Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine

PubMed Central

Zhang, Yan; Wang, Lei; Schultz, Peter G.; Wilson, Ian A.

2005-01-01

The Methanococcus jannaschii tRNATyr/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-l-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 Å, respectively, for comparison with the published structure of TyrRS complexed with tRNATyr and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257–263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through π-stacking and hydrogen bonding interactions. Loop 133–143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNATyr. Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133–143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over l-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids. PMID:15840835

Isoprene synthase genes form a monophyletic clade of acyclic terpene synthases in the TPS-B terpene synthase family.

PubMed

Sharkey, Thomas D; Gray, Dennis W; Pell, Heather K; Breneman, Steven R; Topper, Lauren

2013-04-01

Many plants emit significant amounts of isoprene, which is hypothesized to help leaves tolerate short episodes of high temperature. Isoprene emission is found in all major groups of land plants including mosses, ferns, gymnosperms, and angiosperms; however, within these groups isoprene emission is variable. The patchy distribution of isoprene emission implies an evolutionary pattern characterized by many origins or many losses. To better understand the evolution of isoprene emission, we examine the phylogenetic relationships among isoprene synthase and monoterpene synthase genes in the angiosperms. In this study we identify nine new isoprene synthases within the rosid angiosperms. We also document the capacity of a myrcene synthase in Humulus lupulus to produce isoprene. Isoprene synthases and (E)-β-ocimene synthases form a monophyletic group within the Tps-b clade of terpene synthases. No asterid genes fall within this clade. The chemistry of isoprene synthase and ocimene synthase is similar and likely affects the apparent relationships among Tps-b enzymes. The chronology of rosid evolution suggests a Cretaceous origin followed by many losses of isoprene synthase over the course of evolutionary history. The phylogenetic pattern of Tps-b genes indicates that isoprene emission from non-rosid angiosperms likely arose independently. © 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.

Biochemical and Physical Properties of the Methanococcus jannaschii 20S Proteasome and PAN, a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome†

PubMed Central

Wilson, Heather L.; Ou, Mark S.; Aldrich, Henry C.; Maupin-Furlow, Julie

2000-01-01

The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to ∼50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In addition, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by in vitro assembly of the α and β subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100°C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of β-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Δ1–73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80°C for the full-length protein to 65°C for PAN(Δ1–73). Both PAN protein complexes were stable in the absence of ATP and were inhibited by N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively high Vmax for ATP and CTP hydrolysis of 3.5 and 5.8 μmol of Pi per min per mg of protein as well as a relatively low affinity for CTP and ATP with Km values of 307 and 497 �

In Planta Recapitulation of Isoprene Synthase Evolution from Ocimene Synthases

PubMed Central

Li, Mingai; Xu, Jia; Algarra Alarcon, Alberto; Carlin, Silvia; Barbaro, Enrico; Cappellin, Luca; Velikova, Violeta; Vrhovsek, Urska; Loreto, Francesco; Varotto, Claudio

2017-01-01

Abstract Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated. We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated invivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause invivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms. Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance. PMID:28637270

In Planta Recapitulation of Isoprene Synthase Evolution from Ocimene Synthases.

PubMed

Li, Mingai; Xu, Jia; Algarra Alarcon, Alberto; Carlin, Silvia; Barbaro, Enrico; Cappellin, Luca; Velikova, Violeta; Vrhovsek, Urska; Loreto, Francesco; Varotto, Claudio

2017-10-01

Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated. We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated invivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause invivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms. Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

HAEM SYNTHASE AND COBALT PORPHYRIN SYNTHASE IN VARIOUS MICRO-ORGANISMS.

PubMed

PORRA, R J; ROSS, B D

1965-03-01

1. The preparation of a crude extract of Clostridium tetanomorphum containing cobalt porphyrin synthase but little haem-synthase activity is described. 2. The properties of cobalt porphyrin synthase in the clostridial extracts is compared with the properties of a haem synthase present in crude extracts of the yeast Torulopsis utilis. 3. Cobalt porphyrin synthase in extracts of C. tetanomorphum inserts Co(2+) ions into the following dicarboxylic porphyrins in descending order of rate of insertion: meso-, deutero- and proto-porphyrins. Esterification renders meso- and deutero-porphyrins inactive as substrates. Neither the tetracarboxylic (coproporphyrin III) nor the octacarboxylic (uroporphyrin III) compounds are converted into cobalt porphyrins by the extract, but the non-enzymic incorporation of Co(2+) ions into these two porphyrins is rapid. These extracts are unable to insert Mn(2+), Zn(2+), Mg(2+) or Cu(2+) ions into mesoporphyrin. 4. Crude extracts of T. utilis readily insert both Co(2+) and Fe(2+) ions into deutero-, meso, and proto-porphyrins. Unlike the extracts of C. tetanomorphum, these preparations catalyse the insertion of Co(2+) ions into deuteroporphyrin more rapidly than into mesoporphyrin. This parallels the formation of haems by the T. utilis extract. 5. Cobalt porphyrin synthase is present in the particulate fraction of the extracts of C. tetanomorphum but requires a heat-stable factor present in the soluble fraction. This soluble factor can be replaced by GSH. 6. Cobalt porphyrin synthase in the clostridial extract is inhibited by iodoacetamide and to a smaller extent by p-chloromercuribenzoate and N-ethylmaleimide. The haem synthases of T. utilis and Micrococcus denitrificans are also inhibited by various thiol reagents.

Sandalwood Fragrance Biosynthesis Involves Sesquiterpene Synthases of Both the Terpene Synthase (TPS)-a and TPS-b Subfamilies, including Santalene Synthases*

PubMed Central

Jones, Christopher G.; Moniodis, Jessie; Zulak, Katherine G.; Scaffidi, Adrian; Plummer, Julie A.; Ghisalberti, Emilio L.; Barbour, Elizabeth L.; Bohlmann, Jörg

2011-01-01

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus. PMID:21454632

Distribution of Callose Synthase, Cellulose Synthase, and Sucrose Synthase in Tobacco Pollen Tube Is Controlled in Dissimilar Ways by Actin Filaments and Microtubules1[W

PubMed Central

Cai, Giampiero; Faleri, Claudia; Del Casino, Cecilia; Emons, Anne Mie C.; Cresti, Mauro

2011-01-01

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules. PMID:21205616

Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases.

PubMed

Jones, Christopher G; Moniodis, Jessie; Zulak, Katherine G; Scaffidi, Adrian; Plummer, Julie A; Ghisalberti, Emilio L; Barbour, Elizabeth L; Bohlmann, Jörg

2011-05-20

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Geranyl diphosphate synthase from mint

DOEpatents

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

1999-01-01

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

Geranyl diphosphate synthase from mint

DOEpatents

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

1999-03-02

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

Monoterpene synthases from common sage (Salvia officinalis)

DOEpatents

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

1999-01-01

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

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

PubMed Central

Borejsza-Wysocki, W.; Hrazdina, G.

