than in α1-subunit, resulting in strong absorption near 280 nm observed in the UV absorption spectrum. Based on the DSC analysis, it was found that the glass transition temperatures of gelatin, α1-subunit and α2-subunit were 136.48 °C, 126.77 °C and 119.43 °C, respectively. Moreover, the reduced viscosity and denaturation temperature of α1-subunit were higher than those of α2-subunit, and the reduced viscosity reached the highest when α-subunits were mixed with α1/α2 ratio of approximately 2, suggesting that α1-subunit plays a more important role in the thermostability of gelatin than α2-subunit. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. 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

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

    Indian Academy of Sciences (India)

    Division of Plant Biotechnology, Institute of Forest Genetics and Tree Breeding, P.B. No. 1061 ... known as forest red gum has an extensive natural distribu- ...... mechanical stress. Plant J. 22, 495–502. Wu A., Hu J. S. and Liu J. 2009 Functional analysis of a cotton cel- lulose synthase A4 gene promoter in transgenic tobacco ...

  8. Beta-Glucan Synthase Gene Expression in Pleurotus sp

    International Nuclear Information System (INIS)

    Azhar Mohamad; Nie, H.J.

    2016-01-01

    Pleurotus sp. is a popular edible mushroom, containing various functional component, in particular, Beta-glucan. Beta-glucans is a part of glucan family of polysaccharides and supposedly contribute to medicinal and nutritional value of Pleurotus.sp. In order to understand the distribution of Beta-glucan in Pleurotus.sp, the Beta-glucan synthase gene expression was determined and compared in different part of Pleurotus, namely mycelium, stripe and cap. The Pleurotus.sp RNA was extracted using commercial kit, employing Tissuelyser ll (Qiagen, USA) to disrupt the cell walls. Then the RNA was quantified by Nano drop (Thermo Fisher, USA) and visualized using denaturing agarose gel. RNA with good OD 260.280 reading (∼2.0) was chosen and converted to cDNA. Using Laccase synthase gene as home keeping gene, Beta-glucan synthase gene expression was quantified using CFX 96 Real Time PCR detection system (Biorad, USA). Preliminary result shows that Beta-glucan synthase was relatively expressed the most in stripe, followed by mycelium and barely in cap. (author)

  9. 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 ...

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

    Indian Academy of Sciences (India)

    2014-12-26

    Dec 26, 2014 ... RESEARCH ARTICLE. Aldosterone synthase C-344T, angiotensin II type 1 receptor A1166C and 11-β hydroxysteroid dehydrogenase G534A gene polymorphisms and essential hypertension in the population of Odisha, India. MANISHA PATNAIK1,5, PALLABI PATI1, SURENDRA N. SWAIN2, MANOJ K.

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

    Indian Academy of Sciences (India)

    coumaroyl-CoA with three acetyl-CoA units, followed by folding, and initial cyclization; all catalyzed by a single enzyme, chalcone synthase (CHS). Further ring closure catalyzed by chalcone isomerase (CHI) yields naringenin. Coupling follows a ...

  12. Contribution of granule bound starch synthase in kernel modification

    African Journals Online (AJOL)

    ACSS

    The stability of both GBSS and zein proteins coupled with the use of SDS-PAGE implied that only one was more reliable and required further validation. It was clear from this study, that kernel modification was regulated by complex genetic interactions. Fairly distinct systems such as the starch synthases, zein proteins and ...

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

    OpenAIRE

    Helfrich Eric J N; Piel Jörn

    2016-01-01

    This review discusses the biosynthesis of natural products that are generated by trans AT polyketide synthases a family of catalytically versatile enzymes that represents one of the major group of proteins involved in the production of bioactive polyketides. The article includes 609 references and covers the literature from 2009 through June 2015.

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

    Science.gov (United States)

    Helfrich, Eric J N; Piel, Jörn

    2016-02-01

    This review discusses the biosynthesis of natural products that are generated by trans-AT polyketide synthases, a family of catalytically versatile enzymes that represents one of the major group of proteins involved in the production of bioactive polyketides. The article includes 609 references and covers the literature from 2009 through June 2015.

  15. 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 ...

  16. 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.

  17. 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 ...

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

    African Journals Online (AJOL)

    Isopenicillin N synthase (IPNS) related Non-haem iron-dependent oxygenases and oxidases (NHIDOX) demonstrated a striking structural conservativeness, even with low protein sequence homology. It is evident that these enzymes have an architecturally similar catalytic centre with active ligands lining the reactive pocket.

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

    Indian Academy of Sciences (India)

    This study was undertaken to investigate the association of aldosterone synthase C-344T, angiotensin II type I receptor A1166C and 11- hydroxysteroid dehydrogenase type 2 G534A polymorphisms with essential hypertension in the population of Odisha, India. A total of 246 hypertensive subjects (males, 159; females, ...

  20. 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

  1. 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

  2. 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

  3. 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 ...

  4. Functional plasticity of paralogous diterpene synthases involved in conifer defense.

    Science.gov (United States)

    Keeling, Christopher I; Weisshaar, Sabrina; Lin, Roy P C; Bohlmann, Jörg

    2008-01-22

    The diversity of terpenoid compounds produced by plants plays an important role in mediating various plant-herbivore, plant-pollinator, and plant-pathogen interactions. This diversity has resulted from gene duplication and neofunctionalization of the enzymes that synthesize and subsequently modify terpenes. Two diterpene synthases in Norway spruce (Picea abies), isopimaradiene synthase and levopimaradiene/abietadiene synthase, provide the hydrocarbon precursors for most of the diterpene resin acids found in the defensive oleoresin of conifers. Although these paralogous enzymes are 91% identical at the amino acid level, one is a single-product enzyme, whereas the other is a multiproduct enzyme that forms completely different products. We used a rational approach of homology modeling, protein sequence comparison, domain swapping, and a series of reciprocal site-directed mutagenesis to identify the specific residues that direct the different product outcomes. A one-amino acid mutation switched the levopimaradiene/abietadiene synthase into producing isopimaradiene and sandaracopimaradiene and none of its normal products. Four mutations were sufficient to reciprocally reverse the product profiles for both of these paralogous enzymes while maintaining catalytic efficiencies similar to the wild-type enzymes. This study illustrates how neofunctionalization can result from relatively minor changes in protein sequence, increasing the diversity of secondary metabolites important for conifer defense.

  5. 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 ...

  6. Cloning and expression analysis of chalcone synthase gene from ...

    Indian Academy of Sciences (India)

    Chalcone synthase (CHS) catalyses the first committed step of flavonoid biosynthetic pathway. Full-length cDNA, showing homology with plantCHS gene was isolated from leaves of C. forskohlii and named CfCHS (GenBank accession no. KF643243). Theoretical translation of CfCHS nucleotide sequence shows that it ...

  7. 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

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

    African Journals Online (AJOL)

    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 were selected ...

  9. Molecular cloning and characterization of strictosidine synthase, a ...

    African Journals Online (AJOL)

    Mitragynine is one of the most dominant indole alkaloids present in the leaves of Mitragyna speciosa, a species of Rubiaceae. This alkaloid is believed to be synthesized via condensation of the amino acid derivative, tryptamine and secologanine by the action of strictosidine synthase (STR). The cDNA clone encoding STR ...

  10. Identification of the trehalose-6-phosphate synthase gene family in ...

    Indian Academy of Sciences (India)

    2015-03-04

    Mar 4, 2015 ... Abstract. Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in plants. Trehalose contents are poten- tially modulated by trehalose-6-phosphate synthase (TPS), which is a key enzyme in the trehalose biosynthetic pathway. Using available wheat expressed sequence tag ...

  11. 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

  12. 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 ...

  13. Dynamic properties of motor proteins with two subunits

    International Nuclear Information System (INIS)

    Kolomeisky, Anatoly B; III, Hubert Phillips

    2005-01-01

    The dynamics of motor protein molecules consisting of two subunits is investigated using simple discrete stochastic models. Exact steady-state analytical expressions are obtained for velocities and dispersions for any number of intermediate states and conformations between the corresponding binding states of proteins. These models enable us to provide a detailed description and comparison of two different mechanisms of the motion of motor proteins along the linear tracks: the hand-over-hand mechanism, when the motion of subunits alternate; and the inchworm mechanism, when one subunit is always trailing another one. It is shown that the proteins in the hand-over-hand mechanism move faster and fluctuate more than the molecules in the inchworm mechanism. The effect of external forces on dynamic properties of motor proteins is also discussed. Finally, a quantitative method, based on experimental observations for single motor proteins, is proposed for distinguishing between two mechanisms of motion

  14. Novel subunit structure observed for noncooperative hemoglobin from Urechis caupo.

    Science.gov (United States)

    Kolatkar, P R; Meador, W E; Stanfield, R L; Hackert, M L

    1988-03-05

    Tetrameric hemoglobin from the "fat innkeeper" worm Urechis caupo possesses a novel subunit arrangement having an "inside out" quaternary structure in that the G/H helices are located on the outer surface of the tetramer. A 5-A resolution crystal structure reveals that although the individual subunits are beta-like, having a distinct D helix and the general myoglobin fold, the subunit contacts are very different from those previously observed for hemoglobins. Furthermore, the hemoglobin from U. caupo is also quite different from the unusual hemoglobin tetramer from clam which also has its G/H helices on the outer surface but with the hemes in close proximity through E-F helical contacts (Royer, W. E., Jr., Love, W. E., and Fenderson, F. F. (1985) Nature 316, 277-280).

  15. CSNAP Is a Stoichiometric Subunit of the COP9 Signalosome

    Directory of Open Access Journals (Sweden)

    Shelly Rozen

    2015-10-01

    Full Text Available The highly conserved COP9 signalosome (CSN complex is a key regulator of all cullin-RING-ubiquitin ligases (CRLs, the largest family of E3 ubiquitin ligases. Until now, it was accepted that the CSN is composed of eight canonical components. Here, we report the discovery of an additional integral and stoichiometric subunit that had thus far evaded detection, and we named it CSNAP (CSN acidic protein. We show that CSNAP binds CSN3, CSN5, and CSN6, and its incorporation into the CSN complex is mediated through the C-terminal region involving conserved aromatic residues. Moreover, depletion of this small protein leads to reduced proliferation and a flattened and enlarged morphology. Finally, on the basis of sequence and structural properties shared by both CSNAP and DSS1, a component of the related 19S lid proteasome complex, we propose that CSNAP, the ninth CSN subunit, is the missing paralogous subunit of DSS1.

  16. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    Directory of Open Access Journals (Sweden)

    Keegan J. Baldauf

    2015-03-01

    Full Text Available Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT, which consists of two subunits: the A subunit (CTA and the B subunit (CTB. CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

  17. Dengue vaccine: an update on recombinant subunit strategies.

    Science.gov (United States)

    Martin, J; Hermida, L

    2016-03-01

    Dengue is an increasing public health problem worldwide, with the four serotypes of the virus infecting over 390 million people annually. There is no specific treatment or antiviral drug for dengue, and prevention is largely limited to controlling the mosquito vectors or disrupting the human-vector contact. Despite the considerable progress made in recent years, an effective vaccine against the virus is not yet available. The development of a dengue vaccine has been hampered by many unique challenges, including the need to ensure the absence of vaccine-induced enhanced severity of disease. Recombinant protein subunit vaccines offer a safer alternative to other vaccine approaches. Several subunit vaccine candidates are presently under development, based on different structural and non-structural proteins of the virus. Novel adjuvants or immunopotentiating strategies are also being tested to improve their immunogenicity. This review summarizes the current status and development trends of subunit dengue vaccines.

  18. Isolation and properties of a succinylated subunit of human thyroglobulin.

    Science.gov (United States)

    Smith, D J; Shulman, S

    1978-03-01

    A subunit of succinylated (40:1 molar ratio, succinic anhydride:lysine residues) human thyroglobulin (Tg) was prepared by gel filtration on 6% and 4% agarose. This subunit (S-8) had a sdegrees20, w of 7.6S, Ddegrees20, w of 2.96 x 10(7) and an equilibrium molecular weight of 165,000 after correction for bound succinic anhydride (SA). The S-8 component was suggested to be a quarter unit of intact Tg. The S-8 and intact Tg had nearly identical amino acid compositions with differences only in glutamic acid, glycine and proline. The iodine content of both components was similar. The succinylated S-8 subunit, as well as material which sedimented with a value of 2S or less, retained some but not all heteroantigenic and autoantigenic sites in immunodiffusion and inhibition of passive hemagglutination.

  19. Progesterone modulation of transmembrane helix-helix interactions between the α-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane

    Directory of Open Access Journals (Sweden)

    Askari Amir

    2010-05-01

    Full Text Available Abstract Background Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the α-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the α1-subunit. Results Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020 successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME, can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the α1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. Conclusion We propose that progesterone binding to the first external loop of the α1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions.

  20. Partial agonists and subunit selectivity at NMDA receptors

    DEFF Research Database (Denmark)

    Risgaard, Rune; Hansen, Kasper Bø; Clausen, Rasmus Prætorius

    2010-01-01

    -methyl-D-aspartic acid (NMDA) receptor class. Development of these ligands seems to be a difficult task because of the conserved region in the binding site of the NMDA receptor subunits. A few scaffolds have been developed showing potential to differentiate between the NMDA receptors.......Subunit-selective ligands for glutamate receptors remains an area of interest as glutamate is the major excitatory neurotransmitter in the brain and involved in a number of diseased states in the central nervous system (CNS). Few subtype-selective ligands are known, especially among the N...

  1. 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.

  2. Pseudouridine synthase 1: a site-specific synthase without strict sequence recognition requirements

    Science.gov (United States)

    Sibert, Bryan S.; Patton, Jeffrey R.

    2012-01-01

    Pseudouridine synthase 1 (Pus1p) is an unusual site-specific modification enzyme in that it can modify a number of positions in tRNAs and can recognize several other types of RNA. No consensus recognition sequence or structure has been identified for Pus1p. Human Pus1p was used to determine which structural or sequence elements of human tRNASer are necessary for pseudouridine (Ψ) formation at position 28 in the anticodon stem-loop (ASL). Some point mutations in the ASL stem of tRNASer had significant effects on the levels of modification and compensatory mutation, to reform the base pair, restored a wild-type level of Ψ formation. Deletion analysis showed that the tRNASer TΨC stem-loop was a determinant for modification in the ASL. A mini-substrate composed of the ASL and TΨC stem-loop exhibited significant Ψ formation at position 28 and a number of mutants were tested. Substantial base pairing in the ASL stem (3 out of 5 bp) is required, but the sequence of the TΨC loop is not required for modification. When all nucleotides in the ASL stem other than U28 were changed in a single mutant, but base pairing was retained, a near wild-type level of modification was observed. PMID:22102571

  3. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

    Science.gov (United States)

    Plecitá-Hlavatá, Lydie; Engstová, Hana; Alán, Lukáš; Špaček, Tomáš; Dlasková, Andrea; Smolková, Katarína; Špačková, Jitka; Tauber, Jan; Strádalová, Vendula; Malínský, Jan; Lessard, Mark; Bewersdorf, Joerg; Ježek, Petr

    2016-05-01

    The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J

  4. 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.

  5. Tetrahydrobiopterin, but not L-arginine, decreases NO synthase uncoupling in cells expressing high levels of endothelial NO synthase

    NARCIS (Netherlands)

    Bevers, LM; Braam, B; Post, JA; van Zonneveld, AJ; Rabelink, TJ; Koomans, HA; Verhaar, MC; Joles, JA

    Endothelial NO synthase (eNOS) produces superoxide when depleted of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-arginine by uncoupling the electron flow from NO production. High expression of eNOS has been reported to have beneficial effects in atherosclerotic arteries after relatively short

  6. Discovery and characterization of a novel lachrymatory factor synthase in Petiveria alliacea and its influence on alliinase-mediated formation of biologically active organosulfur compounds.

    Science.gov (United States)

    Musah, Rabi A; He, Quan; Kubec, Roman

    2009-11-01

    A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is a heterotetrameric glycoprotein comprised of two alpha-subunits (68.8 kD each), one gamma-subunit (22.5 kD), and one delta-subunit (11.9 kD). The two alpha-subunits are glycosylated and connected by a disulfide bridge. The LFS has an isoelectric point of 5.2. It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-l-cysteine sulfoxide (petiveriin). Depending on its concentration relative to that of P. alliacea alliinase, the LFS sequesters, to varying degrees, the sulfenic acid intermediate formed by alliinase-mediated breakdown of petiveriin. At LFS:alliinase of 5:1, LFS sequesters all of the sulfenic acid formed by alliinase action on petiveriin, and converts it entirely to (Z)-phenylmethanethial S-oxide. However, starting at LFS:alliinase of 5:2, the LFS is unable to sequester all of the sulfenic acid produced by the alliinase, with the result that sulfenic acid that escapes the action of the LFS condenses with loss of water to form S-benzyl phenylmethanethiosulfinate (petivericin). The results show that the LFS and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the LFS acts. The results also show that the LFS modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the LFS and the alliinase. These observations suggest that manipulation of LFS-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules.

  7. How the nucleus and mitochondria communicate in energy production during stress: nuclear MtATP6, an early-stress responsive gene, regulates the mitochondrial F₁F₀-ATP synthase complex.

    Science.gov (United States)

    Moghadam, Ali Asghar; Ebrahimie, Eemaeil; Taghavi, Seyed Mohsen; Niazi, Ali; Babgohari, Mahbobeh Zamani; Deihimi, Tahereh; Djavaheri, Mohammad; Ramezani, Amin

    2013-07-01

    A small number of stress-responsive genes, such as those of the mitochondrial F1F0-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F0 part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F1F0-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.

  8. C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7.

    OpenAIRE

    Sadhale, P P; Woychik, N A

    1994-01-01

    We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often ...

  9. Subunit compensation and plasticity of synaptic GABAA receptors induced by ethanol in α4 subunit knockout mice

    Directory of Open Access Journals (Sweden)

    Asha eSuryanarayanan

    2011-09-01

    Full Text Available There is considerable evidence that ethanol (EtOH potentiates γ-aminobutyric acid type A receptor (GABAAR action, but only GABAARs containing δ subunits appear sensitive to low mM EtOH. The α4 and δ subunits co-assemble into GABAARs which are relatively highly expressed at extrasynaptic locations in the dentate gyrus where they mediate tonic inhibition. We previously demonstrated reversible- and time-dependent changes in GABAAR function and subunit composition in rats after single-dose EtOH intoxication. We concluded that early tolerance to EtOH occurs by over-activation and subsequent internalization of EtOH-sensitive extrasynaptic α4βδ-GABAARs. Based on this hypothesis, any highly EtOH-sensitive GABAARs should be subject to internalization following exposure to suitably high EtOH doses. To test this, we studied the GABAARs in mice with a global deletion of the α4 subunit (KO. The dentate granule cells (DGCs of these mice exhibited greatly reduced tonic currents and greatly reduced potentiation by acutely applied EtOH, whereas synaptic currents showed heightened sensitivity to low EtOH concentrations. The hippocampus of naive KO mice showed reduced δ subunit protein levels, but increased α2, and γ2 levels compared to wild-type (WT controls, suggesting at least partial compensation by these subunits in synaptic, highly EtOH-sensitive GABAARs of KO mice. In WT mice, cross-linking and Western blot analysis at 1 h after an EtOH challenge (3.5 g/kg, i.p. revealed increased intracellular fraction of the α1, α4 and δ, but not α2, α5 or γ2 subunits. By contrast, we observed significant internalization of α1, α2, δ, and γ2 subunits after a similar EtOH challenge in KO mice. Synaptic currents from naïve KO mice were more sensitive to potentiation by zolpidem (0.3 μM, requiring α1/α2, inactive at α4/5 GABAARs than those from naïve WT mice. At 1 h after EtOH, synaptic currents of WT mice were unchanged, whereas those of KO mice

  10. 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.

  11. α-4 subunit of nicotinic acetylcholine receptor polymorphisms exhibit ...

    African Journals Online (AJOL)

    Background: Smoking behavior is influenced by both genetic and environmental factors. Nicotine is the major addictive substance in cigarettes. Nicotinic acetylcholine receptors (nAChRs) are thought to play an important role in nicotine addiction of smokers. One of the genes, α-4 subunit of nicotinic acetylcholine receptor ...

  12. Thermostable Subunit Vaccines for Pulmonary Delivery: How Close Are We?

    DEFF Research Database (Denmark)

    Foged, Camilla

    2016-01-01

    , such as influenza, tuberculosis, and Ebola, for which no good universal vaccines exist. At least two pharmaceutical improvements are expected to help filling this gap: i) The development of thermostable vaccine dosage forms, and ii) the full exploitation of the adjuvant technology for subunit vaccines to potentiate...