1996-01-01

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. PMID:12226219

Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soriano, Erika V.; McCloskey, Diane E.; Kinsland, Cynthia

2008-04-01

The crystal structures of two arginine decarboxylase mutant proteins provide insights into the mechanisms of pyruvoyl-group formation and the decarboxylation reaction. Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and themore » three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction.« less

[Advances in isoprene synthase research].

PubMed

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.

Biochemical Characterization and Homology Modeling of Methylbutenol Synthase and Implications for Understanding Hemiterpene Synthase Evolution in Plants*

PubMed Central

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

2011-01-01

2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ∼90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K+, whereas isoprene production is inhibited by K+ such that, at physiologically relevant [K+], little or no isoprene emission should be detected from MBO-emitting trees. The Km of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site. PMID:21504898

The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer.

PubMed

Cathcart, Mary-Clare; Reynolds, John V; O'Byrne, Kenneth J; Pidgeon, Graham P

2010-04-01

Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI(2)/TXA(2) ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer.

ATP synthase.

PubMed

Junge, Wolfgang; Nelson, Nathan

2015-01-01

Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

Polyester synthases: natural catalysts for plastics.

PubMed Central

Rehm, Bernd H A

2003-01-01

Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with

Molecular docking studies to map the binding site of squalene synthase inhibitors on dehydrosqualene synthase of Staphylococcus aureus.

PubMed

Kahlon, Amandeep Kaur; Roy, Sudeep; Sharma, Ashok

2010-10-01

Dehydrosqualene synthase of Staphylococcus aureus is involved in the synthesis of golden carotenoid pigment staphyloxanthin. This pigment of S. aureus provides the antioxidant property to this bacterium to survive inside the host cell. Dehydrosqualene synthase (CrtM) is having structural similarity with the human squalene synthase enzyme which is involved in the cholesterol synthesis pathway in humans (Liu et al., 2008). Cholesterol lowering drugs were found to have inhibitory effect on dehydrosqualene synthase enzyme of S. aureus. The present study attempts to focus on squalene synthase inhibitors, lapaquistat acetate and squalestatins reported as cholesterol lowering agents in vitro and in vivo but not studied in context to dehydrosqualene synthase of S. aureus. Mode of binding of lapaquistat acetate and squalestatin analogs on dehydrosqualene synthase (CrtM) enzyme of S. aureus was identified by performing docking analysis with Scigress Explorer Ultra 7.7 docking software. Based on the molecular docking analysis, it was found that the His18, Arg45, Asp48, Asp52, Tyr129, Gln165, Asn168 and Asp172 residues interacted with comparatively high frequency with the inhibitors studied. Comparative docking study with Discovery studio 2.0 also confirmed the involvement of these residues of dehydrosqualene synthase enzyme with the inhibitors studied. This further confirms the importance of these residues in the enzyme function. In silico ADMET analysis was done to predict the ADMET properties of the standard drugs and test compounds. This might provide insights to develop new drugs to target the virulence factor, dehydrosqualene synthase of S. aureus.

Volatile profiles of members of the USDA Geneva Malus Core Collection: utility in evaluation of a hypothesized biosynthetic pathway for esters derived from 2-methylbutanoate and 2-methylbutan-1-ol.

PubMed

Sugimoto, Nobuko; Forsline, Philip; Beaudry, Randolph

2015-02-25

The volatile ester and alcohol profiles of ripening apple fruit from 184 germplasm lines in the USDA Malus Germplasm Repository at the New York Agricultural Experiment Station in Geneva, NY, USA, were evaluated. Cluster analysis suggested biochemical relationships exist between several ester classes. A strong linkage was revealed between 2-methylbutanoate, propanoate, and butanoate esters, suggesting the influence of the recently proposed "citramalic acid pathway" in apple fruit. Those lines with a high content of esters formed from 2-methylbutan-1-ol and 2-methylbutanoate (2MB) relative to straight-chain (SC) esters (high 2MB/SC ratio) exhibited a marked increase in isoleucine and citramalic acid during ripening, but those lines with a low content did not. Thus, the data were consistent with the existence of the hypothesized citramalic acid pathway and suggest that the Geneva Malus Germplasm Repository, appropriately used, could be helpful in expanding our understanding of mechanisms for fruit volatile synthesis and other aspects of secondary metabolism.

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

PubMed

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.

Geranyl diphosphate synthase large subunit, and methods of use

DOEpatents

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

2001-10-16

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Inhibition of glycogen-synthase kinase 3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3-L1 adipocytes.

PubMed

Oreña, S J; Torchia, A J; Garofalo, R S

2000-05-26

The role of glycogen-synthase kinase 3 (GSK3) in insulin-stimulated glucose transport and glycogen synthase activation was investigated in 3T3-L1 adipocytes. GSK3 protein was clearly present in adipocytes and was found to be more abundant than in muscle and liver cell lines. The selective GSK3 inhibitor, LiCl, stimulated glucose transport and glycogen synthase activity (20 and 65%, respectively, of the maximal (1 microm) insulin response) and potentiated the responses to a submaximal concentration (1 nm) of insulin. LiCl- and insulin-stimulated glucose transport were abolished by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin; however, LiCl stimulation of glycogen synthase was not. In contrast to the rapid stimulation of glucose transport by insulin, transport stimulated by LiCl increased gradually over 3-5 h reaching 40% of the maximal insulin-stimulated level. Both LiCl- and insulin-stimulated glycogen synthase activity were maximal at 25 min. However, insulin-stimulated glycogen synthase activity returned to basal after 2 h, coincident with reactivation of GSK3. After a 2-h exposure to insulin, glycogen synthase was refractory to restimulation with insulin, indicating selective desensitization of this pathway. However, LiCl could partially stimulate glycogen synthase in desensitized cells. Furthermore, coincubation with LiCl during the 2 h exposure to insulin completely blocked desensitization of glycogen synthase activity. In summary, inhibition of GSK3 by LiCl: 1) stimulated glycogen synthase activity directly and independently of PI3-kinase, 2) stimulated glucose transport at a point upstream of PI3-kinase, 3) stimulated glycogen synthase activity in desensitized cells, and 4) prevented desensitization of glycogen synthase due to chronic insulin treatment. These data are consistent with GSK3 playing a central role in the regulation of glycogen synthase activity and a contributing factor in the regulation of glucose transport in 3T3-L1

Intersubunit structure within heterodimers of medium-chain prenyl diphosphate synthases. Formation of a hybrid-type heptaprenyl diphosphate synthase.

PubMed

Koike-Takeshita, A; Koyama, T; Ogura, K

1998-10-01

Among prenyltransferases that catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphate to produce prenyl diphosphates with various chain lengths and stereochemistries, medium-chain prenyl diphosphate synthases are exceptional in that they comprise two dissociable heteromeric protein components. These components exist without binding with each other under physiological conditions, and neither of them has any prenyltransferase activity by itself. In order to elucidate the precise molecular mechanism underlying expression of the catalytic function by such a unique two-component system, we examined the possibility of forming a hybrid between two of the components of three different medium-chain prenyl diphosphate synthases, components I and II of heptaprenyl diphosphate synthase from Bacillus subtilis, components I' and II' of heptaprenyl diphosphate synthase from Bacillus stearothermophilus, and components A and B of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26. As a result, only the hybrid-type combination of component I and component II' gave distinct prenyltransferase activity. The hybrid-type enzyme catalyzed the synthesis of heptaprenyl diphosphate and showed moderate heat stability, which lay between those of the natural enzymes from B. subtilis and B. stearothermophilus. There is no possibility of forming a hybrid between the heptaprenyl and hexaprenyl diphosphate synthases.

Glycogen synthase activation by sugars in isolated hepatocytes.

PubMed

Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

1988-07-01

We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

CTP synthase forms cytoophidia in the cytoplasm and nucleus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gou, Ke-Mian; State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193; Chang, Chia-Chun

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 synthasemore » 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.« less

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

PubMed

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.

Molecular architectures of benzoic acid-specific type III polyketide synthases

PubMed Central

Stewart, Charles; Woods, Kate; Macias, Greg; Allan, Andrew C.; Noel, Joseph P.

2017-01-01

Biphenyl synthase and benzophenone synthase constitute an evolutionarily distinct clade of type III polyketide synthases (PKSs) that use benzoic acid-derived substrates to produce defense metabolites in plants. The use of benzoyl-CoA as an endogenous substrate is unusual for type III PKSs. Moreover, sequence analyses indicate that the residues responsible for the functional diversification of type III PKSs are mutated in benzoic acid-specific type III PKSs. In order to gain a better understanding of structure–function relationships within the type III PKS family, the crystal structures of biphenyl synthase from Malus × domestica and benzophenone synthase from Hypericum androsaemum were compared with the structure of an archetypal type III PKS: chalcone synthase from Malus × domestica. Both biphenyl synthase and benzophenone synthase contain mutations that reshape their active-site cavities to prevent the binding of 4-coumaroyl-CoA and to favor the binding of small hydrophobic substrates. The active-site cavities of biphenyl synthase and benzophenone synthase also contain a novel pocket associated with their chain-elongation and cyclization reactions. Collectively, these results illuminate structural determinants of benzoic acid-specific type III PKSs and expand the understanding of the evolution of specialized metabolic pathways in plants. PMID:29199980

Sesquiterpene Synthase-3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Fusion Protein Responsible for Hirsutene Biosynthesis in Stereum hirsutum.

PubMed

Flynn, Christopher M; Schmidt-Dannert, Claudia

2018-06-01

The wood-rotting mushroom Stereum hirsutum is a known producer of a large number of namesake hirsutenoids, many with important bioactivities. Hirsutenoids form a structurally diverse and distinct class of sesquiterpenoids. No genes involved in hirsutenoid biosynthesis have yet been identified or their enzymes characterized. Here, we describe the cloning and functional characterization of a hirsutene synthase as an unexpected fusion protein of a sesquiterpene synthase (STS) with a C-terminal 3-hydroxy-3-methylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-CoA) synthase (HMGS) domain. Both the full-length fusion protein and truncated STS domain are highly product-specific 1,11-cyclizing STS enzymes with kinetic properties typical of STSs. Complementation studies in Saccharomyces cerevisiae confirmed that the HMGS domain is also functional in vivo Phylogenetic analysis shows that the hirsutene synthase domain does not form a clade with other previously characterized sesquiterpene synthases from Basidiomycota. Comparative gene structure analysis of this hirsutene synthase with characterized fungal enzymes reveals a significantly higher intron density, suggesting that this enzyme may be acquired by horizontal gene transfer. In contrast, the HMGS domain is clearly related to other fungal homologs. This STS-HMGS fusion protein is part of a biosynthetic gene cluster that includes P450s and oxidases that are expressed and could be cloned from cDNA. Finally, this unusual fusion of a terpene synthase to an HMGS domain, which is not generally recognized as a key regulatory enzyme of the mevalonate isoprenoid precursor pathway, led to the identification of additional HMGS duplications in many fungal genomes, including the localization of HMGSs in other predicted sesquiterpenoid biosynthetic gene clusters. IMPORTANCE Hirsutenoids represent a structurally diverse class of bioactive sesquiterpenoids isolated from fungi. Identification of their biosynthetic pathways will provide

Unusual features of a recombinant apple alpha-farnesene synthase.

PubMed

Green, Sol; Friel, Ellen N; Matich, Adam; Beuning, Lesley L; Cooney, Janine M; Rowan, Daryl D; MacRae, Elspeth

2007-01-01

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.

L-cysteine desulfidase: an [4Fe-4S] enzyme isolated from Methanocaldococcus jannaschii that catalyzes the breakdown of L-cysteine into pyruvate, ammonia, and sulfide.

PubMed

Tchong, Shih-I; Xu, Huimin; White, Robert H

2005-02-08

A [4Fe-4S] enzyme that decomposes L-cysteine to hydrogen sulfide, ammonia, and pyruvate has been isolated and characterized from Methanocaldococcus jannaschii. The sequence of the isolated enzyme demonstrated that the protein was the product of the M. jannaschii MJ1025 gene. The protein product of this gene was recombinantly produced in Escherichia coli and purified to homogeneity. Both the isolated and recombinant enzymes are devoid of pyridoxal phosphate (PLP) and are rapidly inactivated upon exposure to air. The air-inactivated enzyme is activated by reaction with Fe2+ and dithiothreitol in the absence of air. The air-inactivated enzyme contains 3 mol of iron per subunit (43 kDa, SDS gel electrophoresis), and the native enzyme has a measured molecular mass of 135 kDa (gel filtration), indicating it is a trimer. The enzyme is very specific for L-cysteine, with no activity being detected with D-cysteine, L-homocysteine, 3-mercaptopropionic acid (cysteine without the amino group), cysteamine (cysteine without the carboxylic acid), or mercaptolactate (the hydroxyl analogue of cysteine). The activity of the enzyme was stimulated by 40% when the enzyme was assayed in the presence of methyl viologen (4 mM) and inhibited by 70% when the enzyme was assayed in the presence of EDTA (7.1 mM). Preincubation of the enzyme with iodoacetamide (17 mM) completely abolishes activity. The enzymatic activity has a half-life of 8 or 12 min when the enzyme is treated at room temperature with 0.42 mM N-ethylmaleimide (NEM) or 0.42 mM iodoacetamide, respectively. MALDI analysis of the NEM-inactivated enzyme showed Cys25 as the site of alkylation. Site-directed mutagenesis of each of four of the cysteines conserved in the orthologues of the enzyme reduced the catalytic efficiency and thermal stability of the enzyme. The enzyme was found to catalyze exchange of the C-2 hydrogen of the L-cysteine with solvent. These results are consistent with three of the conserved cysteines being

Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle

PubMed Central

Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G.; Köllner, Tobias G.

2016-01-01

Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon–intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors. PMID:26936952

Studies of UMP synthase in orotic aciduria fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perry, M.E.; Jones, M.E.