  13. Allelic variation of HMW glutenin subunits of Ethiopian bread wheat ...

    African Journals Online (AJOL)

    There were highly significant differences between genotypes and banding patterns for the SDS-sedimentation test, mixograph development time, alveograph strength and loaf volume; but not for protein content. The frequency of subunits 5+10 among genotypes was 73%. The accumulation of high scoring alleles in our ...

  14. Editing modifies the GABA(A) receptor subunit alpha3

    DEFF Research Database (Denmark)

    Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David

    2007-01-01

    to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor...

  15. Development of a Subunit Vaccine for Contagious Bovine ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Their work has set the stage for commercial development of a sub-unit vaccine. Field testing and production This scale-up project will ... The research team will conduct a large field trial involving 1,500 commercial cattle to assess the vaccine's safety and efficacy. They will define the regulatory pathway for the vaccine to be ...

  16. weight glutenin subunits and waxy alleles on dough-mix

    Indian Academy of Sciences (India)

    weight glutenin subunits and waxy alleles on dough-mixing properties in common wheat. Zhiying Deng, Shuna Hu, Feifei Zheng, Junnan Chen, Xinye Zhang, Jiansheng Chen, Cailing Sun,. Yongxiang Zhang, Shouyi Wang and Jichun Tian. J. Genet. 92, 69–79. Table 1. The data of the mixing properties of the RIL population ...

  17. Evaluation of subunit vaccines against feline immunodeficiency virus infection

    NARCIS (Netherlands)

    Horzinek, M.C.; Verschoor, E.J.; Willemse, M.J.; Stam, J.G.; Vliet, A.L.W. van; Pouwels, H.; Chalmers, S.K.; Sondermeijer, P.J.; Hesselink, W.; Ronde, A. de

    1996-01-01

    Subunit vaccines prepared against feline immunodeficiency virus (FIV) infection were evaluated in two trials. First, cats were immunized with bacterial expression products of an envelope fragment that contained the V3 neutralization domain of the FIV surface protein fused to either galactokinase

  18. Characterization of low-molecular-weight glutenin subunit genes of ...

    Indian Academy of Sciences (India)

    2015-09-16

    Sep 16, 2015 ... species. Materials and methods. Plant materials. A total of 13 accessions from five species of Sitopsis were used for gene cloning (table 1 in electronic supplementary. Keywords. wheat; Aegilops; low-molecular-weight glutenin subunits; gene cloning; B genome; Triticum aestivum. Journal of Genetics, Vol.

  19. Purification of subunits from human chorionic gonadotropin and radioimmunoassay

    International Nuclear Information System (INIS)

    Okumura, Hajime; Sudo, Tadamitsu; Fujisawa, Munetoshi; Namba, Shuichi; Matsushima, Sanae

    1976-01-01

    A crude hCG with an activity of about 3,000 IU per mg was purified to 10,000-15,000 IU per mg of dry weight using Amberlite CG-50 chromatography combined with DEAE-Sephadex A-50 and Sephadex G-75. The alpha and beta subunits of hCG were prepared by urea-treatment of the hormone and were isolated by DEAE-Sephadex A-50 chromatography. Further purification of the subunits was achieved by gel filtration on a Sephadex G-57 column. For radioimmunoassay, hCG was iodinated by the DMSO-chloramine T method. Iodination of hCG with non-radioactive iodine revealed that the addition of DMSO to the iodination mixture seemed to reduce the iodination damage to the antigenic activity of the hormone. The hCG iodinated with non-radioactive iodine (accomplished by the DMSO-chloramine T method) was 1.5 times more immunoreactive in the hCG radioimmunoassay then the hCG iodinated by the usual chloramine T method. The radioimmunoassay of the hCG-beta subunit developed in our laboratory was satisfactory with respect to specificity. The hLH, hFSH, hTSH and hCG-alpha subunits tested cross-reacted very poorly in our assay system. Desialylated-hCG and subunits, in which the biologic potency was almost zero, also exhibited decreased immunoreactivity, about 30% of the native hormone with grossly unimpaired parallelism in their respective homologous radioimmunoassays. The concentrations of hCG and the subunits were determined on human sera from pregnant patients. The hCG levels reached to a peak at the first trimester of the pregnancy, but the hCG-beta subunits varied poorly in their concentrations throughout the periods of pregnancy. The hCG-alpha levels, on the other hand, showed two distinct peaks, in the early period and at the term of pregnancy. (J.P.N.)

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

    Science.gov (United States)

    2016-01-01

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

  1. Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study.

    Science.gov (United States)

    Bharadwaj, Vivek S; Dean, Anthony M; Maupin, C Mark

    2013-08-21

    The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.

  2. Molecular characterization of Als1, an acetohydroxyacid synthase mutation conferring resistance to sulfonylurea herbicides in soybean.

    Science.gov (United States)

    Ghio, Cecilia; Ramos, María Laura; Altieri, Emiliano; Bulos, Mariano; Sala, Carlos A

    2013-12-01

    The AHAS gene family in soybean was characterized. The locus Als1 for sulfonylurea resistance was mapped and the resistant allele was characterized at the molecular level. Sulfonylurea (SU) resistance in soybean is controlled by Als1, a semi-dominant allele obtained by EMS mutagenesis over the cultivar Williams 82. The overall objective of this research was to map Als1 in the soybean genome and to determine the nucleotidic changes conferring resistance to SU. Four nucleotide sequences (GmAhas1-4) showing high homology with the Arabidopsis thaliana acetohydroxyacid synthase (AHAS, EC 4.1.3.18) gene sequence were identified by in silico analysis, PCR-amplified from the SU-resistant line BTK323STS and sequenced. Expression analysis showed that GmAhas1, located on chromosome 4 by in silico analysis, is the most expressed sequence in true leaves. F2:3 families derived from the cross between susceptible and resistant lines were evaluated for SU resistance. Mapping results indicate that the locus als1 is located on chromosome 4. Sequence comparison of GmAhas1 between BTK323STS and Williams 82 showed a single nucleotide change from cytosine to thymine at position 532. This transversion generates an amino acid change from proline to serine at position 197 (A. thaliana nomenclature) of the AHAS catalytic subunit. An allele-specific marker developed for the GmAhas1 mutant sequence cosegregated with SU resistance in the F2 population. Taking together, the mapping, expression and sequencing results indicate that the GmAhas1 sequence corresponds to the Als1 gene sequence controlling SU resistance in soybean. The molecular breeding tools described herein create the basis to speed up the identification of new mutations in soybean AHAS leading to enhanced levels of resistance to SU or to other families of AHAS inhibitor herbicides.

  3. Complexes with mixed primary and secondary cellulose synthases are functional in Arabidopsis thaliana plants

    Energy Technology Data Exchange (ETDEWEB)

    Carroll, Andrew; Mansoori, N; Li, Shundai; Lei, Lei; Vernhettes, Samantha; Visser, Richard G. F.; Somerville, Chris R; Gu, Ying; Trindade, Luisa M.

    2012-10-01

    In higher plants, cellulose is synthesized by so-called rosette protein complexes with cellulose synthases (CESAs) as catalytic subunits of the complex. The CESAs are divided into two distinct families, three of which are thought to be specialized for the primary cell wall and three for the secondary cell wall. In this article, the potential of primary and secondary CESAs forming a functional rosette complex has been investigated. The membrane-based yeast two-hybrid and biomolecular fluorescence systems were used to assess the interactions between three primary (CESA1, CESA3, CESA6), and three secondary (CESA4, CESA7, CESA8) Arabidopsis (Arabidopsis thaliana) CESAs. The results showed that all primary CESAs can physically interact both in vitro and in planta with all secondary CESAs. Although CESAs are broadly capable of interacting in pairwise combinations, they are not all able to form functional complexes in planta. Analysis of transgenic lines showed that CESA7 can partially rescue defects in the primary cell wall biosynthesis in a weak cesa3 mutant. Green fluorescent protein-CESA protein fusions revealed that when CESA3 was replaced by CESA7 in the primary rosette, the velocity of the mixed complexes was slightly faster than the native primary complexes. CESA1 in turn can partly rescue defects in secondary cell wall biosynthesis in a cesa8ko mutant, resulting in an increase of cellulose content relative to cesa8ko. These results demonstrate that sufficient parallels exist between the primary and secondary complexes for cross-functionality and open the possibility that mixed complexes of primary and secondary CESAs may occur at particular times.

  4. 7-ketocholesterol inhibits Na,K-ATPase activity by decreasing expression of its α1-subunit and membrane fluidity in human endothelial cells.

    Science.gov (United States)

    Duran, M J; Pierre, S V; Lesnik, P; Pieroni, G; Bourdeaux, M; Dignat-Georges, F; Sampol, J; Maixent, J M

    2010-11-09

    As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 μg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.

  5. Effect of high and low molecular weight glutenin subunits, and subunits of gliadin on physicochemical parameters of different wheat genotypes

    Directory of Open Access Journals (Sweden)

    Mariana Souza Costa

    2013-02-01

    Full Text Available Identification of functional properties of wheat flour by specific tests allows genotypes with appropriate characteristics to be selected for specific industrial uses. The objective of wheat breeding programs is to improve the quality of germplasm bank in order to be able to develop wheat with suitable gluten strength and extensibility for bread making. The aim of this study was to evaluate 16 wheat genotypes by correlating both glutenin subunits of high and low molecular weight and gliadin subunits with the physicochemical characteristics of the grain. Protein content, sedimentation volume, sedimentation index, and falling number values were analyzed after the grains were milled. Hectoliter weight and mass of 1000 seeds were also determined. The glutenin and gliadin subunits were separated using polyacrylamide gel in the presence of sodium dodecyl sulfate. The data were evaluated using variance analysis, Pearson's correlation, principal component analysis, and cluster analysis. The IPR 85, IPR Catuara TM, T 091015, and T 091069 genotypes stood out from the others, which indicate their possibly superior grain quality with higher sedimentation volume, higher sedimentation index, and higher mass of 1000 seeds; these genotypes possessed the subunits 1 (Glu-A1, 5 + 10 (Glu-D1, c (Glu-A3, and b (Glu-B3, with exception of T 091069 genotype that possessed the g allele instead of b in the Glu-B3.

  6. In vitro biochemical characterization of all barley endosperm starch synthases

    DEFF Research Database (Denmark)

    Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian

    2016-01-01

    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...... 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...

  7. 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.

  8. 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.

  9. 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...

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

    OpenAIRE

    Kim Jinki; Yi Gwan-Su

    2012-01-01

    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 polyke...

  11. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

    OpenAIRE

    Yan Ping Lim; Maybelle K. Go; Wen Shan Yew

    2016-01-01

    Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs) using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalc...

  12. ASMPKS: an analysis system for modular polyketide synthases

    OpenAIRE

    Kong Eun-Bae; Tae Hongseok; Park Kiejung

    2007-01-01

    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(PKS)s, 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 rapi...

  13. Deficiency of mitochondrial ATP synthase of nuclear genetic origin

    Czech Academy of Sciences Publication Activity Database

    Sperl, W.; Ješina, Pavel; Zeman, J.; Mayr, J. A.; DeMeirleir, L.; VanCoster, R.; Pícková, Andrea; Hansíková, H.; Houšťková, H.; Krejčík, Zdeněk; Koch, J.; Smet, J.; Muss, W.; Holme, E.; Houštěk, Josef

    2006-01-01

    Roč. 16, č. 11 (2006), s. 821-829 ISSN 0960-8966 R&D Projects: GA MZd(CZ) NR7790; GA MŠk(CZ) 1M0520 Grant - others:CZ-AT(CZ) 6-06-3 Institutional research plan: CEZ:AV0Z50110509 Keywords : mitochondria * ATP synthase * disease Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.615, year: 2006

  14. Trypanosoma brucei solanesyl-diphosphate synthase localizes to the mitochondrion

    Czech Academy of Sciences Publication Activity Database

    Lai, D.-H.; Bontempi, E. J.; Lukeš, Julius

    2012-01-01

    Roč. 183, č. 2 (2012), s. 189-192 ISSN 0166-6851 R&D Projects: GA ČR(CZ) GAP305/11/2179 Institutional support: RVO:60077344 Keywords : Trypanosoma brucei * Sleeping sickness * Ubiquinone * Solanesyl-diphosphate synthase * Digitonin permeabilization * In situ tagging Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.734, year: 2012 http://www.sciencedirect.com/science/article/pii/S0166685112000539

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

    Energy Technology Data Exchange (ETDEWEB)

    Pichersky, E.

    1998-12-15

    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. 5 figs.

  16. 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

  17. Nitric oxide synthase isoforms in spontaneous and salt hypertension

    Czech Academy of Sciences Publication Activity Database

    Hojná, Silvie; Kuneš, Jaroslav; Zicha, Josef

    2007-01-01

    Roč. 25, Suppl. 2 (2007), S 338-S 338 ISSN 0263-6352. [European Meeting on Hypertension /17./. 15.06.2007-19.06.2007, Milan] R&D Projects: GA MŠk(CZ) 1M0510 Institutional research plan: CEZ:AV0Z50110509 Keywords : nitric oxide synthase isoforms * spontaneous and salt hypertension Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

  18. The cellulose synthase superfamily in fully sequenced plants and algae

    Directory of Open Access Journals (Sweden)

    Xu Ying

    2009-07-01

    Full Text Available Abstract Background The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl families and one cellulose synthase (CesA family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. Results A single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ, providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome. Conclusion Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.

  19. Iterative polyketide biosynthesis by modular polyketide synthases in bacteria.

    Science.gov (United States)

    Chen, Haotong; Du, Liangcheng

    2016-01-01

    Modular polyketide synthases (type I PKSs) in bacteria are responsible for synthesizing a significant percentage of bioactive natural products. This group of synthases has a characteristic modular organization, and each module within a PKS carries out one cycle of polyketide chain elongation; thus each module is non-iterative in function. It was possible to predict the basic structure of a polyketide product from the module organization of the PKSs, since there generally existed a co-linearity between the number of modules and the number of chain elongations. However, more and more bacterial modular PKSs fail to conform to the canonical rules, and a particularly noteworthy group of non-canonical PKSs is the bacterial iterative type I PKSs. This review covers recent examples of iteratively used modular PKSs in bacteria. These non-canonical PKSs give rise to a large array of natural products with impressive structural diversity. The molecular mechanism behind the iterations is often unclear, presenting a new challenge to the rational engineering of these PKSs with the goal of generating new natural products. Structural elucidation of these synthase complexes and better understanding of potential PKS-PKS interactions as well as PKS-substrate recognition may provide new prospects and inspirations for the discovery and engineering of new bioactive polyketides.

  20. Regulation of expression, activity and localization of fungal chitin synthases

    Science.gov (United States)

    Rogg, Luise E.; Fortwendel, Jarrod R.; Juvvadi, Praveen R.; Steinbach, William J.

    2013-01-01

    The fungal cell wall represents an attractive target for pharmacologic inhibition, as many of the components are fungal-specific. Though targeted inhibition of β-glucan synthesis is effective treatment for certain fungal infections, the ability of the cell wall to dynamically compensate via the cell wall integrity pathway may limit overall efficacy. To date, chitin synthesis inhibitors have not been successfully deployed in the clinical setting. Fungal chitin synthesis is a complex and highly regulated process. Regulation of chitin synthesis occurs on multiple levels, thus targeting of these regulatory pathways may represent an exciting alternative approach. A variety of signaling pathways have been implicated in chitin synthase regulation, at both transcriptional and post-transcriptional levels. Recent research suggests that localization of chitin synthases likely represents a major regulatory mechanism. However, much of the regulatory machinery is not necessarily shared among different chitin synthases. Thus, an in depth understanding of the precise roles of each protein in cell wall maintenance and repair will be essential to identifying the most likely therapeutic targets. PMID:21526913

  1. The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

    Science.gov (United States)

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations. PMID:24069347

  2. Longevity in vivo of primary cell wall cellulose synthases.

    Science.gov (United States)

    Hill, Joseph Lee; Josephs, Cooper; Barnes, William J; Anderson, Charles T; Tien, Ming

    2018-02-01

    Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell. Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

  3. Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge

    CSIR Research Space (South Africa)

    Mallajosyula, JK

    2014-03-01

    Full Text Available Recombinant subunit vaccines are an efficient strategy to meet the demands of a possible influenza pandemic, because of rapid and scalable production. However, vaccines made from recombinant hemagglutinin (HA) subunit protein are often of low...

  4. Subunit composition and chromophore content of R-phycoerythrin from Porphyra haitanensis

    Science.gov (United States)

    Gao, Hong-Feng; Ji, Ming-Hou; Cao, Wen-Da

    1996-03-01

    R-phycoerythrin from Porphyra haitanensis exists in two aggregation states with different molecular weights. A more highly aggregated form, RPE I, was chromatographed on Bio-Rex 70 column with urea solution (pH 3.0) as eluent, and the molecular weights of the 3 subunits (α, β, γ) obtained were determined on SDS-PAGE at 18000, 19200 and 30000, respectively. α subunit carried two phycoerythrobilin (PEB); β subunit, three PEB and one phycourobilin (PUB); γ subunit, one PEB and three PUB chromophores. The molar ratio of α, β, and γ subunits of RPE I was 6: 6: 1, and their subunit composition was confired to be (αβ)6γ on account of the molecular weight of RPE I, 232000. A lower aggregated form, RPE II, contained α and β subunits similar to those of RPE I, but its subunit composition was the (αβ) monomer of RPE.

  5. Regulation of glycogen synthase kinase-3{beta} (GSK-3{beta}) after ionizing radiation; Regulation der Glykogen Synthase Kinase-3{beta} (GSK-3{beta}) nach ionisierender Strahlung

    Energy Technology Data Exchange (ETDEWEB)

    Boehme, K.A.

    2006-12-15

    Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3{beta} by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3{beta} at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3{beta} serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKB{beta}, is required for phosphorylation of GSK- 3{beta} at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3{beta} at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3{beta} in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer

  6. 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.

  7. 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.

  8. 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

  9. 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 Brandt; Von Wettstein-Knowles, Penny

    2007-01-01

    Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual...... 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...... degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic...

  10. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function

    Science.gov (United States)

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K.; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V.; Craig, Patricio O.; Millar, Neil S.; Bouzat, Cecilia

    2017-01-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits. PMID:28069778

  11. Differential expression of G protein alpha and ß subunit genes during development of Phytophthora infestans

    NARCIS (Netherlands)

    Laxalt, A.M.; Latijnhouwers, M.; Hulten, van M.; Govers, F.

    2002-01-01

    A G protein subunit gene (pigpa1) and a G protein subunit gene (pigpb1) were isolated from the oomycete Phytophthora infestans, the causal agent of potato late blight. Heterotrimeric G proteins are evolutionary conserved GTP-binding proteins that are composed of ,, and subunits and participate in

  12. 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.

  13. A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria).

    Science.gov (United States)

    Formighieri, Cinzia; Melis, Anastasios

    2015-11-01

    Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow Kcat (low Vmax) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g(-1) dry cell weight (dcw) versus the 0.03 mg g(-1)dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  14. Testing experimental subunit furunculosis vaccines for rainbow trout

    DEFF Research Database (Denmark)

    Marana, Moonika H.; Chettri, Jiwan Kumar; Skov, Jakob

    2016-01-01

    Aeromonas salmonicida subsp. salmonicida (AS) is the etiological agent of typical furunculosis in salmonid fish. The disease causes bacterial septicemia and is a major fish health problem in salmonid aquaculture worldwide, inducing high morbidity and mortality. In this study we vaccinated rainbow...... trout with subunit vaccines containing protein antigens that were selected based on an in silico antigen discovery approach. Thus, the proteome of AS strain A449 was analyzed by an antigen discovery platform and its proteins consequently ranked by their predicted ability to evoke protective immune...... response against AS. Fourteen proteins were prepared in 3 different experimental subunit vaccine combinations and used to vaccinate rainbow trout by intraperitoneal (i.p.) injection. We tested the proteins for their ability to elicit antibody production and protection. Thus, fish were exposed to virulent...

  15. Architecture of the large subunit of the mammalian mitochondrial ribosome.

    Science.gov (United States)

    Greber, Basil J; Boehringer, Daniel; Leitner, Alexander; Bieri, Philipp; Voigts-Hoffmann, Felix; Erzberger, Jan P; Leibundgut, Marc; Aebersold, Ruedi; Ban, Nenad

    2014-01-23

    Mitochondrial ribosomes synthesize a number of highly hydrophobic proteins encoded on the genome of mitochondria, the organelles in eukaryotic cells that are responsible for energy conversion by oxidative phosphorylation. The ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution, including ribosomal RNA shortening and acquisition of mitochondria-specific ribosomal proteins. Here we present the three-dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo-electron microscopy at 4.9 Å resolution. The structure, combined with data from chemical crosslinking and mass spectrometry experiments, reveals the unique features of the 39S subunit at near-atomic resolution and provides detailed insight into the architecture of the polypeptide exit site. This region of the mitochondrial ribosome has been considerably remodelled compared to its bacterial counterpart, providing a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain.