UMP synthase catalyzes the final two reactions of de novo pyrimidine biosynthesis in mammals. UMP synthase activities are low in fibroblasts from a patient with hereditary orotic aciduria, but increase 80-100 fold to normal levels when the cells are incubated in the presence of 6-azauridine (6-azaU). Normal fibroblasts exhibit at most a two-fold increase in UMP synthase activities in response to 6-azaU. The increase in mutant cell enzyme activity is accompanied by increased UMP synthase protein in immunoprecipitates from (/sup 3//sub 5/S)-methionine-labeled cell extracts. This 6-azaU-dependent protein is precipitated by several monoclonal antibodies and polyclonal antibody raised against pure humanmore » UMP synthase. UMP synthase from normal and mutant fibroblasts comigrate on SDS gels and are stable for at least 2 1/2 hrs at 37/sup 0/C in the presence of a substrate, OMP. However, in the absence of substrate, at 57/sup 0/C, they have different inactivation patterns. Stability of the enzyme derived from normal cells > that of the enzyme from mutant cells cultured with 6-azaU > that of the enzyme from mutant cells. Southern blots of DNA from normal and mutant cells show identical restriction patterns with five enzymes. These results are consistent with the theory that the low level of UMP synthase in mutant cells reflects an increased susceptibility to proteolytic degradation which can be blocked by administration of 6-azaU to the cells in culture.« less

Clearing the skies over modular polyketide synthases.

PubMed

Sherman, David H; Smith, Janet L

2006-09-19

Modular polyketide synthases (PKSs) are large multifunctional proteins that synthesize complex polyketide metabolites in microbial cells. A series of recent studies confirm the close protein structural relationship between catalytic domains in the type I mammalian fatty acid synthase (FAS) and the basic synthase unit of the modular PKS. They also establish a remarkable similarity in the overall organization of the type I FAS and the PKS module. This information provides important new conclusions about catalytic domain architecture, function, and molecular recognition that are essential for future efforts to engineer useful polyketide metabolites with valuable biological activities.

Isolation and functional characterization of a τ-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia.

PubMed

Jullien, Frédéric; Moja, Sandrine; Bony, Aurélie; Legrand, Sylvain; Petit, Cécile; Benabdelkader, Tarek; Poirot, Kévin; Fiorucci, Sébastien; Guitton, Yann; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis

2014-01-01

In this paper we characterize three sTPSs: a germacrene D (LaGERDS), a (E)-β-caryophyllene (LaCARS) and a τ-cadinol synthase (LaCADS). τ-cadinol synthase is reported here for the first time and its activity was studied in several biological models including transiently or stably transformed tobacco species. Three dimensional structure models of LaCADS and Ocimum basilicum γ-cadinene synthase were built by homology modeling using the template structure of Gossypium arboreum δ-cadinene synthase. The depiction of their active site organization provides evidence of the global influence of the enzymes on the formation of τ-cadinol: instead of a unique amino-acid, the electrostatic properties and solvent accessibility of the whole active site in LaCADS may explain the stabilization of the cadinyl cation intermediate. Quantitative PCR performed from leaves and inflorescences showed two patterns of expression. LaGERDS and LaCARS were mainly expressed during early stages of flower development and, at these stages, transcript levels paralleled the accumulation of the corresponding terpene products (germacrene D and (E)-β-caryophyllene). By contrast, the expression level of LaCADS was constant in leaves and flowers. Phylogenetic analysis provided informative results on potential duplication process leading to sTPS diversification in lavender.

A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

PubMed

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.

A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase

PubMed Central

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

2017-01-01

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 phy-tochemicals is based on tradition or word of mouth with few evidence-based studies. Moreo-ver, 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 perti-nent. 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 die-tary phytochemicals are known to inhibit ATP synthase. Structural modifications of phyto-chemicals have been shown to increase the inhibitory potency and extent of inhibition. Site-directed 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 re-sult in selective binding and inhibition of microbial ATP synthase. In this review, the therapeu-tic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective target-ing of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. PMID:28831918

Divinyl ether synthase gene and protein, and uses thereof

DOEpatents

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.

Divinyl ether synthase gene, and protein and uses thereof

DOEpatents

Howe, Gregg A.; Itoh, Aya

2006-12-26

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.

Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus.

PubMed

Tsuge, Takeharu; Hyakutake, Manami; Mizuno, Kouhei

2015-08-01

This review highlights the recent investigations of class IV polyhydroxyalkanoate (PHA) synthases, the newest classification of PHA synthases. Class IV synthases are prevalent in organisms of the Bacillus genus and are composed of a catalytic subunit PhaC (approximately 40 kDa), which has a PhaC box sequence ([GS]-X-C-X-[GA]-G) at the active site, and a second subunit PhaR (approximately 20 kDa). The representative PHA-producing Bacillus strains are Bacillus megaterium and Bacillus cereus; the nucleotide sequence of phaC and the genetic organization of the PHA biosynthesis gene locus are somewhat different between these two strains. It is generally considered that class IV synthases favor short-chain-length monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization, but can polymerize some unusual monomers as minor components. In Escherichia coli expressing PhaRC from B. cereus YB-4, the biosynthesized PHA undergoes synthase-catalyzed alcoholytic cleavage using endogenous and exogenous alcohols. This alcoholysis is thought to be shared among class IV synthases, and this reaction is useful not only for the regulation of PHA molecular weight but also for the modification of the PHA carboxy terminus. The novel properties of class IV synthases will open up the possibility for the design of new PHA materials.

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

PubMed Central

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

2016-01-01

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

The chloroplast ATP synthase features the characteristic redox regulation machinery.

PubMed

Hisabori, Toru; Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

2013-11-20

Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system.

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

PubMed Central

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

2017-01-01

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

Cellulose in Cyanobacteria. Origin of Vascular Plant Cellulose Synthase?

PubMed Central

Nobles, David R.; Romanovicz, Dwight K.; Brown, R. Malcolm

2001-01-01

Although cellulose biosynthesis among the cyanobacteria has been suggested previously, we present the first conclusive evidence, to our knowledge, of the presence of cellulose in these organisms. Based on the results of x-ray diffraction, electron microscopy of microfibrils, and cellobiohydrolase I-gold labeling, we report the occurrence of cellulose biosynthesis in nine species representing three of the five sections of cyanobacteria. Sequence analysis of the genomes of four cyanobacteria revealed the presence of multiple amino acid sequences bearing the DDD35QXXRW motif conserved in all cellulose synthases. Pairwise alignments demonstrated that CesAs from plants were more similar to putative cellulose synthases from Anabaena sp. Pasteur Culture Collection 7120 and Nostoc punctiforme American Type Culture Collection 29133 than any other cellulose synthases in the database. Multiple alignments of putative cellulose synthases from Anabaena sp. Pasteur Culture Collection 7120 and N. punctiforme American Type Culture Collection 29133 with the cellulose synthases of other prokaryotes, Arabidopsis, Gossypium hirsutum, Populus alba × Populus tremula, corn (Zea mays), and Dictyostelium discoideum showed that cyanobacteria share an insertion between conserved regions U1 and U2 found previously only in eukaryotic sequences. Furthermore, phylogenetic analysis indicates that the cyanobacterial cellulose synthases share a common branch with CesAs of vascular plants in a manner similar to the relationship observed with cyanobacterial and chloroplast 16s rRNAs, implying endosymbiotic transfer of CesA from cyanobacteria to plants and an ancient origin for cellulose synthase in eukaryotes. PMID:11598227

Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopy

PubMed Central

Li, Wei; Hoffman, David W.

2001-01-01

Translation initiation factor 1A (aIF-1A) from the archaeon Methanococcus jannaschii was expressed in Escherichia coli, purified, and characterized in terms of its structure and dynamics using multidimensional NMR methods. The protein was found to be a member of the OB-fold family of RNA-associated proteins, containing a barrel of five beta-strands, a feature that is shared with the homologous eukaryotic translation initiation factor 1A (eIF-1A), as well as the prokaryotic translation initiation factor IF1. External to the β barrel, aIF-1A contains an α-helix at its C-terminal and a flexible loop at its N-terminal, features that are qualitatively similar to those found in eIF-1A, but not present in prokaryotic IF1. The structural model of aIF-1A, when used in combination with primary sequence information for aIF-1A in divergent species, permitted the most-conserved residues on the protein surface to be identified, including the most likely candidates for direct interaction with the 16S ribosomal RNA and other components of the translational apparatus. Several of the conserved surface residues appear to be unique to the archaea. Nitrogen-15 relaxation and amide exchange rate data were used to characterize the internal motions within aIF-1A, providing evidence that the protein surfaces that are most likely to participate in intermolecular interactions are relatively flexible. A model is proposed, suggesting some specific interactions that may occur between aIF-1A and the small subunit of the archaeal ribosome. PMID:11714910

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

PubMed Central

Assreuy, J.; Cunha, F. Q.; Liew, F. Y.; Moncada, S.

1993-01-01

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly. PMID:7682140

The Tomato Terpene Synthase Gene Family1[W][OA

PubMed Central

Falara, Vasiliki; Akhtar, Tariq A.; Nguyen, Thuong T.H.; Spyropoulou, Eleni A.; Bleeker, Petra M.; Schauvinhold, Ines; Matsuba, Yuki; Bonini, Megan E.; Schilmiller, Anthony L.; Last, Robert L.; Schuurink, Robert C.; Pichersky, Eran

2011-01-01

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far. PMID:21813655

The Chloroplast ATP Synthase Features the Characteristic Redox Regulation Machinery

PubMed Central

Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

2013-01-01

Abstract Significance: Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. Recent Advances: The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Critical Issues: Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. Future Directions: The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system. Antioxid. Redox Signal. 19, 1846–1854. PMID:23145525

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

PubMed

Jastreboff, M M; Todd, M B; Malech, H L; Bertino, J R

1985-01-29

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

ATP Synthase Diseases of Mitochondrial Genetic Origin

PubMed Central

Dautant, Alain; Meier, Thomas; Hahn, Alexander; Tribouillard-Tanvier, Déborah; di Rago, Jean-Paul; Kucharczyk, Roza

2018-01-01

Devastating human neuromuscular disorders have been associated to defects in the ATP synthase. This enzyme is found in the inner mitochondrial membrane and catalyzes the last step in oxidative phosphorylation, which provides aerobic eukaryotes with ATP. With the advent of structures of complete ATP synthases, and the availability of genetically approachable systems such as the yeast Saccharomyces cerevisiae, we can begin to understand these molecular machines and their associated defects at the molecular level. In this review, we describe what is known about the clinical syndromes induced by 58 different mutations found in the mitochondrial genes encoding membrane subunits 8 and a of ATP synthase, and evaluate their functional consequences with respect to recently described cryo-EM structures. PMID:29670542

Generation and Functional Evaluation of Designer Monoterpene Synthases.

PubMed

Srividya, N; Lange, I; Lange, B M

2016-01-01

Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes. © 2016 Elsevier Inc. All rights reserved.

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

PubMed Central

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

The Structural Enzymology of Iterative Aromatic Polyketide Synthases: A Critical Comparison with Fatty Acid Synthases.

PubMed

Tsai, Shiou-Chuan Sheryl

2018-06-20

Polyketides are a large family of structurally complex natural products including compounds with important bioactivities. Polyketides are biosynthesized by polyketide synthases (PKSs), multienzyme complexes derived evolutionarily from fatty acid synthases (FASs). The focus of this review is to critically compare the properties of FASs with iterative aromatic PKSs, including type II PKSs and fungal type I nonreducing PKSs whose chemical logic is distinct from that of modular PKSs. This review focuses on structural and enzymological studies that reveal both similarities and striking differences between FASs and aromatic PKSs. The potential application of FAS and aromatic PKS structures for bioengineering future drugs and biofuels is highlighted.

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

PubMed Central

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

2004-01-01

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. PMID:15550168

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans*

PubMed Central

Mohanty, Soumya; Jobichen, Chacko; Chichili, Vishnu Priyanka Reddy; Velázquez-Campoy, Adrián; Low, Boon Chuan; Hogue, Christopher W. V.; Sivaraman, J.

2015-01-01

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase. PMID:26370083

Natural and engineered polyhydroxyalkanoate (PHA) synthase: key enzyme in biopolyester production.

PubMed

Zou, Huibin; Shi, Mengxun; Zhang, Tongtong; Li, Lei; Li, Liangzhi; Xian, Mo

2017-10-01

With the finite supply of petroleum and increasing concern with environmental issues associated with their harvest and processing, the development of more eco-friendly, sustainable alternative biopolymers that can effectively fill the role of petro-polymers has become a major focus. Polyhydroxyalkanoate (PHA) can be naturally produced by many species of bacteria and the PHA synthase is believed to be key enzyme in this natural pathway. Natural PHA synthases are diverse and can affect the properties of the produced PHAs, such as monomer composition, molecular weights, and material properties. Moreover, recent studies have led to major advances in the searching of PHA synthases that display specific properties, as well as engineering efforts that offer more efficient PHA synthases, increased PHA compound production, or even novel biopolyesters which cannot be naturally produced. In this article, we review the updated information of natural PHA synthases and their engineering strategies for improved performance in polyester production. We also speculate future trends on the development of robust PHA synthases and their application in biopolyester production.

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

PubMed

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

2010-07-01

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

Cloning and Characterization of Inducible Nitric Oxide Synthase from Mouse Macrophages

NASA Astrophysics Data System (ADS)

Xie, Qiao-Wen; Cho, Hearn J.; Calaycay, Jimmy; Mumford, Richard A.; Swiderek, Kristine M.; Lee, Terry D.; Ding, Aihao; Troso, Tiffany; Nathan, Carl

1992-04-01

Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.

Functional Characterization of Novel Sesquiterpene Synthases from Indian Sandalwood, Santalum album

PubMed Central

Srivastava, Prabhakar Lal; Daramwar, Pankaj P.; Krithika, Ramakrishnan; Pandreka, Avinash; Shankar, S. Shiva; Thulasiram, Hirekodathakallu V.