  16. Protein kinase A regulatory subunit distribution in medulloblastoma

    International Nuclear Information System (INIS)

    Mucignat-Caretta, Carla; Denaro, Luca; Redaelli, Marco; D'Avella, Domenico; Caretta, Antonio

    2010-01-01

    Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

  17. ASIC subunit ratio and differential surface trafficking in the brain.

    Science.gov (United States)

    Wu, Junjun; Xu, Yuanyuan; Jiang, Yu-Qing; Xu, Jiangping; Hu, Youjia; Zha, Xiang-ming

    2016-01-08

    Acid-sensing ion channels (ASICs) are key mediators of acidosis-induced responses in neurons. However, little is known about the relative abundance of different ASIC subunits in the brain. Such data are fundamental for interpreting the relative contribution of ASIC1a homomers and 1a/2 heteromers to acid signaling, and essential for designing therapeutic interventions to target these channels. We used a simple biochemical approach and semi-quantitatively determined the molar ratio of ASIC1a and 2 subunits in mouse brain. Further, we investigated differential surface trafficking of ASIC1a, ASIC2a, and ASIC2b. ASIC1a subunits outnumber the sum of ASIC2a and ASIC2b. There is a region-specific variation in ASIC2a and 2b expression, with cerebellum and striatum expressing predominantly 2b and 2a, respectively. Further, we performed surface biotinylation and found that surface ASIC1a and ASIC2a ratio correlates with their total expression. In contrast, ASIC2b exhibits little surface presence in the brain. This result is consistent with increased co-localization of ASIC2b with an ER marker in 3T3 cells. Our data are the first semi-quantitative determination of relative subunit ratio of various ASICs in the brain. The differential surface trafficking of ASICs suggests that the main functional ASICs in the brain are ASIC1a homomers and 1a/2a heteromers. This finding provides important insights into the relative contribution of various ASIC complexes to acid signaling in neurons.

  18. Energetics of subunit assembly and ligand binding in human hemoglobin.

    OpenAIRE

    Ackers, G K

    1980-01-01

    An extensive and self-consistent set of thermodynamic properties has recently been established for the coupled processes of subunit assembly and ligand binding (oxygen and protons) in human hemoglobin. The resulting thermodynamic values permit a consideration of the possible sources of energetic terms accounting for stability of the tetrameric quaternary structures at different stages of ligation, and of the possible sources of cooperative energy. The analysis indicates that: (a) The change i...

  19. Radioimmunoassay of TSH subunits in thyroid diseases and endocrine opthalmopahty

    International Nuclear Information System (INIS)

    Eder, W.

    1982-01-01

    Highly sensitive radioimmunoassays of hTSH sub-units were developed. The hormone preparations were labelled with 125-iodine according to a modified chloramine -T method, and purified by chromatography using biogel P6 and P60. Rabbit antisera were used as antibodies. Separation of the antibody-bound and of the free antigens was carried out via the double antibody method. The antiserum required for this purpose was obtained from a goat. The sensitivity of the assay was influenced by changing the protein content of the buffer, the incubation volume, the tracer amounts, the incubation time and the incubation temperature. For hTSH-α, the lowest detectable limit was found to be 50 pg/ml, for hTSH-#betta# 20 pg/ml. Thus, the sub-units could be determined for 98% of the patients under review. The #betta#-TSH radioimmunoassay is largely specific, TSH cross-reacts to a degree of 5%. The computerized evoluation was carried out by means of Spline approximation using the Siemens 4004 computer. Precision and accurateness are in compliance with generally accpted criteria. The serum levels of α and #betta# sub-units showed no discordancy with regard to TSH. In all groups of patients examined, the levels of the hormone-specific #betta#-chain were found to be exclusively dependent upon the actual thyroid activity. (orig.) [de

  20. 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 c....... solfataricus CTP synthase according to a structural alignment with the E. coli enzyme all have large thermal parameters in the dimeric form. Furthermore, they are seen to undergo substantial movement upon tetramerization....

  1. Clp Protease and OR Directly Control the Proteostasis of Phytoene Synthase, the Crucial Enzyme for Carotenoid Biosynthesis in Arabidopsis.

    Science.gov (United States)

    Welsch, Ralf; Zhou, Xiangjun; Yuan, Hui; Álvarez, Daniel; Sun, Tianhu; Schlossarek, Dennis; Yang, Yong; Shen, Guoxin; Zhang, Hong; Rodriguez-Concepcion, Manuel; Thannhauser, Theodore W; Li, Li

    2018-01-08

    Phytoene synthase (PSY) is the crucial plastidial enzyme in the carotenoid biosynthetic pathway. However, its post-translational regulation remains elusive. Likewise, Clp protease constitutes a central part of the plastid protease network, but its substrates for degradation are not well known. In this study, we report that PSY is a substrate of the Clp protease. PSY was uncovered to physically interact with various Clp protease subunits (i.e., ClpS1, ClpC1, and ClpD). High levels of PSY and several other carotenogenic enzyme proteins overaccumulate in the clpc1, clpp4, and clpr1-2 mutants. The overaccumulated PSY was found to be partially enzymatically active. Impairment of Clp activity in clpc1 results in a reduced rate of PSY protein turnover, further supporting the role of Clp protease in degrading PSY protein. On the other hand, the ORANGE (OR) protein, a major post-translational regulator of PSY with holdase chaperone activity, enhances PSY protein stability and increases the enzymatically active proportion of PSY in clpc1, counterbalancing Clp-mediated proteolysis in maintaining PSY protein homeostasis. Collectively, these findings provide novel insights into the quality control of plastid-localized proteins and establish a hitherto unidentified post-translational regulatory mechanism of carotenogenic enzymes in modulating carotenoid biosynthesis in plants. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  2. Effects of Intracerebroventricularly (ICV) Injected Ghrelin on Cardiac Inducible Nitric Oxide Synthase Activity/Expression in Obese Rats.

    Science.gov (United States)

    Sudar Milovanovic, E; Jovanovic, A; Misirkic-Marjanovic, M; Vucicevic, Lj; Janjetovic, K; Isenovic, E R

    2015-11-01

    The aim of this study was to examine the effects of ghrelin on regulation of cardiac inducible nitric oxide synthase (iNOS) activity/expression in high fat (HF), obese rats.For this study, male Wistar rats fed with HF diet (30% fat) for 4 weeks were injected every 24 h for 5 days intracerebroventricularly (ICV) with ghrelin (0.3 nmol/5 µl) or with an equal volume of phosphate buffered saline (PBS). Control rats were ICV injected with an equal volume of PBS. Glucose, insulin and nitric oxide (NO) concentrations were measured in serum, while arginase activity and citrulline concentrations were measured in heart lysate. Protein iNOS and regulatory subunit of nuclear factor-κB (NFκB-p65), phosphorylation of enzymes protein kinase B (Akt) at Ser(473), and extracellular signal-regulated kinases 1/2 (ERK1/2) at Tyr(202)/Tyr(204) were determined in heart lysate by Western blot. For gene expression of iNOS qRT-PCR was used.Results show significantly (parginase activity (pactivity of cardiac iNOS via Akt phosphorylation followed by NFκB activation in HF rats. © Georg Thieme Verlag KG Stuttgart · New York.

  3. Effect of Phosphate Ion on the Structure of Lumazine Synthase, an Antigen Presentation System From Bacillus anthracis.

    Science.gov (United States)

    Wei, Yangjie; Wahome, Newton; Kumar, Prashant; Whitaker, Neal; Picking, Wendy L; Middaugh, C Russell

    2018-03-01

    Lumazine synthase (LS) is an oligomeric enzyme involved in the biosynthesis of riboflavin in microorganisms, fungi, and plants. LS has become of significant interest to biomedical science because of its critical biological role and attractive structural properties for antigen presentation in vaccines. LS derived from Bacillus anthracis (BaLS) consists of 60 identical subunits forming an icosahedron. Its crystal structure has been solved, but its dynamic conformational properties have not yet been studied. We investigated the conformation of BaLS in response to different stress conditions (e.g., chemical denaturants, pH, and temperature) using a variety of biophysical techniques. The physical basis for these thermal transitions was studied, indicating that a molten globular state was present during chemical unfolding by guanidine HCl. In addition, BaLS showed 2 distinct thermal transitions in phosphate-containing buffers. The first transition was due to the dissociation of phosphate ions from BaLS and the second one came from the dissociation and conformational alteration of its icosahedral structure. A small conformational alteration was induced by the binding/dissociation of phosphate ions to BaLS. This work provides a closer view of the conformational behavior of BaLS and provides important information for the formulation of vaccines which use this protein. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  4. Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

    International Nuclear Information System (INIS)

    Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

    1986-01-01

    Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with [ 3 H]-NaBH 4 , and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P

  5. Complementation analysis of mutants of 1-aminocyclopropane- 1-carboxylate synthase reveals the enzyme is a dimer with shared active sites.

    Science.gov (United States)

    Tarun, A S; Theologis, A

    1998-05-15

    The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway. ACS shares the conservation of 11 invariant residues with a family of aminotransferases that includes aspartate aminotransferase. Site-directed mutagenesis on two of these residues, Tyr-92 and Lys-278, in the tomato isoenzyme Le-ACS2 greatly reduces enzymatic activity, indicating their importance in catalysis. These mutants have been used in complementation experiments either in vivo in Escherichia coli or in an in vitro transcription/translation assay to study whether the enzyme functions as a dimer. When the Y92L mutant is coexpressed with the K278A mutant protein, there is partial restoration of enzyme activity, suggesting that the mutant proteins can dimerize and form active heterodimers. Coexpressing a double mutant with the wild-type protein reduces wild-type activity, indicating that inactive heterodimers are formed between the wild-type and the double mutant protein subunits. Furthermore, hybrid complementation shows that another tomato isoenzyme, Le-ACS4, can dimerize and that Le-ACS2 and Le-ACS4 have limited capacity for heterodimerization. The data suggest that ACS functions as a dimer with shared active sites.

  6. Domains of macrophage N(O) synthase have divergent roles in forming and stabilizing the active dimeric enzyme.

    Science.gov (United States)

    Ghosh, D K; Abu-Soud, H M; Stuehr, D J

    1996-02-06

    The cytokine-inducible NO synthase (iNOS) is a flavin-containing hemeprotein that must dimerize to generate NO. Trypsin cleaves the dimeric enzyme into an oxygenase domain fragment that remains dimeric, contains heme and H4biopterin, and binds L-arginine and a reductase domain fragment that is monomeric, binds NADPH, FAD, FMN, and catalyzes the reduction of cytochrome c [Ghosh, D. K. & Stuehr, D. J. (1995) Biochemistry 34, 801-807]. The current study investigates the isolated oxygenase and reductase domains of iNOS to understand how they form and stabilize the active dimeric enzyme. The dimeric oxygenase domain dissociated into folded, heme-containing monomers when incubated with 2-5 M urea, whereas the reductase domain unfolded under these conditions and lost its ability to catalyze NADPH-dependent cytochrome c reduction. Spectral analysis of the dissociation reaction showed that it caused structural changes within the oxygenase domain and exposed the distal side of the heme to solvent, enabling it to bind dithiothreitol as a sixth ligand. Importantly, the oxygenase domain monomers could reassociate into a dimeric form even in the absence of the reductase domain. The reaction required L-arginine and H4biopterin and completely reversed the structural changes in heme pocket and protein structure that occurred upon dissociating the original dimer. Together, this confirms that the oxygenase domain contains all of the determinants needed for subunit dimerization and indicates that the dimeric structure greatly affects the heme and protein environment in the oxygenase domain.

  7. On the multiple roles of the voltage gated sodium channel β1 subunit in genetic diseases

    Directory of Open Access Journals (Sweden)

    Debora eBaroni

    2015-05-01

    Full Text Available Voltage-gated sodium channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are composed of a pore-forming α-subunit and associated β-subunits. The β1-subunit was the first accessory subunit to be cloned. It can be important for controlling cell excitability and modulating multiple aspects of sodium channel physiology. Mutations of β1 are implicated in a wide variety of inherited pathologies, including epilepsy and cardiac conduction diseases. This review summarizes β1-subunit related channelopathies pointing out the current knowledge concerning their genetic background and their underlying molecular mechanisms.

  8. Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

    OpenAIRE

    Azuma, Y; Yamagishi, M; Ishihama, A

    1993-01-01

    To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomy...

  9. Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents.

    Science.gov (United States)

    Hillel, Z; Wu, C W

    1977-07-26

    The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holenzyme (alpha 2 beta beta' sigma) and core enzyme (alpha 2 beta beta') have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N'-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large beta and beta' subunits constitute the backbone of both core and holoenzyme, while sigma and two alpha subunits interact with this structure along the contact domain of beta and beta' subunits. In holoenzyme, sigma subunit is in the vicinity of at least one alpha subunit. The two alpha subunits are close to each other in holoenzyme, core enzyme, and the isolated alpha 2 beta complex. Cross-linking of the "premature" core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition as well as the mechanism of subunit assembly of the enzyme.

  10. Incorporation of phosphate into glycogen by glycogen synthase.

    Science.gov (United States)

    Contreras, Christopher J; Segvich, Dyann M; Mahalingan, Krishna; Chikwana, Vimbai M; Kirley, Terence L; Hurley, Thomas D; DePaoli-Roach, Anna A; Roach, Peter J

    2016-05-01

    The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of (32)P from [β-(32)P]UDP-glucose to glycogen by glycogen synthase. The (32)P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The (32)P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-(32)P]UDP generated in the reaction. Furthermore, [(32)P]UDP did not bind non-covalently to glycogen. The (32)P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, Kurt; Wrzesinski, Krzysztof; Larsen, Peter Mose

    2003-01-01

    quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting...

  12. Regulation of NF-kappaB activity and inducible nitric oxide synthase by regulatory particle non-ATPase subunit 13 (Rpn13)

    DEFF Research Database (Denmark)

    Mazumdar, Tuhina; Gorgun, F Murat; Sha, Youbao

    2010-01-01

    Human Rpn13, also known as adhesion regulating molecule 1 (ADRM1), was recently identified as a novel 19S proteasome cap-associated protein, which recruits the deubiquitinating enzyme UCH37 to the 26S proteasome. Knockdown of Rpn13 by siRNA does not lead to global accumulation of ubiquitinated ce...

  13. Keragaman Genetik Sekuen Gen ATP Synthase FO Subunit 6 (ATP6 Monyet Hantu (Tarsius Indonesia (GENETIC DIVERSITY STUDY OF ATP6 GENE SEQUENCES OF TARSIERS FROM INDONESIA

    Directory of Open Access Journals (Sweden)

    Rini Widayanti

    2013-07-01

    Full Text Available In a conservation effort, the identification of Tarsier species, on the bases of the morphological andmolecular characteristic is necessary. Up to now, the identification of the animals were based on themorphology and vocalizations, which is extremely difficult to identify each, tarsier species. The objective ofthis research was to study the genetic diversity on ATP6 gene of Tarsius sp. Based on sequencing of PCRproduct using primer ATP6F and ATP6R with 681 nts. PCR product. The sequence of ATP6 fragmentswere aligned with other primates from Gene bank with aid of software Clustal W, and were analyzed usingMEGA program version 4.0. Three different nucleotide sites were found (nucleotide no. 288, 321 and 367.The genetic distance based on nucleotide ATP6 sequence calculated using Kimura 2-parameter modelindicated that the smallest genetic distance 0%, biggest 0.8% and average 0, 2%. The phylogenetic treeusing neighbor joining method based on the sequence of nucleotide ATP6 gene could not be used todifferentiate among T. Dianae (from Central Sulawesi, T. Spectrum (from North Sulawesi, T. bancanus(from lampung, South Sumatera and T.bancanus from West Kalimantan.

  14. Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension.

    Science.gov (United States)

    Afolayan, Adeleye J; Eis, Annie; Alexander, Maxwell; Michalkiewicz, Teresa; Teng, Ru-Jeng; Lakshminrusimha, Satyan; Konduri, Girija G

    2016-01-01

    Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2 , partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen. Copyright © 2016 the American Physiological Society.

  15. Fatty acid synthase inhibitors isolated from Punica granatum L

    International Nuclear Information System (INIS)

    Jiang, He-Zhong; Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing; Fan, Hui-Jin; Ma, Xiao-Feng

    2012-01-01

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC 50 value of 10.3 μmol L -1 . (author)

  16. Fatty acid synthase inhibitors isolated from Punica granatum L

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, He-Zhong [School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, (China); Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing, E-mail: zhaoyx1011@163.com [Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou (China); Fan, Hui-Jin; Ma, Xiao-Feng, E-mail: maxiaofeng@gucas.ac.cn [College of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing (China)

    2012-05-15

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC{sub 50} value of 10.3 {mu}mol L{sup -1}. (author)

  17. Dephosphorylation of the Core Clock Protein KaiC in the Cyanobacterial KaiABC Circadian Oscillator Proceeds via an ATP Synthase Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha; Xu, Yao; Qin, Ximing; Johnson, Carl H. (Vanderbilt)

    2014-10-02

    The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P{sub i} (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the

  18. 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.

  19. 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.

  20. Molecular cloning and spatiotemporal expression of prostaglandin F synthase and microsomal prostaglandin E synthase-1 in porcine endometrium.

    Science.gov (United States)

    Waclawik, Agnieszka; Rivero-Muller, Adolfo; Blitek, Agnieszka; Kaczmarek, Monika M; Brokken, Leon J S; Watanabe, Kikuko; Rahman, Nafis A; Ziecik, Adam J

    2006-01-01

    Endometrial prostaglandins (PGs) and the PGE2/PGF2alpha ratio play an important role in regulating the estrous cycle and establishment of pregnancy. The enzymes downstream of cyclooxygenase-2 may determine the PGE2/PGF2alpha ratio in the porcine uterus. Thus, we have cloned porcine PGF synthase (PGFS) and microsomal PGE synthase-1 (mPGES-1) and characterized their expression in porcine endometrium during the estrous cycle and early pregnancy. PGFS and mPGES-1 amino acid sequences possessed a high degree (>67% and >77%, respectively) of identity with the other mammalian homologs. There was little modulation of mPGES-1 throughout the estrous cycle; however, PGFS expression was highly up-regulated in endometrium around the time of luteolysis. During early pregnancy, PGFS at the protein level showed a time-dependent increase (low on d 10-13, intermediate on d 14-23, and high on d 24-25). In pregnancy, expression of mPGES-1 was intermediate on d 10-11 and low on d 14-17 and then increased after d 22, reaching the maximum on d 24-25. Immunohistochemistry showed localization of PGFS and mPGES-1 proteins mainly in luminal and glandular epithelium. Concluding, the spatiotemporal expression of PGFS throughout the estrous cycle indicates an involvement of PGFS in regulating luteolysis in the pig. The comparison of endometrial PGFS and mPGES-1 expression on d 10-13 of the estrous cycle and pregnancy suggest a supportive role of these enzymes in determining the increase of uterine PGE2/PGF2alpha ratio during maternal recognition of pregnancy. Moreover, high expression of both PG synthases after initiation of implantation may indicate their significant role in placentation.

  1. Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis.

    Science.gov (United States)

    Miyanaga, Akimasa; Funa, Nobutaka; Awakawa, Takayoshi; Horinouchi, Sueharu

    2008-01-22

    Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.

  2. Human mediator subunit MED15 promotes transcriptional activation.

    Science.gov (United States)

    Nakatsubo, Takuya; Nishitani, Saori; Kikuchi, Yuko; Iida, Satoshi; Yamada, Kana; Tanaka, Aki; Ohkuma, Yoshiaki

    2014-10-01

    In eukaryotes, the Mediator complex is an essential transcriptional cofactor of RNA polymerase II (Pol II). In humans, it contains up to 30 subunits and consists of four modules: head, middle, tail, and CDK/Cyclin. One of the subunits, MED15, is located in the tail module, and was initially identified as Gal11 in budding yeast, where it plays an essential role in the transcriptional regulation of galactose metabolism with the potent transcriptional activator Gal4. For this reason, we investigated the function of the human MED15 subunit (hMED15) in transcriptional activation. First, we measured the effect of hMED15 knockdown on cell growth in HeLa cells. The growth rate was greatly reduced. By immunostaining, we observed the colocalization of hMED15 with the general transcription factors TFIIE and TFIIH in the nucleus. We measured the effects of siRNA-mediated knockdown of hMED15 on transcriptional activation using two different transcriptional activators, VP16 and SREBP1a. Treatment with siRNAs reduced transcriptional activation, and this reduction could be rescued by overexpression of HA/Flag-tagged, wild-type hMED15. To investigate hMED15 localization, we treated human MCF-7 cells with the MDM2 inhibitor Nutlin-3, thus inducing p21 transcription. We found that hMED15 localized to both the p53 binding site and the p21 promoter region, along with TFIIE and TFIIH. These results indicate that hMED15 promotes transcriptional activation.

  3. 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.