2015-01-01

Indian Sandalwood, Santalum album L. is highly valued for its fragrant heartwood oil and is dominated by a blend of sesquiterpenes. Sesquiterpenes are formed through cyclization of farnesyl diphosphate (FPP), catalyzed by metal dependent terpene cyclases. This report describes the cloning and functional characterization of five genes, which encode two sesquisabinene synthases (SaSQS1, SaSQS2), bisabolene synthase (SaBS), santalene synthase (SaSS) and farnesyl diphosphate synthase (SaFDS) using the transcriptome sequencing of S. album. Using Illumina next generation sequencing, 33.32 million high quality raw reads were generated, which were assembled into 84,094 unigenes with an average length of 494.17 bp. Based on the transcriptome sequencing, five sesquiterpene synthases SaFDS, SaSQS1, SaSQS2, SaBS and SaSS involved in the biosynthesis of FPP, sesquisabinene, β-bisabolene and santalenes, respectively, were cloned and functionally characterized. Novel sesquiterpene synthases (SaSQS1 and SaSQS2) were characterized as isoforms of sesquisabinene synthase with varying kinetic parameters and expression levels. Furthermore, the feasibility of microbial production of sesquisabinene from both the unigenes, SaSQS1 and SaSQS2 in non-optimized bacterial cell for the preparative scale production of sesquisabinene has been demonstrated. These results may pave the way for in vivo production of sandalwood sesquiterpenes in genetically tractable heterologous systems. PMID:25976282

Functional Characterization of Novel Sesquiterpene Synthases from Indian Sandalwood, Santalum album.

PubMed

Srivastava, Prabhakar Lal; Daramwar, Pankaj P; Krithika, Ramakrishnan; Pandreka, Avinash; Shankar, S Shiva; Thulasiram, Hirekodathakallu V

2015-05-15

Indian Sandalwood, Santalum album L. is highly valued for its fragrant heartwood oil and is dominated by a blend of sesquiterpenes. Sesquiterpenes are formed through cyclization of farnesyl diphosphate (FPP), catalyzed by metal dependent terpene cyclases. This report describes the cloning and functional characterization of five genes, which encode two sesquisabinene synthases (SaSQS1, SaSQS2), bisabolene synthase (SaBS), santalene synthase (SaSS) and farnesyl diphosphate synthase (SaFDS) using the transcriptome sequencing of S. album. Using Illumina next generation sequencing, 33.32 million high quality raw reads were generated, which were assembled into 84,094 unigenes with an average length of 494.17 bp. Based on the transcriptome sequencing, five sesquiterpene synthases SaFDS, SaSQS1, SaSQS2, SaBS and SaSS involved in the biosynthesis of FPP, sesquisabinene, β-bisabolene and santalenes, respectively, were cloned and functionally characterized. Novel sesquiterpene synthases (SaSQS1 and SaSQS2) were characterized as isoforms of sesquisabinene synthase with varying kinetic parameters and expression levels. Furthermore, the feasibility of microbial production of sesquisabinene from both the unigenes, SaSQS1 and SaSQS2 in non-optimized bacterial cell for the preparative scale production of sesquisabinene has been demonstrated. These results may pave the way for in vivo production of sandalwood sesquiterpenes in genetically tractable heterologous systems.

[BIOINFORMATIC SEARCH AND PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENES OF FLAX (LINUM USITATISSIMUM)].

PubMed

Pydiura, N A; Bayer, G Ya; Galinousky, D V; Yemets, A I; Pirko, Ya V; Podvitski, T A; Anisimova, N V; Khotyleva, L V; Kilchevsky, A V; Blume, Ya B

2015-01-01

A bioinformatic search of sequences encoding cellulose synthase genes in the flax genome, and their comparison to dicots orthologs was carried out. The analysis revealed 32 cellulose synthase gene candidates, 16 of which are highly likely to encode cellulose synthases, and the remaining 16--cellulose synthase-like proteins (Csl). Phylogenetic analysis of gene products of cellulose synthase genes allowed distinguishing 6 groups of cellulose synthase genes of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7 and CesA8. Paralogous sequences within classes CesA1/10 and CesA5/6/2/9 which are associated with the primary cell wall formation are characterized by a greater similarity within these classes than orthologous sequences. Whereas the genes controlling the biosynthesis of secondary cell wall cellulose form distinct clades: CesA4, CesA7, and CesA8. The analysis of 16 identified flax cellulose synthase gene candidates shows the presence of at least 12 different cellulose synthase gene variants in flax genome which are represented in all six clades of cellulose synthase genes. Thus, at this point genes of all ten known cellulose synthase classes are identify in flax genome, but their correct classification requires additional research.

Anaerobic Coculture of Microalgae with Thermosipho globiformans and Methanocaldococcus jannaschii at 68°C Enhances Generation of n-Alkane-Rich Biofuels after Pyrolysis

PubMed Central

Matsuyama, Shigeru; Igarashi, Kensuke; Utsumi, Motoo; Shiraiwa, Yoshihiro; Kuwabara, Tomohiko

2013-01-01

We tested different alga-bacterium-archaeon consortia to investigate the production of oil-like mixtures, expecting that n-alkane-rich biofuels might be synthesized after pyrolysis. Thermosipho globiformans and Methanocaldococcus jannaschii were cocultured at 68°C with microalgae for 9 days under two anaerobic conditions, followed by pyrolysis at 300°C for 4 days. Arthrospira platensis (Cyanobacteria), Dunaliella tertiolecta (Chlorophyta), Emiliania huxleyi (Haptophyta), and Euglena gracilis (Euglenophyta) served as microalgal raw materials. D. tertiolecta, E. huxleyi, and E. gracilis cocultured with the bacterium and archaeon inhibited their growth and CH4 production. E. huxleyi had the strongest inhibitory effect. Biofuel generation was enhanced by reducing impurities containing alkanenitriles during pyrolysis. The composition and amounts of n-alkanes produced by pyrolysis were closely related to the lipid contents and composition of the microalgae. Pyrolysis of A. platensis and D. tertiolecta containing mainly phospholipids and glycolipids generated short-carbon-chain n-alkanes (n-tridecane to n-nonadecane) and considerable amounts of isoprenoids. E. gracilis also produced mainly short n-alkanes. In contrast, E. huxleyi containing long-chain (31 and 33 carbon atoms) alkenes and very long-chain (37 to 39 carbon atoms) alkenones, in addition to phospholipids and glycolipids, generated a high yield of n-alkanes of various lengths (n-tridecane to n-pentatriacontane). The gas chromatography-mass spectrometry (GC-MS) profiles of these n-alkanes were similar to those of native petroleum crude oils despite containing a considerable amount of n-hentriacontane. The ratio of phytane to n-octadecane was also similar to that of native crude oils. PMID:23183975

Effects and mechanism of acid rain on plant chloroplast ATP synthase.

PubMed

Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

2016-09-01

Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

Oxidation of thymidylate synthase by inorganic compounds.