  4. Stereochemical course of enzyme-catalyzed aminopropyl transfer: spermidine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Kullberg, D.W.; Orr, G.R.; Coward, J.K.

    1986-05-01

    The R and S enantionmers of S-adenosyl-3-(/sup 2/H)3-(methylthio)-1-propylamine (decarboxylated S-adenosylmethionine), previously synthesized in this laboratory, were incubated with (1,4-/sup 2/H/sub 4/)-putrescine in the presence of spermidine synthase from E. coli. The resulting chiral (/sup 2/H/sub 5/)spermidines were isolated and converted to their N/sub 1/,N/sub 7/-dibocspermidine-N/sub 4/-(1S,4R)-camphanamides. The derivatives were analyzed by 500 MHz /sup 1/H-NMR and the configuration of the chiral center assigned by correlation with the spectra of synthetic chiral (/sup 2/H/sub 3/)dibocspermidine camphanamide standards. The enzyme-catalyzed aminopropyl transfer was shown to occur with net retention of configuration, indicative of a double-displacement mechanism. This result concurs with that of a previous steady-state kinetics study of spermidine synthase isolated from E. coli, but contradicts the single-displacement mechanism suggested by a stereochemical analysis of chiral spermidines biosynthesized in E. coli treated with chirally deuterated methionines. It also indicates that this aminopropyltransferase is mechanistically distinct from the methyltransferases, which have been shown to act via a single-displacement mechanism (net inversion at -CH/sub 3/) in all cases studied to date.

  5. 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.

  6. Cryptic polyketide synthase genes in non-pathogenic Clostridium SPP.

    Directory of Open Access Journals (Sweden)

    Swantje Behnken

    Full Text Available Modular type I polyketide synthases (PKS produce a vast array of bacterial metabolites with highly diverse biological functions. Notably, all known polyketides were isolated from aerobic bacteria, and yet no example has been reported for strict anaerobes. In this study we explored the diversity and distribution of PKS genes in the genus Clostridium. In addition to comparative genomic analyses combined with predictions of modular type I polyketide synthase (PKS gene clusters in sequenced genomes of Clostridium spp., a representative selection of other species inhabiting a variety of ecological niches was investigated by PCR screening for PKS genes. Our data reveal that all studied pathogenic Clostridium spp. are devoid of putative PKS genes. In stark contrast, cryptic PKS genes are widespread in genomes of non-pathogenic Clostridium species. According to phylogenetic analyses, the Clostridium PKS genes have unusual and diverse origins. However, reverse transcription quantitative PCR demonstrates that these genes are silent under standard cultivation conditions, explaining why the related metabolites have been overlooked until now. This study presents clostridia as a putative source for novel bioactive polyketides.

  7. 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

  8. Chromosomal localization of the human and mouse hyaluronan synthase genes

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  9. Crystal structure of the human Polϵ B-subunit in complex with the C-terminal domain of the catalytic subunit.

    Science.gov (United States)

    Baranovskiy, Andrey G; Gu, Jianyou; Babayeva, Nigar D; Kurinov, Igor; Pavlov, Youri I; Tahirov, Tahir H

    2017-09-22

    The eukaryotic B-family DNA polymerases include four members: Polα, Polδ, Polϵ, and Polζ, which share common architectural features, such as the exonuclease/polymerase and C-terminal domains (CTDs) of catalytic subunits bound to indispensable B-subunits, which serve as scaffolds that mediate interactions with other components of the replication machinery. Crystal structures for the B-subunits of Polα and Polδ/Polζ have been reported: the former within the primosome and separately with CTD and the latter with the N-terminal domain of the C-subunit. Here we present the crystal structure of the human Polϵ B-subunit (p59) in complex with CTD of the catalytic subunit (p261 C ). The structure revealed a well defined electron density for p261 C and the phosphodiesterase and oligonucleotide/oligosaccharide-binding domains of p59. However, electron density was missing for the p59 N-terminal domain and for the linker connecting it to the phosphodiesterase domain. Similar to Polα, p261 C of Polϵ contains a three-helix bundle in the middle and zinc-binding modules on each side. Intersubunit interactions involving 11 hydrogen bonds and numerous hydrophobic contacts account for stable complex formation with a buried surface area of 3094 Å 2 Comparative structural analysis of p59-p261 C with the corresponding Polα complex revealed significant differences between the B-subunits and CTDs, as well as their interaction interfaces. The B-subunit of Polδ/Polζ also substantially differs from B-subunits of either Polα or Polϵ. This work provides a structural basis to explain biochemical and genetic data on the importance of B-subunit integrity in replisome function in vivo . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Structure of protein kinase CK2: dimerization of the human beta-subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Mietens, U; Issinger, O G

    1996-01-01

    Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits. Us......Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta......-subunits. Using the two-hybrid system we could show that the alpha- or alpha'-subunits of CK2 can interact with the beta-subunits of CK2, but not with other alpha- or alpha'-subunits. By comparison, the beta-subunit of CK2 can interact with another beta-subunit. Important amino acids for successful dimerization...... of the beta-subunit were localized between amino acid residues 156 and 165. Furthermore, we identified residues between amino acid 170 and 180 which antagonize the dimerization....

  11. N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression.

    Science.gov (United States)

    Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M; Shi, Shujie; Chen, Jingxin; Blobner, Brandon M; Buck, Teresa M; Brodsky, Jeffrey L; Hughey, Rebecca P; Kleyman, Thomas R

    2018-03-01

    Epithelial Na + channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na + . The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

  12. Stable incorporation versus dynamic exchange of β subunits in a native Ca2+ channel complex.

    Science.gov (United States)

    Campiglio, Marta; Di Biase, Valentina; Tuluc, Petronel; Flucher, Bernhard E

    2013-05-01

    Voltage-gated Ca(2+) channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation-contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary β subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether β subunits can reversibly interact with, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged α1 and β subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca(2+) channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle β1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle β2a and β4b isoforms dynamically interacted with both α1 subunits. Neither replacing the I-II loop of CaV1.1 with that of CaV2.1, nor deletions in the proximal I-II loop, known to change the orientation of β relative to the α1 subunit, altered the specific dynamic properties of the β subunits. In contrast, a single residue substitution in the α interaction pocket of β1aM293A increased the FRAP rate threefold. Taken together, these findings indicate that in skeletal muscle triads the homologous β1a subunit forms a stable complex, whereas the heterologous β2a and β4b subunits form dynamic complexes with the Ca(2+) channel. The distinct binding properties are not determined by differences in the I-II loop sequences of the α1 subunits, but are intrinsic properties of the β subunit isoforms.

  13. 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

    , 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...

  14. Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells

    NARCIS (Netherlands)

    Verschuur, A. C.; van Gennip, A. H.; Muller, E. J.; Voûte, P. A.; Vreken, P.; van Kuilenburg, A. B.

    1999-01-01

    Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood

  15. Expression of prostaglandin synthases (pgds and pges) duringzebrafishgonadal differentiation

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Nielsen, John E.; Nielsen, Betina F.

    2010-01-01

    The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E-2 synthase (pges) and especially the lipocalin-type prostaglandin D-2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found ...

  16. Use of octaketide synthases to produce kermesic acid and flavokermesic acid

    DEFF Research Database (Denmark)

    2016-01-01

    A method for producing an octaketide derived aromatic compound of interest (e.g. carminic acid), wherein the method comprises (I): heterologous expression of a recombinantly introduced Type III polyketide synthase (PKS) gene encoding an octaketide synthase (OKS) to obtain non-reduced octaketide...

  17. 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 ...

  18. The localization of chitin synthase in membranous vesicles (chitosomes) in Neurospora crassa

    NARCIS (Netherlands)

    Sietsma, JH; Din, AB; Ziv, [No Value; Sjollema, KA; Yarden, O

    Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neurospora crassa. A single band of approximately 110 kDa was observed in Western blots of total protein extracts

  19. 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

    ) required inorganic phosphate for activity, whereas the others were phosphate independent. PRPP synthase isozymes 2 and 3 contained 76 and 87 amino acid extensions, respectively, at their N-terminal ends in comparison with other PRPP synthases. Isozyme 2 was synthesized in vitro and shown to be imported...

  20. 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

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

    OpenAIRE

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

    2011-01-01

    The levopimaradiene/abietadiene synthase from Norway spruce (Picea abies; PaLAS) has previously been reported to produce a mixture of four diterpene hydrocarbons when incubated with geranylgeranyl diphosphate as the substrate: levopimaradiene, abietadiene, neoabietadiene, and palustradiene. However, variability in the assay products observed by GC-MS of this and orthologous conifer diterpene synthases over the past 15 years suggested that these diterpenes may not be the initial enzyme assay p...

  2. Regulation of glycogen synthase kinase-3β (GSK-3β) after ionizing radiation

    International Nuclear Information System (INIS)

    Boehme, K.A.

    2006-12-01

    Glycogen Synthase Kinase-3β (GSK-3β) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3β by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3β at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3β serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKBβ, is required for phosphorylation of GSK- 3β at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3β at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3β in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer able to degrade p53 which in

  3. Methanogenic Paraffin Biodegradation: Alkylsuccinate Synthase Gene Quantification and Dicarboxylic Acid Production.

    Science.gov (United States)

    Oberding, Lisa K; Gieg, Lisa M

    2018-01-01

    Paraffinic n -alkanes (>C 17 ) that are solid at ambient temperature comprise a large fraction of many crude oils. The comparatively low water solubility and reactivity of these long-chain alkanes can lead to their persistence in the environment following fuel spills and pose serious problems for crude oil recovery operations by clogging oil production wells. However, the degradation of waxy paraffins under the anoxic conditions characterizing contaminated groundwater environments and deep subsurface energy reservoirs is poorly understood. Here, we assessed the ability of a methanogenic culture enriched from freshwater fuel-contaminated aquifer sediments to biodegrade the model paraffin n -octacosane (C 28 H 58 ). Compared with that in controls, the consumption of n -octacosane was coupled to methane production, demonstrating its biodegradation under these conditions. Smithella was postulated to be an important C 28 H 58 degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identified assA gene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from other Smithella organisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays for assA demonstrated significant increases in the abundance and expression of this gene in C 28 H 58 -degrading cultures compared with that in controls, suggesting n -octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the C 28 H 58 -degrading cultures, a novel observation providing clues as to how methanogenic consortia access waxy hydrocarbons. The results of this study broaden our understanding of how waxy paraffins can be biodegraded in anoxic environments with an application toward bioremediation and improved oil recovery. IMPORTANCE Understanding the methanogenic biodegradation of different classes of hydrocarbons has important

  4. cDNA isolation, functional expression, and characterization of (+)-alpha-pinene synthase and (-)-alpha-pinene synthase from loblolly pine (Pinus taeda): stereocontrol in pinene biosynthesis.

    Science.gov (United States)

    Phillips, Michael A; Wildung, Mark R; Williams, David C; Hyatt, David C; Croteau, Rodney

    2003-03-15

    The complex mixture of monoterpenes, sesquiterpenes, and diterpenes that comprises oleoresin provides the primary defense of conifers against bark beetles and their associated fungal pathogens. Monoterpene synthases produce the turpentine fraction of oleoresin, which allows mobilization of the diterpene resin acid component (rosin) and is also toxic toward invading insects; this is particularly the case for alpha-pinene, a prominent bicyclic monoterpene of pine turpentine. The stereochemistry of alpha-pinene is a critical determinant of host defense capability and has implications for host selection, insect pheromone biosynthesis, and tritrophic-level interactions. Pines produce both enantiomers of alpha-pinene, which appear to arise through antipodal reaction mechanisms by distinct enzymes. Using a cDNA library constructed with mRNA from flushing needles of loblolly pine (Pinus taeda), we employed a homology-based cloning strategy to isolate, and confirm by functional expression, the genes encoding (+)-(3R:5R)-alpha-pinene synthase, (-)-(3S:5S)-alpha-pinene synthase, and several other terpene synthases. The pinene synthases, which produce mirror-image products, share only 66% amino acid identity (72% similarity) but are similar in general properties to other monoterpene synthases of gymnosperms. The stereochemical control of monoterpene cyclization reactions, the evolution of "antipodal" enzymes, and the implications of turpentine composition in ecological interactions are discussed.

  5. 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.

  6. Riboflavin accumulation and characterization of cDNAs encoding lumazine synthase and riboflavin synthase in bitter melon (Momordica charantia).

    Science.gov (United States)

    Tuan, Pham Anh; Kim, Jae Kwang; Lee, Sanghyun; Chae, Soo Cheon; Park, Sang Un

    2012-12-05

    Riboflavin (vitamin B2) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotide--cofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that McLS and McRS share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that McLS and McRS were constitutively expressed in M. charantia, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. McLS and McRS were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from M. charantia may aid the metabolic engineering of vitamin B2 in crops.

  7. Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.

    Science.gov (United States)

    Brodelius, Maria; Lundgren, Anneli; Mercke, Per; Brodelius, Peter E

    2002-07-01

    A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.

  8. 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.

  9. Measurement and charactereistics of hCG β-subunit

    International Nuclear Information System (INIS)

    Isojima, Shinzo; Koyama, Koji; Naga, Osamu.

    1975-01-01

    The authors studied an hCG-radioimmunoassay method in which the anti-hCG β-subunit antibody- 125 I-hCG β-subunit system was used, and they determined that in this method the antibody reacted much more specifically against hCG than in the usual radioimmunoassay method in which the anti-hCG antibody- 125 I-hCG has been used. In addition, they explained that a specific method for hCG-radioimmunoassay can be established in which the reaction system does not depend on the amount of physiological LH secretion. The significance of the clinical application of this method was considered and it was proved that the method was necessary, especially for studies of the hCG secretion pattern in patients with villous tumors having a low level of LH. Furthermore, the phenomenon of the cellular effect, namely a temporary increase of the hCG value which was thought to be caused by the administration of anticancerous agents, was proved to be due to hCG hypersecretion, not merely to LH variation. (Ichikawa, K.)

  10. Thermostable cross-protective subunit vaccine against Brucella species.

    Science.gov (United States)

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. The TFIIH subunit Tfb3 regulates cullin neddylation

    Science.gov (United States)

    Rabut, Gwenaël; Le Dez, Gaëlle; Verma, Rati; Makhnevych, Taras; Knebel, Axel; Kurz, Thimo; Boone, Charles; Deshaies, Raymond J.; Peter, Matthias

    2011-01-01

    Summary Cullin proteins are scaffolds for the assembly of multi-subunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely-varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING-domain of Hrt1 nor did it require the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient neddylation involves the RING-domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 as well as the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING-domain protein Tfb3 controls activation of a subset of cullins. PMID:21816351

  12. Binding of ATP by pertussis toxin and isolated toxin subunits

    International Nuclear Information System (INIS)

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

    1990-01-01

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [ 3 H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of [ 3 H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [ 3 H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site

  13. Binding of ATP by pertussis toxin and isolated toxin subunits

    Energy Technology Data Exchange (ETDEWEB)

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. (Center for Biologics Evaluation and Research, Bethesda, MD (USA))

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  14. Kinetic mechanism of luciferase subunit folding and assembly.

    Science.gov (United States)

    Clark, A C; Raso, S W; Sinclair, J F; Ziegler, M M; Chaffotte, A F; Baldwin, T O

    1997-02-18

    The kinetic mechanism in vitro of the folding and assembly of the heterodimeric flavin monooxygenase bacterial luciferase has been defined by a unique set of rate constants which describe both the productive refolding pathway and competing off-pathway reactions in 50 mM phosphate, pH 7.0 at 18 degrees C. The individual alpha and beta subunits fold independently to form heterodimerization-competent species, alpha i and beta i. The alpha i beta i species can interact to form an inactive heterodimeric intermediate, [alpha beta ]i, which isomerizes to form the active alpha beta structure; the structure of the enzyme has been determined to 1.5 A resolution [Fisher, A. J., Thompson, T. B., Thoden, J. B., Baldwin, T. O., & Rayment, I. (1996) J. Biol. Chem. 271, 21956-21968]. In the absence of alpha i, beta i can form a kinetically trapped homodimer, beta 2, with a second-order rate constant of about 180 M-1 s-1 [Sinclair, J. F., Ziegler, M. M., & Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320-326]; the structure of beta 2 has recently been reported [Thoden. J. B., Holden, H. M., Fisher, A. J., Sinclair. J. F., Wesenberg, G., Baldwin, T.O., & Rayment, I. (1997) Protein Sci. 6, 13-23]. The beta i species, or some other form that precedes beta i on the refolding pathway, can also undergo a first-order conversion into a form (designated beta x) that cannot associate with alpha i to form the native enzyme. The rate constant for this process, assigned here, accounts well for the previously observed dependence of final yield on concentration of refolding species [Ziegler, M.M., Goldberg, M.E., Chaffotte, A. F., & Baldwin, T. O. (1993) J. Biol. Chem. 268, 10760-10765]. In simulations of the refolding reaction, all processes associated with the refolding of the individual subunits were combined into single first-order rate constants for each subunit which were consistent with the rate constants determined from stopped-flow circular dichroism studies. The first-order rate constant

  15. Efficient expression of functional (α6β22β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

    Directory of Open Access Journals (Sweden)

    Carson Kai-Kwong Ley

    Full Text Available Human (α6β2(α4β2β3 nicotinic acetylcholine receptors (AChRs are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β22β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β22β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

  16. Compensatory expression of human -Acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma

    OpenAIRE

    Pohl , Sandra; Tiede , Stephan; Castrichini , Monica; Cantz , Michael; Gieselmann , Volkmar; Braulke , Thomas

    2009-01-01

    Abstract The N-Acetylglucosaminyl-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate (M6P) recognition markers essential for efficient transport of lysosomal hydrolases to lysosomes. The phosphotransferase is composed of six subunits (?2, ?2, ?2). The ?- and ?-subunits are catalytically active and encoded by a single gene, GNPTAB, whereas the ?-subunit encoded by GNPTG is proposed to recognize conformational structures common to lysosomal enzymes. Defects in GN...

  17. Cloning and sequencing of the casein kinase 2 alpha subunit from Zea mays

    DEFF Research Database (Denmark)

    Dobrowolska, G; Boldyreff, B; Issinger, O G

    1991-01-01

    The nucleotide sequence of the cDNA coding for the alpha subunit of casein kinase 2 of Zea mays has been determined. The cDNA clone contains an open reading frame of 996 nucleotides encoding a polypeptide comprising 332 amino acids. The primary amino acid sequence exhibits 75% identity to the alpha...... subunit and 71% identity to the alpha' subunit of human casein kinase 2....

  18. Beta-Subunit of Human Chorionic Gonadotropin in Malignant Lymphoma : An Immunohistochemical Study

    OpenAIRE

    Senba, Masachika; Watanabe, Masami

    1991-01-01

    We present a rare case of a 77-year-old Japanese man with malignant lymphoma associated with production of beta-subunit of human chorionic gonadotropin in the cytoplasms of lymphoma cells in the lymph nodes. By immunoperoxidase staining, numerous tumor cells were reacted with beta-subunit of human chorionic gonadotropin. To the best of our knowledge, production of beta-subunit of human chorionic gonadotropin in the cytoplasm of lymphoma cells has not been reported. This patient evidences that...

  19. Characterisation by nuclear magnetic resonance of the β catalytic subunit of the chloroplastic coupling factor

    International Nuclear Information System (INIS)

    Andre, Francois

    1986-09-01

    This academic work addressed the use of nuclear magnetic resonance (NMR) for the structural and dynamic study of the catalytic sub-unit of the extrinsic section of a membrane complex, the chloroplastic H+-ATPase. This work included the development of a protocol of preparation and quantitative purification of β subunits isolated from the CF1 for the elaboration of a concentrated sample for NMR, and then the study of the β subunit by using proton NMR

  20. 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

  1. 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

  2. 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.

  3. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

    Science.gov (United States)

    Wieczorek, Anna; McHenry, Charles S

    2006-05-05

    The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

  4. NMR Crystallography of Enzyme Active Sites: Probing Chemically-Detailed, Three-Dimensional Structure in Tryptophan Synthase

    Science.gov (United States)

    Dunn, Michael F.

    2013-01-01

    crystallography for application to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the β-subunit of tryptophan synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in tryptophan synthase and its surprising role in directing the next step in the catalysis of L-Trp formation. PMID:23537227

  5. Translation activity of chimeric ribosomes composed of Escherichia coli and Bacillus subtilis or Geobacillus stearothermophilus subunits

    Directory of Open Access Journals (Sweden)

    Sayaka Tsuji

    2017-07-01

    Full Text Available Ribosome composition, consisting of rRNA and ribosomal proteins, is highly conserved among a broad range of organisms. However, biochemical studies focusing on ribosomal subunit exchangeability between organisms remain limited. In this study, we show that chimeric ribosomes, composed of Escherichia coli and Bacillus subtilis or E. coli and Geobacillus stearothermophilus subunits, are active for β-galactosidase translation in a highly purified E. coli translation system. Activities of the chimeric ribosomes showed only a modest decrease when using E. coli 30 S subunits, indicating functional conservation of the 50 S subunit between these bacterial species.

  6. Specific radioimmunoassay of HCG and its α and β subunits: methods and results

    International Nuclear Information System (INIS)

    Reuter, A.M.; Schoonbrood, J.; Franchimont, P.

    1976-01-01

    To create antisera that are specific for the radioimmunoassay of HCG and its subunits, the antisera are neutralized by incubation with LH or HCG. For each RIA system the inhibition curves of HCG and its subunits LH, FSH, TSH and STH are obtained. The 125 I labelled hormones HCG, α and β subunits and LH were chromatographed over a Sephadex G 100 column. Serum of menopausal and pregnant women were chromatographed in the same way and the fractions subjected to RIA. HCG and its subunits were determined by RIA in the sera of patients with different kinds of cancer

  7. Thermal aggregation behaviour of soy protein: characteristics of different polypeptides and sub-units.

    Science.gov (United States)

    He, Xiu-Ting; Yuan, De-Bao; Wang, Jin-Mei; Yang, Xiao-Quan

    2016-03-15

    Due to the differences in structure and composition of glycinin and β-conglycinin, they exhibit different characteristics during heat treatment. In present study, the thermal aggregation behaviour of glycinin, β-conglycinin and their isolated sub-units was investigated at pH 7.0. Acidic polypeptides, basic polypeptides, αα' and β sub-units of soy protein were denatured during the isolation process. The degree of aggregation of protein fractions after heat treatment was in the order: denatured basic polypeptides > native glycinin > denatured β sub-unit > native β-conglycinin > denatured acidic polypeptides > denatured αα' sub-units. Glycinin, β-conglycinin, acidic polypeptides and αα'/β sub-units exhibited different changing trends of surface hydrophobicity with increasing temperature. The αα' sub-units showed higher ability to suppress thermal aggregation of basic polypeptides than β sub-units during heat treatment. The β sub-units were shown to form soluble aggregates with glycinin after heating. The interaction mechanism of αα' and β sub-units heated with basic polypeptides was proposed. For the β sub-units-basic polypeptides mixed system, more hydrophobic chains were binding together and buried inside during heat treatment, which resulted in lower surface hydrophobicity. The αα' sub-units-basic polypeptides mixed system was considered to be a stable system with higher surface hydrophobicity after being heated. © 2015 Society of Chemical Industry.

  8. Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans.

    Science.gov (United States)

    Ahuja, Manmeet; Chiang, Yi-Ming; Chang, Shu-Lin; Praseuth, Mike B; Entwistle, Ruth; Sanchez, James F; Lo, Hsien-Chun; Yeh, Hsu-Hua; Oakley, Berl R; Wang, Clay C C

    2012-05-16

    Genome sequencing has revealed that fungi have the ability to synthesize many more natural products (NPs) than are currently known, but methods for obtaining suitable expression of NPs have been inadequate. We have developed a successful strategy that bypasses normal regulatory mechanisms. By efficient gene targeting, we have replaced, en masse, the promoters of nonreducing polyketide synthase (NR-PKS) genes, key genes in NP biosynthetic pathways, and other genes necessary for NR-PKS product formation or release. This has allowed us to determine the products of eight NR-PKSs of Aspergillus nidulans, including seven novel compounds, as well as the NR-PKS genes required for the synthesis of the toxins alternariol (8) and cichorine (19).

  9. Type III Polyketide Synthases: Functional Classification and Phylogenomics.

    Science.gov (United States)

    Shimizu, Yugo; Ogata, Hiroyuki; Goto, Susumu

    2017-01-03

    Polyketide synthases (PKSs) catalyze the sequential condensation of simple acetate units to produce a large class of natural products, including pharmacologically valuable compounds. PKSs are classified into three types on the basis of their domain structures; type III PKSs have the simplest domain structure, although their products have various structures and functions. The sequence-function relationship is fundamental for predicting enzyme functions, but it has not been well investigated in type III PKSs to date. Consequently, the current methods for predicting type III PKS functions are still immature in comparison with those that target type I/II PKSs. In this review we summarize the current functional and phylogenomic knowledge about type III PKSs and propose a new classification of their enzymatic reactions. We also discuss possible directions for the development of better computational tools for functional prediction of type III PKS homologues. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Noncovalent Intermediate of Thymidylate Synthase: Fact or Fiction?

    Science.gov (United States)

    Kholodar, Svetlana A; Kohen, Amnon

    2016-07-06

    Thymidylate synthase is an attractive target for antibiotic and anticancer drugs due to its essential role in the de novo biosynthesis of the DNA nucleotide thymine. The enzymatic reaction is initiated by a nucleophilic activation of the substrate via formation of a covalent bond to an active site cysteine. The traditionally accepted mechanism is then followed by a series of covalently bound intermediates, where that bond is only cleaved upon product release. Recent computational and experimental studies suggest that the covalent bond between the protein and substrate is actually quite labile. Importantly, these findings predict the existence of a noncovalently bound bisubstrate intermediate, not previously anticipated, which could be the target of a novel class of drugs inhibiting DNA biosynthesis. Here we report the synthesis of the proposed intermediate and findings supporting its chemical and kinetic competence. These findings substantiate the predicted nontraditional mechanism and the potential of this intermediate as a new drug lead.

  11. 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.

  12. 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.

  13. Atomic model for the dimeric FOregion of mitochondrial ATP synthase.

    Science.gov (United States)

    Guo, Hui; Bueler, Stephanie A; Rubinstein, John L

    2017-11-17

    Mitochondrial adenosine triphosphate (ATP) synthase produces the majority of ATP in eukaryotic cells, and its dimerization is necessary to create the inner membrane folds, or cristae, characteristic of mitochondria. Proton translocation through the membrane-embedded F O region turns the rotor that drives ATP synthesis in the soluble F 1 region. Although crystal structures of the F 1 region have illustrated how this rotation leads to ATP synthesis, understanding how proton translocation produces the rotation has been impeded by the lack of an experimental atomic model for the F O region. Using cryo-electron microscopy, we determined the structure of the dimeric F O complex from Saccharomyces cerevisiae at a resolution of 3.6 angstroms. The structure clarifies how the protons travel through the complex, how the complex dimerizes, and how the dimers bend the membrane to produce cristae. Copyright © 2017, American Association for the Advancement of Science.

  14. Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering.

    Science.gov (United States)

    Zerbe, Philipp; Bohlmann, Jörg

    2015-07-01

    Plants produce thousands of diterpenoid natural products; some of which are of significant industrial value as biobased pharmaceuticals (taxol), fragrances (sclareol), food additives (steviosides), and commodity chemicals (diterpene resin acids). In nature, diterpene synthase (diTPS) enzymes are essential for generating diverse diterpene hydrocarbon scaffolds. While some diTPSs also form oxygenated compounds, more commonly, oxygenation is achieved by cytochrome P450-dependent mono-oxygenases. Recent genome-, transcriptome-, and metabolome-guided gene discovery and enzyme characterization identified novel diTPS functions that form the core of complex modular pathway systems. Insights into diterpene metabolism may translate into the development of new bioengineered microbial and plant-based production systems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Two Cycloartenol Synthases for Phytosterol Biosynthesis in Polygala tenuifolia Willd

    Directory of Open Access Journals (Sweden)

    Mei Lan Jin

    2017-11-01

    Full Text Available Oxidosqualene cyclases (OSCs are enzymes that play a key role in control of the biosynthesis of phytosterols and triterpene saponins. In order to uncover OSC genes from Polygala tenuifolia seedlings induced by methyl jasmonate (MeJA, RNA-sequencing analysis was performed using the Illumina sequencing platform. A total of 148,488,632 high-quality reads from two samples (control and the MeJA treated were generated. We screened genes related to phytosterol and triterpene saponin biosynthesis and analyzed the transcriptional changes of differentially expressed unigene (DEUG values calculated by fragments per kilobase million (FPKM. In our datasets, two full-length cDNAs of putative OSC genes, PtCAS1, and PtCAS2, were found, in addition to the PtBS (β-amyrin synthase gene reported in our previous studies and the two cycloartenol synthase genes of P. tenuifolia. All genes were isolated and characterized in yeast cells. The functional expression of the two PtCAS genes in yeast cells showed that the genes all produce a cycloartenol as the sole product. When qRT-PCR analysis from different tissues was performed, the expressions of PtCAS1 and PtCAS2 were highest in flowers and roots, respectively. After MeJA treatment, the transcripts of PtCAS1 and PtCAS2 genes increased by 1.5- and 2-fold, respectively. Given these results, we discuss the potential roles of the two PtCAS genes in relation to triterpenoid biosynthesis.

  16. Glycogen Synthase in Sertoli Cells: More Than Glycogenesis?

    Science.gov (United States)

    Maldonado, Rodrigo; Mancilla, Héctor; Villarroel-Espíndola, Franz; Slebe, Felipe; Slebe, Juan Carlos; Méndez, Raúl; Guinovart, Joan J; Concha, Ilona I

    2016-11-01

    Sertoli cell metabolism actively maintains the nutritional needs of germ cells. It has been described that after glucose incorporation in Sertoli cells, less than 1% is converted to glycogen suggesting low levels of glycogen synthase activity. Phosphorylation of muscle glycogen synthase (MGS) at serine 640 (pS640MGS) decreases its activity, and this form of the enzyme was discovered as a non-ribosomal protein that modulates the translation of a subset of transcripts in HeLa cells. The aim of our study was to functionally characterize MGS in cultured Sertoli cells, as well as to explore this new feature related to RNA molecules. We detected MGS in the cytoplasm of Sertoli cells as well as in the nuclei. The activity rates of the enzyme were extremely low indicating that MGS is expressed but almost inactive. Protein targeting to glycogen (PTG) overexpression was performed to activate MGS by dephosphorylation. PTG induced glycogen synthesis massively, confirming that this enzyme is present but inactive. This finding correlates with high levels of pS640MGS, which were assayed by phosphatase treatment. To explore a putative new function for MGS in Sertoli cells, we performed RNA immunoprecipitation coupled to microarray studies. The results revealed that MGS co-immunoprecipitated with the several mRNAs and also rRNAs. These findings indicate that MGS is expressed Sertoli cells but in an inactive form, and also support a possibly novel feature of this metabolic enzyme associated with RNA-related molecules. J. Cell. Biochem. 117: 2597-2607, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

    Science.gov (United States)

    Marroquí, Silvia; Zorreguieta, Angeles; Santamaría, Carmen; Temprano, Francisco; Soberón, Mario; Megías, Manuel; Downie, J. Allan

    2001-01-01

    We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c1 and CycM and a small increase in the amount of cytochrome aa3. In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene. PMID:11208782

  18. Intestinal nitric oxide synthase activity changes during experimental colon obstruction.

    Science.gov (United States)

    Palásthy, Zsolt; Kaszaki, József; Lázár, György; Nagy, Sándor; Boros, Mihály

    2006-08-01

    The experiments in this study were designed to follow the time course of nitric oxide (NO) synthesis in the large bowel during acute mechanical ileus. Occlusion of the mid-transverse colon was maintained for 420 min in anesthetized dogs. Strain-gauge transducers were used to analyze motility changes on the hepatic and lienal flexures, respectively. Constitutive NO synthase (cNOS) and inducible NOS (iNOS) activities were determined in tissue biopsies, and plasma nitrite/nitrate (NOx) level was measured in the portal blood. Following completion of the baseline studies, the animals were treated with either 7-nitroindazole (7-NI, selective neuronal NOS inhibitor), or N-nitro-L-arginine (NNA, non-selective NOS inhibitor). In the sham-operated group the cNOS activities differed significantly in the oral and aboral tissue samples (oral: 102.9; versus aboral: 62.1 fmol/mg protein/min). The obstruction elicited a significant increase in portal NOx and elevated tissue inducible NO synthase (iNOS) activity. NNA treatment decreased the motility index in both intestinal segments for 60 min, but 120 min later the motility index was significantly elevated (2.5-fold increase in the oral part, and 1.8-fold enhancement in the aboral segment, respectively). Treatment with 7-NI decreased the cNOS activity in the oral and aboral parts by approximately 40% and 70%, respectively, and suppressed the motility increase in the aboral colon segment. The motility of the colon was either significantly increased or decreased, depending on the type and selectivity of the NOS inhibitor compounds applied. NO of neuronal origin is a transmitter that stimulates peristaltic activity; but an increased iNOS/nNOS ratio significantly moderates the obstruction-induced motility increase.

  19. Chaperonin Structure - The Large Multi-Subunit Protein Complex

    Directory of Open Access Journals (Sweden)

    Irena Roterman

    2009-03-01

    Full Text Available The multi sub-unit protein structure representing the chaperonins group is analyzed with respect to its hydrophobicity distribution. The proteins of this group assist protein folding supported by ATP. The specific axial symmetry GroEL structure (two rings of seven units stacked back to back - 524 aa each and the GroES (single ring of seven units - 97 aa each polypeptide chains are analyzed using the hydrophobicity distribution expressed as excess/deficiency all over the molecule to search for structure-to-function relationships. The empirically observed distribution of hydrophobic residues is confronted with the theoretical one representing the idealized hydrophobic core with hydrophilic residues exposure on the surface. The observed discrepancy between these two distributions seems to be aim-oriented, determining the structure-to-function relation. The hydrophobic force field structure generated by the chaperonin capsule is presented. Its possible influence on substrate folding is suggested.

  20. HCG-subunits in choriocarcinoma and in pregnancy

    International Nuclear Information System (INIS)

    Talas, M.; Fingerova, H.; Kudela, M.

    1979-01-01

    Pure HCG-α and HCG-β and anti HCG-β serum were used in the study of HCG-subunits. The following RIA systems were studied: HCG* - anti HCG, HCG* - anti HCG-β, HCG-β* - anti HCG-β, HCG-β* - anti HCG and HCG-α* - anti HCG. HCG-α, HCG-β and the 2nd International Standard of HCG were used as standard preparations. The sensitivity and parallelism of individual inhibition curves were compared. Samples obtained from women with choriocarcinoma and in pregnancy were estimated in RIA systems studied. It was found out that the values of HCG in women treated for choriocarcinoma as obtained in the system HCG* - anti HCG-β were higher than those obtained in the system HCG* - anti HCG. (author)

  1. Determination of hCG-alpha subunit in threatened pregnancy

    International Nuclear Information System (INIS)

    Talas, M.; Pohanka, J.; Fingerova, H.; Janouskova, M.; Krikal, Z.; Prasilova, J.; Zupkova, H.

    1987-01-01

    Radioimmunoassay of the hCG-alpha subunit was made using an antibody anti hCG-alpha serum, highly purified hCG-alpha for 125 I-labelling and the standard hCG-alpha. Sera of healthy pregnant women sampled throughout the whole pregnancies were used to determine x-bar±S.D. of hCG-alpha for 14-day intervals. Included in the study were groups of women with high risk of premature labor, late toxemia of pregnancy, twins and fetal hypotrophy. It was shown that increased hCG-alpha is found in pregnant women in whom signs of late toxemia of pregnancy are combined with high risk of premature labor, or with twin pregnancies, while in those with fetal hypotrophy hCG-alpha is within normal limits. (author). 3 figs., 7 refs

  2. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Directory of Open Access Journals (Sweden)

    Ariel Avila

    2013-08-01

    Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

  3. The subunit structure of the extracellular hemoglobin of Biomphalaria glabrata

    International Nuclear Information System (INIS)

    Arndt, Marcio H.L.; Naves, Cristiani F.; Xavier, Luciana P.; Santoro, Marcelo M.

    1997-01-01

    Full text. The hemoglobin of Biomphalaria glabrata was purified to homogeneity by a two step purification protocol using a gel filtration column (Superose 6 HR/Pharmacia ) followed by an anion exchange chromatography (MONO-Q Sepharose/Pharmacia). The dissociation products were analysed by a 5 - 15 % Polyacrylamide gel electrophoresis containing Sodium Dodecyl Sulfate (SDS-PAGE) giving a band of 270 K Daltons and a band of 180 K Daltons after reduction with β-mercaptoethanol. The same profile was obtained in a 3.5 % Agarose gel electrophoresis containing SDS (SDS-AGE) showing additional bands of higher molecular weight. These bands were proposed to be monomers, dimers and trimers and, after reduction in a Bidimensional SDS-AGE, the proposed monomers and dimers were decomposed in two and four bands that were interpreted as 1 - 4 chains. The hemoglobin was digested by four different proteases ( Thrombin, Trypsin, Chymotrypsin and Subtilisin ) showing several equivalent fragments with molecular weights multiples of its minimum molecular weight ( 17.7 K Daltons). The circular dichroism spectrum of the protein showed a characteristic high α-helix content. We proposed that this hemoglobin is a pentamer of approx. 360 K Daltons subunits each formed by two 180 K Daltons chains linked in pairs by disulfide bridges and each of these chains comprises ten Heme binding domains. These data were compared to other Planorbidae extracellular hemoglobins. Up to now, the quaternary structure of this hemoglobin (shape and disposition of the subunits) is unknown. It is intended to elucidate its structure by Small Angle X-Ray Scattering in Brazilian National Laboratory of Synchrotron Light (LNLS). (author)

  4. Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, T.; Weintraub, B.D.

    1985-04-01

    The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing (/sup 14/C)alanine and (/sup 3/H) glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, (/sup 14/C)alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. (/sup 3/H)Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function.

  5. Copolymer semiconductors comprising thiazolothiazole or benzobisthiazole, or benzobisoxazole electron acceptor subunits, and electron donor subunits, and their uses in transistors and solar cells

    Science.gov (United States)

    Jenekhe, Samson A; Subramaniyan, Selvam; Ahmed, Eilaf; Xin, Hao; Kim, Felix Sunjoo

    2014-10-28

    The inventions disclosed, described, and/or claimed herein relate to copolymers comprising copolymers comprising electron accepting A subunits that comprise thiazolothiazole, benzobisthiazole, or benzobisoxazoles rings, and electron donating subunits that comprise certain heterocyclic groups. The copolymers are useful for manufacturing organic electronic devices, including transistors and solar cells. The invention also relates to certain synthetic precursors of the copolymers. Methods for making the copolymers and the derivative electronic devices are also described.

  6. A photocrosslinking assay for reporting protein interactions in polyketide and fatty acid synthases.

    Science.gov (United States)

    Ye, Zhixia; Bair, Morgan; Desai, Hemant; Williams, Gavin J

    2011-11-01

    Understanding protein-protein interactions that occur between ACP and KS domains of polyketide synthases and fatty acid synthases is critical to improving the scope and efficiency of combinatorial biosynthesis efforts aimed at producing non-natural polyketides. Here, we report a facile strategy for rapidly reporting such ACP-KS interactions based on the incorporation of an amino acid with photocrosslinking functionality. Crucially, this photocrosslinking strategy can be applied to any polyketide or fatty acid synthase regardless of substrate specificity, and can be adapted to a high-throughput format for directed evolution studies. This journal is © The Royal Society of Chemistry 2011

  7. 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....

  8. Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

    Science.gov (United States)

    Yeo, Yun-Soo; Nybo, S Eric; Chittiboyina, Amar G; Weerasooriya, Aruna D; Wang, Yan-Hong; Góngora-Castillo, Elsa; Vaillancourt, Brieanne; Buell, C Robin; DellaPenna, Dean; Celiz, Mary Dawn; Jones, A Daniel; Wurtele, Eve Syrkin; Ransom, Nick; Dudareva, Natalia; Shaaban, Khaled A; Tibrewal, Nidhi; Chandra, Suman; Smillie, Troy; Khan, Ikhlas A; Coates, Robert M; Watt, David S; Chappell, Joe

    2013-02-01

    Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

  9. Functional Identification of Valerena-1,10-diene Synthase, a Terpene Synthase Catalyzing a Unique Chemical Cascade in the Biosynthesis of Biologically Active Sesquiterpenes in Valeriana officinalis*

    Science.gov (United States)

    Yeo, Yun-Soo; Nybo, S. Eric; Chittiboyina, Amar G.; Weerasooriya, Aruna D.; Wang, Yan-Hong; Góngora-Castillo, Elsa; Vaillancourt, Brieanne; Buell, C. Robin; DellaPenna, Dean; Celiz, Mary Dawn; Jones, A. Daniel; Wurtele, Eve Syrkin; Ransom, Nick; Dudareva, Natalia; Shaaban, Khaled A.; Tibrewal, Nidhi; Chandra, Suman; Smillie, Troy; Khan, Ikhlas A.; Coates, Robert M.; Watt, David S.; Chappell, Joe

    2013-01-01

    Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [13C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes. PMID:23243312

  10. [Nature of the heterogeneity of the 30S ribosomal subunits in vitro. II. Two types of inactivation of the 30S subunits of Escherichia coli ribosomes].