PubMed

Aull, J L; Ivery, T C; Daron, H H

1984-10-01

Thymidylate synthase from methotrexate-resistant Lactobacillus casei was rapidly and completely inactivated by low concentrations of permanganate, periodate, or potassium triiodide at 0 degree C. The enzyme was not inactivated to any appreciable extent by iodate, iodide, ferricyanate, iodosobenzoate, or hydrogen peroxide. The inactivation by permanganate was retarded by the substrate 2'-deoxyuridylate and, to a lesser extent, by phosphate. Titration of enzyme activity with permanganate showed that two moles of permanganate were required to completely inactivate one mole of thymidylate synthase.

Identification of a Fungal 1,8-Cineole Synthase from Hypoxylon sp. with Specificity Determinants in Common with the Plant Synthases*

PubMed Central

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

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

PubMed

Lather, Amit; Sharma, Sunil; Khatkar, Anurag

2018-01-01

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

Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus

PubMed Central

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

2009-01-01

SUMMARY Fungi are a rich source of bioactive secondary metabolites and mushroom-forming fungi (Agaricomycetes) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared to the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpene oxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5, functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae. Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as a α-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes δ-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homolog, but instead was found to catalyze highly specific the synthesis of α-cuprenene. Co-expression of cop6 and the two monooxygenase genes next to it yields oxygenated α-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species. PMID:19400802

Characterization of the human gene (TBXAS1) encoding thromboxane synthase.

PubMed

Miyata, A; Yokoyama, C; Ihara, H; Bandoh, S; Takeda, O; Takahashi, E; Tanabe, T

1994-09-01

The gene encoding human thromboxane synthase (TBXAS1) was isolated from a human EMBL3 genomic library using human platelet thromboxane synthase cDNA as a probe. Nucleotide sequencing revealed that the human thromboxane synthase gene spans more than 75 kb and consists of 13 exons and 12 introns, of which the splice donor and acceptor sites conform to the GT/AG rule. The exon-intron boundaries of the thromboxane synthase gene were similar to those of the human cytochrome P450 nifedipine oxidase gene (CYP3A4) except for introns 9 and 10, although the primary sequences of these enzymes exhibited 35.8% identity each other. The 1.2-kb of the 5'-flanking region sequence contained potential binding sites for several transcription factors (AP-1, AP-2, GATA-1, CCAAT box, xenobiotic-response element, PEA-3, LF-A1, myb, basic transcription element and cAMP-response element). Primer-extension analysis indicated the multiple transcription-start sites, and the major start site was identified as an adenine residue located 142 bases upstream of the translation-initiation site. However, neither a typical TATA box nor a typical CAAT box is found within the 100-b upstream of the translation-initiation site. Southern-blot analysis revealed the presence of one copy of the thromboxane synthase gene per haploid genome. Furthermore, a fluorescence in situ hybridization study revealed that the human gene for thromboxane synthase is localized to band q33-q34 of the long arm of chromosome 7. A tissue-distribution study demonstrated that thromboxane synthase mRNA is widely expressed in human tissues and is particularly abundant in peripheral blood leukocyte, spleen, lung and liver. The low but significant levels of mRNA were observed in kidney, placenta and thymus.

ATP Synthase: A Molecular Therapeutic Drug Target for Antimicrobial and Antitumor Peptides

PubMed Central

Ahmad, Zulfiqar; Okafor, Florence; Azim, Sofiya; Laughlin, Thomas F.

2015-01-01

In this review we discuss the role of ATP synthase as a molecular drug target for natural and synthetic antimi-crobial/antitumor peptides. We start with an introduction of the universal nature of the ATP synthase enzyme and its role as a biological nanomotor. Significant structural features required for catalytic activity and motor functions of ATP synthase are described. Relevant details regarding the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it a potential drug target with respect to antimicrobial peptides and other inhibitors such as dietary polyphenols, is also reviewed. ATP synthase is known to have about twelve discrete inhibitor binding sites including peptides and other inhibitors located at the interface of α/β subunits on the F1 sector of the enzyme. Molecular interaction of peptides at the β DEELSEED site on ATP synthase is discussed with specific examples. An inhibitory effect of other natural/synthetic inhibitors on ATP is highlighted to explore the therapeutic roles played by peptides and other inhibitors. Lastly, the effect of peptides on the inhibition of the Escherichia coli model system through their action on ATP synthase is presented. PMID:23432591

Sucrose synthase in wild tomato Lycopersicon chmielewskii and tomato fruit sink strength

Treesearch

Shi-Jean S. Sung; T. Loboda; S.S. Sung; C.C. Black

1992-01-01

Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers.In mature, nongrowing fruits, sucrose synthase activities approached nil values.Therefore, sucrose synthase...

Functional reconstitution of cellulose synthase in Escherichia coli.

PubMed

Imai, Tomoya; Sun, Shi-Jing; Horikawa, Yoshiki; Wada, Masahisa; Sugiyama, Junji

2014-11-10

Cellulose is a high molecular weight polysaccharide of β1 → 4-d-glucan widely distributed in nature-from plant cell walls to extracellular polysaccharide in bacteria. Cellulose synthase, together with other auxiliary subunit(s) in the cell membrane, facilitates the fibrillar assembly of cellulose polymer chains into a microfibril. The gene encoding the catalytic subunit of cellulose synthase is cesA and has been identified in many cellulose-producing organisms. Very few studies, however, have shown that recombinant CesA protein synthesizes cellulose polymer, but the mechanism by which CesA protein synthesizes cellulose microfibrils is not known. Here we show that cellulose-synthesizing activity is successfully reconstituted in Escherichia coli by expressing the bacterial cellulose synthase complex of Gluconacetobacter xylinus: CesA and CesB (formerly BcsA and BcsB, respectively). Cellulose synthase activity was, however, only detected when CesA and CesB were coexpressed with diguanyl cyclase (DGC), which synthesizes cyclic-di-GMP (c-di-GMP), which in turn activates cellulose-synthesizing activity in bacteria. Direct observation by electron microscopy revealed extremely thin fibrillar structures outside E. coli cells, which were removed by cellulase treatment. This fiber structure is not likely to be the native crystallographic form of cellulose I, given that it was converted to cellulose II by a chemical treatment milder than ever described. We thus putatively conclude that this fine fiber is an unprecedented structure of cellulose. Despite the inability of the recombinant enzyme to synthesize the native structure of cellulose, the system described in this study, named "CESEC (CEllulose-Synthesizing E. Coli)", represents a useful tool for functional analyses of cellulose synthase and for seeding new nanomaterials.

Isolation and structural determination of squalene synthase inhibitor from Prunus mume fruit.