    Science.gov (United States)

    Peshin, N N; Kirillov, S V

    1979-01-01

    The influence of concentration of monovalent cations on the binding constant of Phe-tRNAPhe to 30S.poly(U) complex was studied. Two types of inactivation of the 30S subunits by ammonium ions at the low magnesium concentration (1 mM) were found. The first type of inactivation was observed at high concentrations of NH4+ ions (from 0.5 to 1.5 M) and due to the dissociation of ribosomal proteins from 30S subunits. This inactivation only decreased the binding constant of Phe-tRNAPhe to 30S.poly(U) complex up to 50 times but all 30S subunits were equally achieved in Phe-tRNAPhe binding. This type of inactivation was reversible, addition of S-proteins restored the association constant to the original value. At low concentration of NH4+ ions (below 100 mM) about half of the 30S subunits is irreversibly inactivated (the binding constant of Phe-tRNAPhe decreased below detectable level) probably as a result of conformational changes in ribosomal RNA. Both types of inactivation of the 30S subunits can take place during the preparation of isolated subunits of ribosomes.

  11. Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea.

    OpenAIRE

    Ruggero, D; Londei, P

    1996-01-01

    Hybrid ribosomes obtained by mixing the ribosomal subunits of the extremely thermophilic archaea Sulfolobus solfataricus and Desulfurococcus mobilis were tested for their sensitivity to selected antibiotics. It is shown that structural differences in the large ribosomal subunits determine qualitatively and quantitatively the patterns of response to alpha-sarcin and paromomycin in these species.

  12. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposited...

  13. Isoforms of human cytochrome-c oxidase. Subunit composition and steady-state kinetic properties

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; Dekker, H. L.; van den Bogert, C.; Nieboer, P.; van Gelder, B. F.; Muijsers, A. O.

    1991-01-01

    The subunit pattern and the steady-state kinetics of cytochrome-c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome-c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in

  14. Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation

    NARCIS (Netherlands)

    Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

    The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A. NR2B, and NR2D subunit transcripts

  15. Human heart cytochrome c oxidase subunit VIII. Purification and determination of the complete amino acid sequence

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; Muijsers, A. O.; Demol, H.; Dekker, H. L.; van Beeumen, J. J.

    1988-01-01

    Subunit VIII was purified from a preparation of the human heart cytochrome c oxidase and its complete amino acid sequence was determined. The sequence proved to be much more related to that of the bovine liver oxidase subunit VIII than to that found in bovine heart. Our finding of a 'liver-type'

  16. Lack of NMDA receptor subunit exchange alters Purkinje cell dendritic morphology in cerebellar slice cultures

    NARCIS (Netherlands)

    Metzger, F; Pieri, [No Value; Eisel, ULM; Pieri, Isabelle

    2005-01-01

    Early postnatal developmental changes in N-methyl-D-aspartate (NMDA) receptor (NR) subunits regulate cerebellar granule cell maturation and potentially Purkinje cell development. We therefore investigated Purkinje cell morphology in slice cultures from mice with genetic subunit exchange from NR2C to

  17. Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

    Science.gov (United States)

    Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

    1995-02-01

    The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

  18. Expression of the scaffolding subunit A of protein phosphatase 2A during rat testicular development

    NARCIS (Netherlands)

    van den Ham, R.; van Dissel-Emiliani, F. M. F.; van Pelt, A. M. M.

    2003-01-01

    Previously, we found that the poly(A)+ RNA of the scaffolding subunit A (alpha isoform) of protein phosphatase 2A (PP2A-Aalpha) was clearly expressed by fetal gonocytes but weakly expressed by adult single (As), paired (Apr), and aligned (Aal) A spermatogonia. The scaffolding subunit A of PP2A

  19. Regulation of KV channel voltage-dependent activation by transmembrane β subunits

    Directory of Open Access Journals (Sweden)

    Xiaohui eSun

    2012-04-01

    Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

  20. Visualization of subunit interactions and ternary complexes of protein phosphatase 2A in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Shu-Ting Mo

    Full Text Available Protein phosphatase 2A (PP2A is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET. Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells.

  1. The A and B variants of the alpha 3 integrin subunit: tissue distribution and functional characterization

    NARCIS (Netherlands)

    de Melker, A. A.; Sterk, L. M.; Delwel, G. O.; Fles, D. L.; Daams, H.; Weening, J. J.; Sonnenberg, A.

    1997-01-01

    The alpha subunits of the laminin-binding integrins alpha 3 beta 1, alpha 6 beta 1, and alpha 7 beta 1 have homologous sequences and are similar in structure. Two cytoplasmic variants, A and B, have been identified for each of these alpha subunits, although the alpha 3B splice variant has been

  2. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposite...

  3. Purification of the alpha and beta subunits of phosphorylase kinase for structural studies

    International Nuclear Information System (INIS)

    Sotiroudis, T.G.; Heilmeyer, L.M.G. Jr.; Crabb, J.W.

    1987-01-01

    Structural analysis of the alpha (Mr, 132,000) and beta (Mr, 113,000) subunits of phosphorylase kinase may provide clues to their yet unknown functions however purification remains problematic. Preparative RP-HPLC procedures yield inconveniently large, dilute solutions and concentration steps are required prior to subunit modification and fragmentation. Concentration of the β subunit usually results in significant losses due to insolubility. Using preparative SDS-polyacrylamide gel electrophoresis, they have purified the α, 7 , and β subunits from rabbit muscle phosphorylase kinase in a soluble and concentrated form suitable for structural studies. Phosphorylase kinase labelled with fluorescein isothiocyanate in the α and α' subunits and fully 14 C-S-carboxymethylated was fractionated on a 5% acrylamide Laemmli slab gel. The subunit bands were visualized by fluorescence and by SDS precipitation then excised and electroeluted in the presence of SDS using an ELUTRAP device. From 4.5 mg of enzyme applied to a 4.5 mm thick gel about 70% of the α subunit and about 90% of the β subunit were typically recovered in less than 1 ml with overnight elution

  4. Elastic deformations of the rotary double motor of single F(o)F(1)-ATP synthases detected in real time by Förster resonance energy transfer.

    Science.gov (United States)

    Ernst, Stefan; Düser, Monika G; Zarrabi, Nawid; Dunn, Stanley D; Börsch, Michael

    2012-10-01

    Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to a 3-stepped F(1) motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F(O)F(1)-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Facilitation by the beta2a subunit of pore openings in cardiac Ca2+ channels.

    Science.gov (United States)

    Costantin, J; Noceti, F; Qin, N; Wei, X; Birnbaumer, L; Stefani, E

    1998-02-15

    1. Single channel recordings were performed on the cardiac calcium channel (alpha1C) in order to study the effect of coexpression of the accessory beta2a subunit. On-cell patch clamp recordings were performed after expression of these channels in Xenopus oocytes. 2. The alpha1C subunit, when expressed alone, had similar single channel properties to native cardiac channels. Slow transitions between low and high open probability (Po) gating modes were found as well as fast gating transitions between the open and closed states. 3. Coexpression of the beta2a subunit caused changes in the fast gating during high Po mode. In this mode, open time distributions reveal at least three open states and the beta2a subunit favours the occupancy of the longest, 10-15 ms open state. No effect of the beta2a subunit was found when the channel was gating in the low Po mode. 4. Slow gating transitions were also affected by the beta2a subunit. The high Po mode was maintained for the duration of the depolarizing pulse in the presence of the beta2a subunit; while the alpha1C channel when expressed alone, frequently switched into and out of the high Po mode during the course of a sweep. 5. The beta2a subunit also affected mode switching that occurred between sweeps. Runs analysis revealed that the alpha1C subunit has a tendency toward non-random mode switching. The beta2a subunit increased this tendency. A chi2 analysis of contingency tables indicated that the beta2a subunit caused the alpha1C channel to gain 'intrinsic memory', meaning that the mode of a given sweep can be non-independent of the mode of the previous sweep. 6. We conclude that the beta2a subunit causes changes to the alpha1C channel in both its fast and slow gating behaviour. The beta2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the beta2a subunit decreases the slow switching between low and high Po modes.

  6. Pattern of secretion of immunoreactive inhibin/activin subunits by avian granulosa cells.

    Science.gov (United States)

    Johnson, P A; Brooks, C F; Davis, A J

    2005-05-01

    The messenger RNA expression for the inhibin/activin subunits in the granulosa layer of avian follicles of different developmental stages has previously been reported. In the present study, we examined the pattern of secretion of these protein subunits from cultured granulosa cells (GC) of avian follicles of defined maturity. Laying hens were euthanized and the F1, F2, F3, F4, small yellow follicles (SYF; 6-10 mm) and large white follicles (LWF; 3-5 mm) were removed. GC were isolated from the follicles, plated by size at a density of 6.25 x 10(5)cells per well (3 wells per follicle size) and cultured for 48 h in medium 199 with 5% FBS, antibiotics, and 1.0 microg/ml bovine insulin. After 48 h, the cultures were terminated and the media were saved (n = 6 replications). Proteins were precipitated from media, reconstituted for electrophoresis (SDS-PAGE), and analyzed by Western blot. Progesterone was also measured in the medium. For detection of the inhibin alpha-subunit, a rabbit antibody against the chicken inhibin alpha-subunit (1-26 aa) was used. The betaA-subunit was detected with rabbit anti-betaA-subunit (81-113 aa) and the betaB-subunit was detected with rabbit anti-betaB-subunit (80-112 aa). Under reduced conditions, GC from the larger follicle sizes (F1-F4) secreted the most (p subunit compared to smaller follicle sizes. Under non-reduced conditions, a band at approximately 32 kDa was detected by both the alpha-subunit antibody and by the betaA-subunit antibody in media from GC of the F1-F4 follicles, suggesting secretion of intact inhibin A. Immunoreactive alpha-subunit and betaB-subunit were detected under reduced conditions in media from the GC of the SYF, suggesting that this follicle population may secrete intact inhibin B. In addition, under non-reduced conditions, cells from the SYF secreted the greatest amount of intact inhibin B (p subunits. Progesterone concentration in the media from the F1 follicle was greatest and was decreased in media from

  7. Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

    DEFF Research Database (Denmark)

    Grankowski, N; Boldyreff, B; Issinger, O G

    1991-01-01

    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...... of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step...... at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 micro...

  8. Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channel.

    Science.gov (United States)

    Gao, Ying; Liu, Shuang-Shuang; Qiu, Shuang; Cheng, Wei; Zheng, Jie; Luo, Jian-Hong

    2007-07-20

    Acid-sensing ion channels (ASICs) are believed to be homo- or heteromeric complexes, which have been verified by classical methods such as co-immunoprecipitation or electrophysiological assays. However, the exact subunit combinations of ASICs in living cells have not been established yet. Here, we apply assays based on fluorescence resonance energy transfer (FRET) between GFP color mutants CFP and YFP to investigate ASIC assembly directly in living cells. Homomerization as well as heteromerization of different combinations of ASIC subunits were found. In addition, our results suggest the formation of heteromeric 1a/2a channels of stoichiometry consisting of at least two 1a subunits and two 2a subunits. Similar stoichiometry was observed from heteromeric 1a/2b and 2a/2b channels. Our results imply that these heteromeric ASIC channels contain at least four subunits.

  9. Submitochondrial distributions and stabilities of subunits 4, 5, and 6 of yeast cytochrome oxidase in assembly defective mutants.

    Science.gov (United States)

    Glerum, D M; Tzagoloff, A

    1997-08-04

    The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.

  10. Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis).

    Science.gov (United States)

    Kwon, Moonhyuk; Cochrane, Stephen A; Vederas, John C; Ro, Dae-Kyun

    2014-12-20

    Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  11. Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Sandrini, Michael; Piskur, Jure

    2001-01-01

    beta -Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamy-beta -alanine as the sole nitrogen source and exhibits diminished beta -alanine synthase...... no pyrimidine catabolic pathway, it enabled growth on N-carbamyl- beta -alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta -alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial...... N- carbamyl amidohydrolases. All three beta -alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N...

  12. Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases.

    Science.gov (United States)

    Schrepfer, Patrick; Buettner, Alexander; Goerner, Christian; Hertel, Michael; van Rijn, Jeaphianne; Wallrapp, Frank; Eisenreich, Wolfgang; Sieber, Volker; Kourist, Robert; Brück, Thomas

    2016-02-23

    Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.

  13. SCREENING OF 6-PYRUVOYL-TETRAHYDROPTERIN SYNTHASE ACTIVITY DEFICIENCY AMONG HYPERP HENYLALANINEMIC PATIENTS

    Directory of Open Access Journals (Sweden)

    DURDI QUJEQ

    1999-10-01

    Full Text Available A deficiency of the phenylalanine hydroxylase activity or its cofactor tetrahydrobiopterin may"nlead to hyperphenylalamnemia and as a result, loss of IQ, poor school performance, and"nbehavior problems occurs. Deficiency in 6-pyruvoyl-tetrahydropterin synthase activity is the"nmajor cause of tetrahydrobiopterin deficient phenylketonuria. In this study, blood specimens"nfrom 165 healthy volunteers and 127 children with phenylketonuria were used to determine"nthe 6-pyruvoyl-tetrahydropterin synthase activity. It was found that the activity of 6-"npyruvoyl- tetrahydropterin synthase was decreased in comparison with control [23.46 +/-"n2.94, (mean +/- SD, mmol/ ml/h, n=I27 vs. 127.63 +/- 4.52, n=165, p<0.05]. Results of"nthis study indicate that examination of 6-pyruvoyl-tetrahydropterin synthase activity is helpful"nand may lead to the diagnosis cause of hyperphenylalaninemia.

  14. The Structure of Sucrose Synthase-1 from Arabidopsis thaliana and Its Functional Implications

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yi; Anderson, Spencer; Zhang, Yanfeng; Garavito, R. Michael (MSU); (NWU)

    2014-10-02

    Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) has been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-{angstrom} resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.

  15. A close look at a ketosynthase from a trans-acyltransferase modular polyketide synthase.

    Science.gov (United States)

    Gay, Darren C; Gay, Glen; Axelrod, Abram J; Jenner, Matthew; Kohlhaas, Christoph; Kampa, Annette; Oldham, Neil J; Piel, Jörn; Keatinge-Clay, Adrian T

    2014-03-04

    The recently discovered trans-acyltransferase modular polyketide synthases catalyze the biosynthesis of a wide range of bioactive natural products in bacteria. Here we report the structure of the second ketosynthase from the bacillaene trans-acyltransferase polyketide synthase. This 1.95 Å resolution structure provides the highest resolution view available of a modular polyketide synthase ketosynthase and reveals a flanking subdomain that is homologous to an ordered linker in cis-acyltransferase modular polyketide synthases. The structure of the cysteine-to-serine mutant of the ketosynthase acylated by its natural substrate provides high-resolution details of how a native polyketide intermediate is bound and helps explain the basis of ketosynthase substrate specificity. The substrate range of the ketosynthase was further investigated by mass spectrometry. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Use of heterologous expressed polyketide synthase and small molecule foldases to make aromatic and cyclic compounds

    DEFF Research Database (Denmark)

    2016-01-01

    A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell.......A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell....

  17. Characterization of a 1-aminocyclopropane-1-carboxylate synthase gene from loblolly pine (Pinus taeda L.).

    Science.gov (United States)

    Barnes, J R; Lorenz, W W; Dean, J F D

    2008-04-30

    1-Aminocyclopropane-1-carboxylate (ACC) synthase catalyzes what is typically the rate-limiting step in the biosynthesis of ethylene, a gaseous plant growth regulator that plays numerous roles in the growth and development of higher plants. Although ACC synthase genes have been characterized from a wide variety of angiosperm plant species, no ACC synthase genes have been described previously for gymnosperms. Evidence suggests that ethylene helps to regulate wood formation in trees, and may also signal for the metabolic shifts that lead to compression wood formation on the undersides of branches and leaning stems in gymnosperm trees. Since compression wood is an inferior feedstock for the manufacturing of most wood products, a better understanding of the factors influencing its formation could lead to substantial economic benefits. This study describes the isolation and characterization of a putative ACC synthase gene, PtaACS1, from loblolly pine (Pinus taeda L.), an important commercial forest tree species. Also described is an apparent splice variant of PtaACS1 (PtaACS1s) that is missing 138 bp from the 5' end of the transcript, including bases that encode a conserved amino acid residue considered critical for ACC synthase activity. The two sequences share interesting homologies with a group of plant aminotransferases, in addition to ACC synthases, but structural models and the conservation of critical catalytic amino acid residues strongly support PtaACS1 as encoding an active ACC synthase. The two transcripts were differentially expressed in various tissues of loblolly pine, as well as in response to perturbations of pine seedling stems. Transcript levels of this ACC synthase gene increased rapidly in response to bending stress but returned to near starting levels within 30 min. It remains unclear to what extent bending-induced expression of this gene product plays a role in compression wood formation.

  18. A domain swapping approach to elucidate differential regiospecific hydroxylation by geraniol and linalool synthases from perilla.

    Science.gov (United States)

    Sato-Masumoto, Naoko; Ito, Michiho

    2014-06-01

    Geraniol and linalool are acyclic monoterpenes found in plant essential oils that have attracted much attention for their commercial use and in pharmaceutical studies. They are synthesized from geranyl diphosphate (GDP) by geraniol and linalool synthases, respectively. Both synthases are very similar at the amino acid level and share the same substrate; however, the position of the GDP to which they introduce hydroxyl groups is different. In this study, the mechanisms underlying the regiospecific hydroxylation of geraniol and linalool synthases were investigated using a domain swapping approach and site-directed mutagenesis in perilla. Sequences of the synthases were divided into ten domains (domains I to IV-4), and each corresponding domain was exchanged between both enzymes. It was shown that different regions were important for the formation of geraniol and linalool, namely, domains IV-1 and -4 for geraniol, and domains III-b, III-d, and IV-4 for linalool. These results suggested that the conformation of carbocation intermediates and their electron localization were seemingly to be different between geraniol and linalool synthases. Further, five amino acids in domain IV-4 were apparently indispensable for the formation of geraniol and linalool. According to three-dimensional structural models of the synthases, these five residues seemed to be responsible for the different spatial arrangement of the amino acid at H524 in the case of geraniol synthase, while N526 is the corresponding residue in linalool synthase. These results suggested that the side-chains of these five amino acids, in combination with several relevant domains, localized the positive charge in the carbocation intermediate to determine the position of the introduced hydroxyl group. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Use of octaketide synthases to produce kermesic acid and flavokermesic acid

    DEFF Research Database (Denmark)

    2016-01-01

    A method for producing an octaketide derived aromatic compound of interest (e.g. carminic acid), wherein the method comprises (I): heterologous expression of a recombinantly introduced Type III polyketide synthase (PKS) gene encoding an octaketide synthase (OKS) to obtain non-reduced octaketide...... in vivo within the recombinant host cell and (II): converting in vivo the non-reduced octaketide of step (I) into a C14-C34 aromatic compound of interest (e.g. carminic acid)....

  20. Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment

    International Nuclear Information System (INIS)

    Tanaka, Hiroaki; Tsurumura, Toshiharu; Aritake, Kosuke; Furubayashi, Naoki; Takahashi, Sachiko; Yamanaka, Mari; Hirota, Erika; Sano, Satoshi; Sato, Masaru; Kobayashi, Tomoyuki; Tanaka, Tetsuo; Inaka, Koji; Urade, Yoshihiro

    2011-01-01

    Crystals of hematopoietic prostaglandin D synthase grown in microgravity show improved quality. Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein

  1. Expression, characterization, and site-directed mutation of a multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) from Pseudomonas sp. Lm10.

    Science.gov (United States)

    Lang, Zhi-Fei; Shen, Jing-Jing; Cai, Shu; Zhang, Jun; He, Jian; Li, Shun-Peng

    2011-08-01

    A multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) gene was cloned from Pseudomonas sp. Lm10. Sequence analysis showed that the rAHAS regulatory subunit was identical to that of Pseudomonas putida KT2440 (sensitive AHAS, sAHAS), whereas six different sites [H134→N (rAHAS→sAHAS), A135→P, S136→T, I210→V, F264→Y, and S486→W] were found in the catalytic subunit. The rAHAS and sAHAS were over expressed, purified and characterized. rAHAS showed higher resistance to four kinds of AHAS-inhibitor herbicides than sAHAS. The resistance factor of rAHAS was 56.0-fold, 12.6-fold, 6.5-fold, and 9.2-fold as compared with sAHAS when metsulfuron-methyl, imazethapyr, flumetsulam, and pyriminobac-methyl used as inhibitor, respectively. The specific activity of rAHAS was lower than that of sAHAS and the K (m) value of rAHAS for pyruvate was approximately onefold higher than the corresponding value for sAHAS. Data from site-directed mutagenesis demonstrated that alteration at A135, F264, and S486 resulted in resistance reduction, while the mutation at H134, S136, and I210 has little effect on the resistance. A135 was mainly responsible for resistance to imidazolinone; F264 conferred resistance to sulfonylurea and triazolopyrimidine sulfonamide; and S486 showed multiple herbicides resistance to the four herbicides.

  2. Effect of the ATPase inhibitor protein IF{sub 1} on H{sup +} translocation in the mitochondrial ATP synthase complex

    Energy Technology Data Exchange (ETDEWEB)

    Zanotti, Franco [Dept. of Medical Biochemistry, Biology and Physics, University of Bari (Italy); Inst. of Biomembranes and Bioenergetics, CNR, Bari (Italy); Gnoni, Antonio; Mangiullo, Roberto [Dept. of Medical Biochemistry, Biology and Physics, University of Bari (Italy); Papa, Sergio, E-mail: papabchm@cimedoc.uniba.it [Dept. of Medical Biochemistry, Biology and Physics, University of Bari (Italy); Inst. of Biomembranes and Bioenergetics, CNR, Bari (Italy)

    2009-06-19

    The H{sup +} F{sub o}F{sub 1}-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F{sub 1} moiety of the complex protrudes at the inner side of the membrane, the F{sub o} sector spans the membrane reaching the outer side. The IF{sub 1} component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F{sub o}F{sub 1}-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF{sub 1} (residue 42-58) to the F{sub 1}-{alpha}/{beta} subunits. We have analyzed the effect of native purified IF{sub 1} the IF{sub 1}-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K{sup +}] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F{sub o}F{sub 1} complex. The results show that IF{sub 1}, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F{sub 1} to the F{sub o} side and in the opposite direction. Cross-linking of IF{sub 1} to F{sub 1}-{alpha}/{beta} subunits inhibits the ATP-driven H{sup +} translocation but enhances H{sup +} conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F{sub o}F{sub 1} complex.

  3. Comparative study of Chalcone synthase promoters across plant families

    Directory of Open Access Journals (Sweden)

    Francisco Buitrago

    2009-07-01

    Full Text Available En la era post-genómica, el entendimiento de la regulación génica se ha convertido en un reto y una prioridad de investigación. En este trabajo realizamos un estudio comparativo de las secuencias reguladoras del gen de la chalcón sintetasa de varias familias botánicas. Veintidós secuencias de promotores de Chalcone Synthase fueron comparados teniendo en cuenta tres elementos Cis reguladores: Caja-G, Caja-H y Caja-TATA, que podrían estar actuando como una sola unidad cooperativa. Nuestra comparación muestra que estos elementos puede que se conserven en algunas especies e inclusive que se conserven a nivel de familia. Sin embargo, en algunas especies no todos los elementos Cis fueron encontrados, mostrando que no todas las especies se regulan bajo los mismos parámetros. Adicionalmente, una comparación entre promotores de una misma especie con una familia de multigenes Chs, mostró que los genes duplicados son variables en la composición del elemento Cis, sugiriendo que estos genes pueden estarse expresando de maneras diferentes.

  4. 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.

  5. 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.

  6. 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.

  7. Structural basis for the recruitment of glycogen synthase by glycogenin.

    Science.gov (United States)

    Zeqiraj, Elton; Tang, Xiaojing; Hunter, Roger W; García-Rocha, Mar; Judd, Andrew; Deak, Maria; von Wilamowitz-Moellendorff, Alexander; Kurinov, Igor; Guinovart, Joan J; Tyers, Mike; Sakamoto, Kei; Sicheri, Frank

    2014-07-15

    Glycogen is a primary form of energy storage in eukaryotes that is essential for glucose homeostasis. The glycogen polymer is synthesized from glucose through the cooperative action of glycogen synthase (GS), glycogenin (GN), and glycogen branching enzyme and forms particles that range in size from 10 to 290 nm. GS is regulated by allosteric activation upon glucose-6-phosphate binding and inactivation by phosphorylation on its N- and C-terminal regulatory tails. GS alone is incapable of starting synthesis of a glycogen particle de novo, but instead it extends preexisting chains initiated by glycogenin. The molecular determinants by which GS recognizes self-glucosylated GN, the first step in glycogenesis, are unknown. We describe the crystal structure of Caenorhabditis elegans GS in complex with a minimal GS targeting sequence in GN and show that a 34-residue region of GN binds to a conserved surface on GS that is distinct from previously characterized allosteric and binding surfaces on the enzyme. The interaction identified in the GS-GN costructure is required for GS-GN interaction and for glycogen synthesis in a cell-free system and in intact cells. The interaction of full-length GS-GN proteins is enhanced by an avidity effect imparted by a dimeric state of GN and a tetrameric state of GS. Finally, the structure of the N- and C-terminal regulatory tails of GS provide a basis for understanding phosphoregulation of glycogen synthesis. These results uncover a central molecular mechanism that governs glycogen metabolism.

  8. Plasmodium falciparum dolichol phosphate mannose synthase represents a novel clade

    International Nuclear Information System (INIS)

    Shams-Eldin, Hosam; Santos de Macedo, Cristiana; Niehus, Sebastian; Dorn, Caroline; Kimmel, Juergen; Azzouz, Nahid; Schwarz, Ralph T.

    2008-01-01

    Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker's yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups

  9. Substrate specificity of Arabidopsis 3-ketoacyl-CoA synthases

    International Nuclear Information System (INIS)

    Blacklock, Brenda J.; Jaworski, Jan G.

    2006-01-01

    The very long chain fatty acids (VLCFA) incorporated into plant lipids are derived from the iterative addition of C2 units provided by malonyl-CoA to an acyl-CoA by the 3-ketoacyl-CoA synthase (KCS) component of a fatty acid elongase (FAE) complex. Mining of the Arabidopsis genome sequence database revealed 20 genes with homology to seed-specific FAE1 KCS. Eight of the 20 putative KCSs were cloned, expressed in yeast, and isolated as (His) 6 fusion proteins. Five of the eight (At1g71160, At1g19440, At1g07720, At5g04530, and At4g34250) had little or no activity with C16 to C20 substrates while three demonstrated activity with C16, C18, and C20 saturated acyl-CoA substrates. At1g01120 KCS (KCS1) and At2g26640 KCS had broad substrate specificities when assayed with saturated and mono-unsaturated C16 to C24 acyl-CoAs while At4g34510 KCS was specific for saturated fatty acyl-CoA substrates

  10. 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.

  11. Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination.

    Science.gov (United States)

    Chemler, Joseph A; Tripathi, Ashootosh; Hansen, Douglas A; O'Neil-Johnson, Mark; Williams, Russell B; Starks, Courtney; Park, Sung Ryeol; Sherman, David H

    2015-08-26

    The structural scaffolds of many complex natural products are produced by multifunctional type I polyketide synthase (PKS) enzymes that operate as biosynthetic assembly lines. The modular nature of these mega-enzymes presents an opportunity to construct custom biocatalysts built in a lego-like fashion by inserting, deleting, or exchanging native or foreign domains to produce targeted variants of natural polyketides. However, previously engineered PKS enzymes are often impaired resulting in limited production compared to native systems. Here, we show a versatile method for generating and identifying functional chimeric PKS enzymes for synthesizing custom macrolactones and macrolides. PKS genes from the pikromycin and erythromycin pathways were hybridized in Saccharomyces cerevisiae to generate hybrid libraries. We used a 96-well plate format for plasmid purification, transformations, sequencing, protein expression, in vitro reactions and analysis of metabolite formation. Active chimeric enzymes were identified with new functionality. Streptomyces venezuelae strains that expressed these PKS chimeras were capable of producing engineered macrolactones. Furthermore, a macrolactone generated from selected PKS chimeras was fully functionalized into a novel macrolide analogue. This method permits the engineering of PKS pathways as modular building blocks for the production of new antibiotic-like molecules.

  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. The Stereochemistry of Complex Polyketide Biosynthesis by Modular Polyketide Synthases

    Directory of Open Access Journals (Sweden)

    David H. Kwan

    2011-07-01

    Full Text Available Polyketides are a diverse class of medically important natural products whose biosynthesis is catalysed by polyketide synthases (PKSs, in a fashion highly analogous to fatty acid biosynthesis. In modular PKSs, the polyketide chain is assembled by the successive condensation of activated carboxylic acid-derived units, where chain extension occurs with the intermediates remaining covalently bound to the enzyme, with the growing polyketide tethered to an acyl carrier domain (ACP. Carboxylated acyl-CoA precursors serve as activated donors that are selected by the acyltransferase domain (AT providing extender units that are added to the growing chain by condensation catalysed by the ketosynthase domain (KS. The action of ketoreductase (KR, dehydratase (DH, and enoylreductase (ER activities can result in unreduced, partially reduced, or fully reduced centres within the polyketide chain depending on which of these enzymes are present and active. The PKS-catalysed assembly process generates stereochemical diversity, because carbon–carbon double bonds may have either cis- or trans- geometry, and because of the chirality of centres bearing hydroxyl groups (where they are retained and branching methyl groups (the latter arising from use of propionate extender units. This review shall cover the studies that have determined the stereochemistry in many of the reactions involved in polyketide biosynthesis by modular PKSs.

  14. Inducible expression of trehalose synthase in Bacillus licheniformis.

    Science.gov (United States)

    Li, Youran; Gu, Zhenghua; Zhang, Liang; Ding, Zhongyang; Shi, Guiyang

    2017-02-01

    Trehalose synthase (TreS) could transform maltose into trehalose via isomerization. It is a crucial enzyme in the process of trehalose enzymatical transformation. In this study, plasmid-based inducible expression systems were constructed to produce Thermomonospora curvata TreS in B. licheniformis. Xylose operons from B. subtilis, B. licheniformis and B. megaterium were introduced to regulate the expression of the gene encoding TreS. It was functionally expressed, and the BlsTs construct yielded the highest enzyme activity (12.1 U/mL). Furthermore, the effect of different cultural conditions on the inducible expression of BlsTs was investigated, and the optimal condition was as follows: 4% maltodextrin, 0.4% soybean powder, 1% xylose added after 10 h of growth and an induction time of 12 h at 37 °C. As a result, the maximal yield reached 24.7 U/mL. This study contributes to the industrial application of B. licheniformis, a GRAS workhorse for enzyme production. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. 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.

  16. Hyperbaric oxygen upregulates cochlear constitutive nitric oxide synthase

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. Inhibition of fatty acid synthase prevents preadipocyte differentiation

    International Nuclear Information System (INIS)

    Schmid, Bernhard; Rippmann, Joerg F.; Tadayyon, Moh; Hamilton, Bradford S.

    2005-01-01

    Inhibition of fatty acid synthase (FAS) reduces food intake in rodents. As adipose tissue expresses FAS, we sought to investigate the effect of reduced FAS activity on adipocyte differentiation. FAS activity was suppressed either pharmacologically or by siRNA during differentiation of 3T3-L1 cells. Cerulenin (10 μM), triclosan (50 μM), and C75 (50 μM) reduced dramatically visible lipid droplet accumulation, while incorporation of [1- 14 C]acetate into lipids was reduced by 75%, 70%, and 90%, respectively. Additionally, the substances reduced FAS, CEBPα, and PPARγ mRNA by up to 85% compared to that of control differentiated cells. Transient transfection with FAS siRNA suppressed FAS mRNA and FAS activity, and this was accompanied by reduction of CEBPα and PPARγ mRNA levels, and complete prevention of lipid accumulation. CD36, a late marker of differentiation, was also reduced. Together, these results suggest that FAS generated signals may be essential to support preadipocyte differentiation

  19. 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...... decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein...... for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent...

  20. 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.

  1. 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.

  2. Phosphorylation Regulates myo-Inositol-3-phosphate Synthase

    Science.gov (United States)

    Deranieh, Rania M.; He, Quan; Caruso, Joseph A.; Greenberg, Miriam L.

    2013-01-01

    myo-Inositol-3-phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate, suggesting that the enzyme is post-translationally regulated. Using 32Pi labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site mutants indicated that the three conserved sites in yeast (Ser-184, Ser-296, and Ser-374) and humans (Ser-177, Ser-279, and Ser-357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phosphodeficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374A caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to valproate. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS. PMID:23902760

  3. 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.

  4. Protein modelling of triterpene synthase genes from mangrove plants using Phyre2 and Swiss-model

    Science.gov (United States)

    Basyuni, M.; Wati, R.; Sulistiyono, N.; Hayati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

    2018-03-01

    Molecular cloning of five oxidosqualene cyclases (OSC) genes from Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa had previously been cloned, characterized, and encoded mono and -multi triterpene synthases. The present study analyzed protein modelling of triterpene synthase genes from mangrove using Phyre2 and Swiss-model. The diversity was noted within protein modelling of triterpene synthases using Phyre2 from sequence identity (38-43%) and residue (696-703). RsM2 was distinguishable from others for template structure; it used lanosterol synthase as a template (PDB ID: w6j.1.A). By contrast, other genes used human lanosterol synthase (1w6k.1.A). The predicted bind sites were correlated with the product of triterpene synthase, the product of BgbAS was β-amyrin, while RsM1 contained a significant amount of β-amyrin. Similarly BgLUS and KcMS, both main products was lupeol, on the other hand, RsM2 with the outcome of taraxerol. Homology modelling revealed that 696 residues of BgbAS, BgLUS, RsM1, and RsM2 (91-92% of the amino acid sequence) had been modelled with 100% confidence by the single highest scoring template using Phyre2. This coverage was higher than Swiss-model (85-90%). The present study suggested that molecular cloning of triterpene genes provides useful tools for studying the protein modelling related regulation of isoprenoids biosynthesis in mangrove forests.

  5. The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

    Science.gov (United States)

    Kovaleva, V. D.; Berezhnaya, E. V.; Uzdensky, A. B.

    2015-03-01

    Nitric oxide (NO) is an important second messenger, involved in the implementation of various cell functions. It regulates various physiological and pathological processes such as neurotransmission, cell responses to stress, and neurodegeneration. NO synthase is a family of enzymes that synthesize NO from L-arginine. The activity of different NOS isoforms depends both on endogenous and exogenous factors. In particular, it is modulated by oxidative stress, induced by photodynamic therapy (PDT). We have studied the possible role of NOS in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Antinecrotic and proapoptotic effects of NO on the glial cells were found using inhibitory analysis. We have shown the role of inducible NO synthase in photoinduced apoptosis and involvement of neuronal NO synthase in photoinduced necrosis of glial cells in the isolated crayfish stretch receptor. The activation of NO synthase was evaluated using NADPH-diaphorase histochemistry, a marker of neurons expressing the enzyme. The activation of NO synthase in the isolated crayfish stretch receptor was evaluated as a function of time after PDT. Photodynamic treatment induced transient increase in NO synthase activity and then slowly inhibited this enzyme.

  6. 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.

  7. Linalool and linalool nerolidol synthases in roses, several genes for little scent.

    Science.gov (United States)

    Magnard, Jean-Louis; Bony, Aurélie Rius; Bettini, Fabienne; Campanaro, Ausilia; Blerot, Bernard; Baudino, Sylvie; Jullien, Frédéric

    2018-03-11

    Roses are widely appreciated for the appearance of their flowers and for their fragrance. This latter character results from the combination of different odorant molecules among which monoterpenes are often prevalent constituents. In this study, we report the cloning and characterization of three rose monoterpene synthases. In vitro functional characterization of these enzymes showed that one is a (-)-(3R)-linalool synthase whereas the others have a dual (+)-(3S)-linalool nerolidol synthase activity. However, given that the characterized rose cultivars were only able to produce the (-)-(3R)-linalool stereoisomer, the linalool nerolidol synthases are probably not active in planta. Furthermore, these three enzymes were also characterized by a weak expression level as assessed by RT-qPCR and by the low abundance of the corresponding sequences in an EST library. This characteristic is likely to explain why linalool is generally a minor constituent in rose flowers' scents. On this basis, we propose that in roses the monoterpene biosynthesis effort is focused on the production of acyclic monoterpenes derived from geraniol through the recently characterized Nudix biosynthesis pathway, at the expense of conventional monoterpene biosynthesis via terpene synthases such as linalool or linalool nerolidol synthases. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  8. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    International Nuclear Information System (INIS)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S.

    1991-01-01

    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-[ 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

  9. 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.

  10. Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.

    Science.gov (United States)

    Beekwilder, Jules; van Houwelingen, Adèle; Cankar, Katarina; van Dijk, Aalt D J; de Jong, René M; Stoopen, Geert; Bouwmeester, Harro; Achkar, Jihane; Sonke, Theo; Bosch, Dirk

    2014-02-01

    Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  11. The Regulation of NF-κB Subunits by Phosphorylation

    Directory of Open Access Journals (Sweden)

    Frank Christian

    2016-03-01

    Full Text Available The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function.

  12. DNA binding properties of the small cascade subunit Csa5.

    Directory of Open Access Journals (Sweden)

    Michael Daume

    Full Text Available CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

  13. NMDA receptor structures reveal subunit arrangement and pore architecture.

    Science.gov (United States)

    Lee, Chia-Hsueh; Lü, Wei; Michel, Jennifer Carlisle; Goehring, April; Du, Juan; Song, Xianqiang; Gouaux, Eric

    2014-07-10

    N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

  14. NMDA receptor structures reveal subunit arrangement and pore architecture

    Science.gov (United States)

    Lee, Chia-Hsueh; Lü, Wei; Michel, Jennifer Carlisle; Goehring, April; Du, Juan; Song, Xianqiang; Gouaux, Eric

    2014-01-01

    Summary N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present x-ray crystal structures of the GluN1/GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino terminal and ligand binding domains. The transmembrane domains harbor a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ~2-fold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors. PMID:25008524

  15. Binding of cholera toxin B subunit to intestinal epithelial cells.

    Science.gov (United States)

    Navolotskaya, Elena V; Sadovnikov, Vladimir B; Lipkin, Valery M; Zav'yalov, Vladimir P

    2018-03-01

    We have prepared 125 I-labeled cholera toxin B subunit ( 125 I-labeled CT-B, a specific activity of 98Ci/mmol) and found that it binds to rat IEC-6 and human Caco-2 intestinal epithelial cells with high affinity (K d 3.6 and 3.7nM, respectively). The binding of labeled protein was completely inhibited by unlabeled thymosin-α 1 (TM-α 1 ), interferon-α 2 (IFN-α 2 ), and the synthetic peptide LKEKK that corresponds to residues 16-20 in TM-α 1 and 131-135 in IFN-α 2 , but was not inhibited by the synthetic peptide KKEKL with inverted amino acid sequence (K i >10μM). Thus, TM-α 1 , IFN-α 2 , and the peptide: LKEKK bind with high affinity and specificity to the cholera toxin receptor on IEC-6 and Caco-2 cells. It was found that CT-B and the peptide: LKEKK at concentrations of 10-1000nM increased in a dose-dependent manner the nitric oxide production and the soluble guanylate cyclase activity in IEC-6 and Caco-2 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Vaccine profile of herpes zoster (HZ/su) subunit vaccine.

    Science.gov (United States)

    Cunningham, Anthony L; Heineman, Thomas

    2017-07-01

    Herpes zoster (HZ) causes an often severe and painful rash in older people and may be complicated by prolonged pain (postherpetic neuralgia; PHN) and by dissemination in immune-compromised patients. HZ results from reactivation of latent varicella-zoster virus (VZV) infection, often associated with age-related or other causes of decreased T cell immunity. A live attenuated vaccine boosts this immunity and provides partial protection against HZ, but this decreases with age and declines over 8 years. Areas covered: A new HZ subunit (HZ/su) vaccine combines a key surface VZV glycoprotein (E) with a T cell-boosting adjuvant system (AS01 B ) and is administered by two intramuscular injections two months apart. Expert commentary: HZ/su showed excellent efficacy of ~90% in immunocompetent adults ≥50 and ≥70 years of age, respectively, in the ZOE-50 and ZOE-70 phase III controlled trials. Efficacy was unaffected by advancing age and persisted for >3 years. Approximately 9.5% of subjects had severe, but transient (1-2 days) injection site pain, swelling or redness. Compliance with both vaccine doses was high (95%). The vaccine will have a major impact on HZ management. Phase I-II trials showed safety and immunogenicity in severely immunocompromised patients. Phase III trial results are expected soon.

  17. Design of a hyperstable 60-subunit protein icosahedron

    Science.gov (United States)

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

    2016-07-01

    The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  18. Fc receptor gamma subunit polymorphisms and systemic lupus erythematosus

    International Nuclear Information System (INIS)

    Al-Ansari, Aliya; Ollier, W.E.; Gonzalez-Gay, Miguel A.; Gul, Ahmet; Inanac, Murat; Ordi, Jose; Teh, Lee-Suan; Hajeer, Ali H.

    2004-01-01

    To investigate the possible association between Fc receptor gamma polymorphisms and systemic lupus erythematosus (SLE). We have investigated the full FcR gamma gene for polymorphisms using polymerase chain reaction (PCR)-single strand confirmational polymorphisms and DNA sequencing .The polymorphisms identified were genotype using PCR-restriction fragment length polymorphism. Systemic lupus erythematosus cases and controls were available from 3 ethnic groups: Turkish, Spanish and Caucasian. The study was conducted in the year 2001 at the Arthritis Research Campaign, Epidemiology Unit, Manchester University Medical School, Manchester, United Kingdom. Five single nucleotide polymorphisms were identified, 2 in the promoter, one in intron 4 and, 2 in the 3'UTR. Four of the 5 single nucleotide polymorphisms (SNPs) were relatively common and investigated in the 3 populations. Allele and genotype frequencies of all 4 investigated SNPs were not statistically different cases and controls. fc receptor gamma gene does not appear to contribute to SLE susceptibility. The identified polymorphisms may be useful in investigating other diseases where receptors containing the FcR gamma subunit contribute to the pathology. (author)

  19. Self-Subunit Swapping Occurs in Another Gene Type of Cobalt Nitrile Hydratase

    Science.gov (United States)

    Xia, Yuanyuan; Cui, Youtian; Kobayashi, Michihiko; Zhou, Zhemin

    2012-01-01

    Self-subunit swapping is one of the post-translational maturation of the cobalt-containing nitrile hydratase (Co-NHase) family of enzymes. All of these NHases possess a gene organization of , which allows the activator protein to easily form a mediatory complex with the α-subunit of the NHase after translation. Here, we discovered that the incorporation of cobalt into another type of Co-NHase, with a gene organization of , was also dependent on self-subunit swapping. We successfully isolated a recombinant NHase activator protein (P14K) of Pseudomonas putida NRRL-18668 by adding a Strep-tag N-terminal to the P14K gene. P14K was found to form a complex [α(StrepP14K)2] with the α-subunit of the NHase. The incorporation of cobalt into the NHase of P. putida was confirmed to be dependent on the α-subunit substitution between the cobalt-containing α(StrepP14K)2 and the cobalt-free NHase. Cobalt was inserted into cobalt-free α(StrepP14K)2 but not into cobalt-free NHase, suggesting that P14K functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, NHase from P. putida was also expressed by a mutant gene that was designed with a order. Our findings expand the general features of self-subunit swapping maturation. PMID:23226397

  20. Separation of ribosomal subunits of Escherichia coli by Sepharose chromatography using reverse salt gradient.

    Science.gov (United States)

    Kirillov, S V; Makhno, V I; Peshin, N N; Semenkov, Y P

    1978-11-01

    A mixture of 30 S and 50 S subunits quantitatively absorbs on a column of Sepharose--4B from the buffer: 0.02 M Tris--HCl, pH 7.5, containing 1.5 M (NH4)2SO4. During elution by reverse gradient of ammonium sulphate (1.5--0.05 M) the subunits are eluted at different salt concentrations. Complete separation of subunits is attained in the absence of Mg2+ ions. The 30 S subunits prepared from 70 S ribosomes according to this procedure are fully active in the codon--dependent binding of a specific aminoacyl--tRNA. After their reassociation with 50 S subunits isolated by zonal centrifugation, the resulting 70 S ribosomes are active in polypeptide synthesis at the same degree as control 70 S ribosomes in which both types of subunits were prepared by zonal centrifugation. The initial 70 S ribosomes for the chromatographic separation into subunits can be obtained by their pelleting from a crude extract with subsequent washing with concentrated solutions of NH4Cl in the ultracentrifuge, or by salt fractionation of the crude extract according to a slightly modified procedure of Kurland.

  1. Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM.

    Science.gov (United States)

    Roh, Soung-Hun; Hryc, Corey F; Jeong, Hyun-Hwan; Fei, Xue; Jakana, Joanita; Lorimer, George H; Chiu, Wah

    2017-08-01

    Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10-12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.

  2. Voltage-Gated Sodium Channel β1/β1B Subunits Regulate Cardiac Physiology and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Nnamdi Edokobi

    2018-04-01

    Full Text Available Cardiac myocyte contraction is initiated by a set of intricately orchestrated electrical impulses, collectively known as action potentials (APs. Voltage-gated sodium channels (NaVs are responsible for the upstroke and propagation of APs in excitable cells, including cardiomyocytes. NaVs consist of a single, pore-forming α subunit and two different β subunits. The β subunits are multifunctional cell adhesion molecules and channel modulators that have cell type and subcellular domain specific functional effects. Variants in SCN1B, the gene encoding the Nav-β1 and -β1B subunits, are linked to atrial and ventricular arrhythmias, e.g., Brugada syndrome, as well as to the early infantile epileptic encephalopathy Dravet syndrome, all of which put patients at risk for sudden death. Evidence over the past two decades has demonstrated that Nav-β1/β1B subunits play critical roles in cardiac myocyte physiology, in which they regulate tetrodotoxin-resistant and -sensitive sodium currents, potassium currents, and calcium handling, and that Nav-β1/β1B subunit dysfunction generates substrates for arrhythmias. This review will highlight the role of Nav-β1/β1B subunits in cardiac physiology and pathophysiology.

  3. Topographic Studies of Torpedo Acetylcholine Receptor Subunits as a Transmembrane Complex

    Science.gov (United States)

    Strader, Catherine D.; Raftery, Michael A.

    1980-10-01

    The exposure of the four subunits of the acetylcholine receptor from Torpedo californica on both the extracellular and cytoplasmic faces of the postsynaptic membranes of the electroplaque cells has been investigated. Sealed membrane vesicles containing no protein components other than the receptor were isolated and were shown to have 95% of their synaptic surfaces facing the medium. The susceptibility of the four receptor subunits in these preparations to hydrolysis by trypsin both from the external and from the internal medium was used to investigate the exposure of the subunits on the synaptic and cytoplasmic surfaces of the membrane. It was shown by sodium dodecyl sulfate gel electrophoresis of the tryptic products that all four subunits are exposed on the extracellular surface to a similar degree. All four subunits are also exposed on the internal surface of the membrane, but the apparent degree of exposure varies with the subunit size, the larger subunits being more exposed. The results are discussed in terms of a possible topographic model of the receptor as a transmembrane protein complex.

  4. Expression of gill vacuolar-type H+-ATPase B subunit, and Na+, K+-ATPase alpha- and beta- subunit messenger RNAs in smolting Salmo salar

    DEFF Research Database (Denmark)

    Seidelin, Michel; Madsen, Steffen; Cutler, Christopher P

    2001-01-01

    Changes in gill vacuolar-type H+-ATPase B subunit, and Na+,K+-ATPase alpha and beta subunit mRNA expression were examined during the course of smoltification in Salmo salar. We cloned and sequenced cDNA fragments of S. salar gill i) vacuolar-type H+-ATPase (V-H+-ATPase) B subunit, ii) Na+,K+-ATPase....... The peak smelt stage was, however, characterized by simultaneously elevated gill Na+,K+-ATPase expression and low V-H+-ATPase expression, and possibly ensures the complete transformation of the gill into a hypo-osmoregulatory organ and hence the development of optimal SW-tolerance of the smolt....... alpha (1) subunit, and iii) Na+,K+-ATPase beta (1) subunit, and used these as Northern blotting probes. During smoltification, the salmon showed a typical increase in gill Na+,K+-ATPase activity and improved hypo-osmoregulatory ability as judged by their ability to regulate plasma [Cl-] in a 24-hr...

  5. 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.

  6. Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase

    Science.gov (United States)

    Srividya, Narayanan; Davis, Edward M.; Croteau, Rodney B.; Lange, B. Markus

    2015-01-01

    Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (−)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed. PMID:25733883

  7. Enhanced (S)-linalool production by fusion expression of farnesyl diphosphate synthase and linalool synthase in Saccharomyces cerevisiae.

    Science.gov (United States)

    Deng, Yu; Sun, Mingxue; Xu, Sha; Zhou, Jingwen

    2016-07-01

    In order to improve the availability of geranyl diphosphate (GPP) in the mevalonate pathway for enhancing (S)-linalool production in Saccharomyces cerevisiae. A (S)-linalool synthase (LIS): AaLS1 from Actinidia arguta was coexpressed with FPPS with different peptide linkers to redirect the flux from geranyl diphosphate (GPP) to (S)-linalool production in S. cerevisiae. The strain with the best peptide linker ((GGGGS)3 ), produced 101·55 ± 2·97 μg l(-1) (S)-linalool, a 69·7% increase compared to those with two independent LIS and FPPS expressed. In a 3-l fermenter, the (S)-linalool titre was further improved to 240·64 ± 5·31 μg l(-1) . The results demonstrate that the fusion proteins catalysing consecutive steps in a metabolic pathway significantly improved the (S)-linalool production with GPP as precursor. The fusion protein strategy co-expressing AaLS1 and FPPS, assembled with a long peptide linker made S. cerevisiae produced the highest reported (S)-Linalool titre to date. © 2016 The Society for Applied Microbiology.

  8. An alternating GluN1-2-1-2 subunit arrangement in mature NMDA receptors.

    Directory of Open Access Journals (Sweden)

    Morgane Riou

    Full Text Available NMDA receptors (NMDARs form glutamate-gated ion channels that play a critical role in CNS physiology and pathology. Together with AMPA and kainate receptors, NMDARs are known to operate as tetrameric complexes with four membrane-embedded subunits associating to form a single central ion-conducting pore. While AMPA and some kainate receptors can function as homomers, NMDARs are obligatory heteromers composed of homologous but distinct subunits, most usually of the GluN1 and GluN2 types. A fundamental structural feature of NMDARs, that of the subunit arrangement around the ion pore, is still controversial. Thus, in a typical NMDAR associating two GluN1 and two GluN2 subunits, there is evidence for both alternating 1/2/1/2 and non-alternating 1/1/2/2 arrangements. Here, using a combination of electrophysiological and cross-linking experiments, we provide evidence that functional GluN1/GluN2A receptors adopt the 1/2/1/2 arrangement in which like subunits are diagonal to one another. Moreover, based on the recent crystal structure of an AMPA receptor, we show that in the agonist-binding and pore regions, the GluN1 subunits occupy a "proximal" position, closer to the central axis of the channel pore than that of GluN2 subunits. Finally, results obtained with reducing agents that differ in their membrane permeability indicate that immature (intracellular and functional (plasma-membrane inserted pools of NMDARs can adopt different subunit arrangements, thus stressing the importance of discriminating between the two receptor pools in assembly studies. Elucidating the quaternary arrangement of NMDARs helps to define the interface between the subunits and to understand the mechanism and pharmacology of these key signaling receptors.

  9. Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation.

    Science.gov (United States)

    Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

    2002-04-01

    The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A, NR2B, and NR2D subunit transcripts were present in both nondifferentiated and neuronally differentiated cultures, while NR2C subunits were expressed only transiently, during the early period of neural differentiation. Several splice variants of NR1 were detected in noninduced progenitors and in RA-induced cells, except the N1 exon containing transcripts that appeared after the fourth day of induction, when neuronal processes were already formed. NR1 and NR2A subunit proteins were detected both in nondifferentiated progenitor cells and in neurons, while the mature form of NR2B subunit protein appeared only at the time of neuronal process elongation. Despite the early presence of NR1 and NR2A subunits, NMDA-evoked responses could be detected in NE-4C neurons only after the sixth day of induction, coinciding in time with the expression of the mature NR2B subunit. The formation of functional NMDA receptors also coincided with the appearance of synapsin I and synaptophysin. The lag period between the production of the subunits and the onset of channel function suggests that subunits capable of channel formation cannot form functional NMDA receptors until a certain stage of neuronal commitment. Thus, the in vitro neurogenesis by NE-4C cells provides a suitable tool to investigate some inherent regulatory processes involved in the initial maturation of NMDA receptor complexes. Copyright 2002 Wiley Periodicals, Inc.

  10. Three human alcohol dehydrogenase subunits: cDNA structure and molecular and evolutionary divergence

    International Nuclear Information System (INIS)

    Ikuta, T.; Szeto, S.; Yoshida, A.

    1986-01-01

    Class I human alcohol dehydrogenase (ADH; alcohol:NAD + oxidoreductase, EC 1.1.1.1) consists of several homo- and heterodimers of α, β, and γ subunits that are governed by the ADH1, ADH2, and ADH3 loci. The authors previously cloned a full length of cDNA for the β subunit, and the complete sequence of 374 amino acid residues was established. cDNAs for the α and γ subunits were cloned and characterized. A human liver cDNA library, constructed in phage λgt11, was screened by using a synthetic oligonucleotide probe that was matched to the γ but not to the β sequence. Clone pUCADHγ21 and clone pUCADHα15L differed from β cDNA with respect to restriction sites and hybridization with the nucleotide probe. Clone pUCADHγ21 contained an insertion of 1.5 kilobase pairs (kbp) and encodes 374 amino acid residues compatible with the reported amino acid sequence of the γ subunit. Clone pUCADHα15L contained an insertion of 2.4 kbp and included nucleotide sequences that encode 374 amino acid residues for another subunit, the γ subunit. In addition, this clone contained the sequences that encode the COOH-terminal part of the β subunit at its extended 5' region. The amino acid sequences and coding regions of the cDNAs of the three subunits are very similar. A high degree of resemblance is observed also in their 3' noncoding regions. However, distinctive differences exist in the vicinity of the Zn-binding cysteine residue at position 46. Based on the cDNA sequences and the deduced amino acid sequences of the three subunits, their structural and evolutionary relationships are discussed

  11. Expression of BK Ca channels and the modulatory beta-subunits in the rat and porcine trigeminal ganglion

    DEFF Research Database (Denmark)

    Johansson, Helle Wulf; Hay-Schmidt, Anders; Poulsen, Asser Nyander

    2009-01-01

    (Ca) channel protein was visualized by western blotting and histochemistry. The presence of the modulatory beta1-beta 4 subunit mRNAs was investigated using RT-PCR. beta1-, beta2- and beta 4-subunit mRNAs were expressed in rat TG whereas beta2- and beta 4-subunits were detected in porcine TG. Western blotting...

  12. 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

  13. Control of ionizable residues in the catalytic mechanism of tryptophan synthase from Salmonella typhimurium.

    Science.gov (United States)

    Raboni, Samanta; Mozzarelli, Andrea; Cook, Paul F

    2007-11-13

    The tryptophan synthase alpha2beta2 complex catalyzes the last two steps in the biosynthesis of l-tryptophan in bacteria, plants, and fungi, the conversion of indole-3-glycerol phosphate and l-serine to l-tryptophan, glyceraldehyde 3-phosphate, and water. The beta-subunit binds pyridoxal 5'-phosphate and catalyzes the beta-replacement reaction with serine and indole. Structural, spectral, and kinetic studies indicate that different monovalent cations stabilize the alternative enzyme conformations and equilibrium distribution of the internal, external, and alpha-aminoacrylate Schiff base. To improve our understanding of the role of monovalent cations, the pH dependence of steady-state and pre-steady-state kinetic parameters and primary kinetic deuterium isotope effects were measured in the presence of l-serine and [alpha-2H]-l-serine in the absence and presence of Na+, K+, and Cs+. For the interpretation of the data obtained in this study, it was necessary to re-interpret a number of results published previously. Overall, data suggest that the enzyme exists in two conformers that equilibrate slowly either in the absence of substrates and monovalent cations or in the presence of K+ or Cs+, whereas they equilibrate faster in the presence of Na+. The rate of interconversion of the conformers increases as a group on the enzyme with a pKa of approximately 8 becomes deprotonated. The pH dependence of deuterium isotope effects is suggestive of a mechanism in which a pH-dependent conformational change that closes the active site precedes the chemical steps, likely a result of formation of one or more salt bridges. As the pH increases, the reaction becomes more committed to proceed to products, which causes the deuterium isotope effect to decrease to a value of unity at high pH. The closure of the site is modulated by the different monovalent cations and is fastest in the presence of Na+, which exhibits the maximum isotope effect of 5.7 (likely the intrinsic effect) on V

  14. Expression of Gill Vacuolar-Type H^+-ATPase B Subunit, and Na^+, K^+-ATPase α_1 and β_1 Subunit Messenger RNAs in Smolting Salmo salar(Physiology)

    OpenAIRE

    Michel, Seidelin; Steffen S., Madsen; Christopher P., Cutler; Gordon, Cramb; Institute of Biology, University of Southern Denmark-Main Campus : Odense University:(Present address)Department of Life Sciences and Chemistry, Roskilde University; Institute of Biology, University of Southern Denmark-Main Campus : Odense University; School of Biomedical Sciences, Bute Medical Buildings, University of St. Andrews; School of Biomedical Sciences, Bute Medical Buildings, University of St. Andrews

    2001-01-01

    Changes in gill vacuolar-type HT-ATPase B subunit, and Na^+,K^+-ATPase α and β subunit mRNA expression were examined during the course of smoltification in Salmo salar. We cloned and sequenced cDNA fragments of S. salar gill i) vacuolar-type H^+-ATPase (V-H^+-ATPase) B subunit, ii) Na^+,K^+-ATPase α_1 subunit, and iii) Na^+,K^+-ATPase β_1 subunit, and used these as Northern blotting probes. During smoltification, the salmon showed a typical increase in gill Na^+,K^+-ATPase activity and improv...

  15. Modulation of nucleotide binding to the catalytic sites of thermophilic F(1)-ATPase by the epsilon subunit: implication for the role of the epsilon subunit in ATP synthesis.

    Science.gov (United States)

    Yasuno, Taichi; Muneyuki, Eiro; Yoshida, Masasuke; Kato-Yamada, Yasuyuki

    2009-12-11

    Effect of epsilon subunit on the nucleotide binding to the catalytic sites of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been tested by using alpha(3)beta(3)gamma and alpha(3)beta(3)gammaepsilon complexes of TF(1) containing betaTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the epsilon subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the epsilon subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.

  16. 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.

  17. Discriminating the reaction types of plant type III polyketide synthases.

    Science.gov (United States)

    Shimizu, Yugo; Ogata, Hiroyuki; Goto, Susumu

    2017-07-01

    Functional prediction of paralogs is challenging in bioinformatics because of rapid functional diversification after gene duplication events combined with parallel acquisitions of similar functions by different paralogs. Plant type III polyketide synthases (PKSs), producing various secondary metabolites, represent a paralogous family that has undergone gene duplication and functional alteration. Currently, there is no computational method available for the functional prediction of type III PKSs. We developed a plant type III PKS reaction predictor, pPAP, based on the recently proposed classification of type III PKSs. pPAP combines two kinds of similarity measures: one calculated by profile hidden Markov models (pHMMs) built from functionally and structurally important partial sequence regions, and the other based on mutual information between residue positions. pPAP targets PKSs acting on ring-type starter substrates, and classifies their functions into four reaction types. The pHMM approach discriminated two reaction types with high accuracy (97.5%, 39/40), but its accuracy decreased when discriminating three reaction types (87.8%, 43/49). When combined with a correlation-based approach, all 49 PKSs were correctly discriminated, and pPAP was still highly accurate (91.4%, 64/70) even after adding other reaction types. These results suggest pPAP, which is based on linear discriminant analyses of similarity measures, is effective for plant type III PKS function prediction. pPAP is freely available at ftp://ftp.genome.jp/pub/tools/ppap/. goto@kuicr.kyoto-u.ac.jp. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  18. Hyaluronan synthase mediates dye translocation across liposomal membranes

    Directory of Open Access Journals (Sweden)

    Medina Andria P

    2012-01-01

    Full Text Available Abstract Background Hyaluronan (HA is made at the plasma membrane and secreted into the extracellular medium or matrix by phospolipid-dependent hyaluronan synthase (HAS, which is active as a monomer. Since the mechanism by which HA is translocated across membranes is still unresolved, we assessed the presence of an intraprotein pore within HAS by adding purified Streptococcus equisimilis HAS (SeHAS to liposomes preloaded with the fluorophore Cascade Blue (CB. Results CB translocation (efflux was not observed with mock-purified material from empty vector control E. coli membranes, but was induced by SeHAS, purified from membranes, in a time- and dose-dependent manner. CB efflux was eliminated or greatly reduced when purified SeHAS was first treated under conditions that inhibit enzyme activity: heating, oxidization or cysteine modification with N-ethylmaleimide. Reduced CB efflux also occurred with SeHAS K48E or K48F mutants, in which alteration of K48 within membrane domain 2 causes decreased activity and HA product size. The above results used liposomes containing bovine cardiolipin (BCL. An earlier study testing many synthetic lipids found that the best activating lipid for SeHAS is tetraoleoyl cardiolipin (TO-CL and that, in contrast, tetramyristoyl cardiolipin (TM-CL is an inactivating lipid (Weigel et al, J. Biol. Chem. 281, 36542, 2006. Consistent with the effects of these CL species on SeHAS activity, CB efflux was more than 2-fold greater in liposomes made with TO-CL compared to TM-CL. Conclusions The results indicate the presence of an intraprotein pore in HAS and support a model in which HA is translocated to the exterior by HAS itself.

  19. 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.

  20. 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.