PubMed

Choi, Sung-Won; Hur, Nam-Yoon; Ahn, Soon-Cheol; Kim, Dong-Seob; Lee, Jae-Kwon; Kim, Dae-Ok; Park, Seung-Kook; Kim, Byung-Yong; Baik, Moo-Yeol

2007-12-01

Squalene synthase plays an important role in the cholesterol biosynthetic pathway. Inhibiting this enzyme in hypercholesterolemia can lower not only plasma cholesterol but also plasma triglyceride levels. A squalene synthase inhibitor was screened from Prunus mume fruit, and then purified via sequential processes of ethanol extraction, HP-20 column chromatography, ethyl acetate extraction, silica gel column chromatography, and crystallization. The squalene synthase inhibitor was identified as chlorogenic acid with a molecular mass of 354 Da and a molecular formula of C16H18O9 based on UV spectrophotometry, 1H and 13C NMRs, and mass spectrometry. Chlorogenic acid inhibited the squalene synthase of pig liver with an IC50 level of 100 nM. Since chlorogenic acid was an effective inhibitor against the squalene synthase of an animal source, it may be a potential therapeutic agent for hypercholesterolemia.

Emerging lipid-lowering drugs: squalene synthase inhibitors.

PubMed

Elsayed, Raghda K; Evans, Jeffery D

2008-06-01

Lapaquistat was the only squalene synthase inhibitor in Phase III clinical trials in Europe and the United States, but was recently discontinued from clinical development. Unlike statins, the inhibition of de novo cholesterol biosynthesis by lapaquistat does not deplete mevalonate, a precursor of isoprenoids. Isoprenoids are critical in cell growth and metabolism. The present review will focus on the chemistry, pharmacology, and lipid-lowering effects of novel squalene synthase inhibitors. A search of Pubmed, IPA, and GoogleScholar for studies (animal and human) and review articles published in English between 1990 and April 2008, using the search terms "squalene synthase inhibitors" or "lapaquistat". All clinical trials identified were then cross-referenced for their citations. All literature identified was then complied for this analysis. Lapaquistat mainly targets LDL-C, but may have some effect on HDL-C and TG. Preliminary reports on Phase II and Phase III associated lapaquistat 100 mg with elevated hepatic enzymes. Hepatotoxicity, possible drug-drug interaction with statins, and the investigation of a statin/coenzyme Q10 combination are among the few challenges that impeded lapaquistat's clinical development.

Heterologous expression of an active chitin synthase from Rhizopus oryzae.

PubMed

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.

Hybrid polyketide synthases

DOEpatents

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.

Purification and Biochemical Properties of Phytochromobilin Synthase from Etiolated Oat Seedlings1

PubMed Central

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

Discovery of DF-461, a Potent Squalene Synthase Inhibitor

PubMed Central

2013-01-01

We report the development of a new trifluoromethyltriazolobenzoxazepine series of squalene synthase inhibitors. Structure–activity studies and pharmacokinetics optimization on this series led to the identification of compound 23 (DF-461), which exhibited potent squalene synthase inhibitory activity, high hepatic selectivity, excellent rat hepatic cholesterol synthesis inhibitory activity, and plasma lipid lowering efficacy in nonrodent repeated dose studies. PMID:24900587

Inhibition of ATP Synthase by Chlorinated Adenosine Analogue

PubMed Central

Chen, Lisa S.; Nowak, Billie J.; Ayres, Mary L.; Krett, Nancy L.; Rosen, Steven T.; Zhang, Shuxing; Gandhi, Varsha

2009-01-01

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; FO intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the substrate, ADP, are known in tight binding (αdpβdp) and loose binding (αtpβtp) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the αtpβtp state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (αdpβdp) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF3 −. Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase. PMID:19477165

ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

PubMed Central

Teixeira, Felipe K.; Sanchez, Carlos G.; Hurd, Thomas R.; Seifert, Jessica R. K.; Czech, Benjamin; Preall, Jonathan B.; Hannon, Gregory J.; Lehmann, Ruth

2015-01-01

The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used1,2. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation. PMID:25915123

The type I fatty acid and polyketide synthases: a tale of two megasynthases

PubMed Central

Tsai, Shiou-Chuan

2008-01-01

This review chronicles the synergistic growth of the fields of fatty acid and polyketide synthesis over the last century. In both animal fatty acid synthases and modular polyketide synthases, similar catalytic elements are covalently linked in the same order in megasynthases. Whereas in fatty acid synthases the basic elements of the design remain immutable, guaranteeing the faithful production of saturated fatty acids, in the modular polyketide synthases, the potential of the basic design has been exploited to the full for the elaboration of a wide range of secondary metabolites of extraordinary structural diversity. PMID:17898897

Differences in the efficiency of reductive activation of methionine synthase and exogenous electron acceptors between the common polymorphic variants of human methionine synthase reductase.

PubMed

Olteanu, Horatiu; Munson, Troy; Banerjee, Ruma

2002-11-12

Methionine synthase reductase (MSR) catalyzes the conversion of the inactive form of human methionine synthase to the active state of the enzyme. This reaction is of paramount physiological importance since methionine synthase is an essential enzyme that plays a key role in the methionine and folate cycles. A common polymorphism in human MSR has been identified (66A --> G) that leads to replacement of isoleucine with methionine at residue 22 and has an allele frequency of 0.5. Another polymorphism is 524C --> T, which leads to the substitution of serine 175 with leucine, but its allele frequency is not known. The I22M polymorphism is a genetic determinant for mild hyperhomocysteinemia, a risk factor for cardiovascular disease. In this study, we have examined the kinetic properties of the M22/S175 and I22/S175 and the I22/L175 and I22/S175 pairs of variants. EPR spectra of the semiquinone forms of variants I22/S175 and M22/S175 are indistinguishable and exhibit an isotropic signal at g = 2.00. In addition, the electronic absorption and reduction stoichiometries with NADPH are identical in these variants. Significantly, the variants activate methionine synthase with the same V(max); however, a 3-4-fold higher ratio of MSR to methionine synthase is required to elicit maximal activity with the M22/S175 and I22/L175 variant versus the I22/S175 enzyme. Differences are also observed between the variants in the efficacies of reduction of the artificial electron acceptors: ferricyanide, 2,6-dichloroindophenol, 3-acetylpyridine adenine dinucleotide phosphate, menadione, and the anticancer drug doxorubicin. These results reveal differences in the interactions between the natural and artificial electron acceptors and MSR variants in vitro, which are predicted to result in less efficient reductive repair of methionine synthase in vivo.

Nitric Oxide Synthase and Neuronal NADPH Diaphorase are Identical in Brain and Peripheral Tissues

NASA Astrophysics Data System (ADS)

Dawson, Ted M.; Bredt, David S.; Fotuhi, Majid; Hwang, Paul M.; Snyder, Solomon H.

1991-09-01

NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.

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

PubMed

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

2012-03-01

Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and β-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/β-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively). Copyright © 2011 Elsevier GmbH. All rights reserved.

Characterization of a monoterpene synthase from Paeonia lactiflora producing α-pinene as its single product.

PubMed

Ma, Xiaohui; Guo, Juan; Ma, Ying; Jin, Baolong; Zhan, Zhilai; Yuan, Yuan; Huang, Luqi

2016-07-01

To identify a terpene synthase that catalyzes the conversion of geranyl pyrophosphate (GPP) to α-pinene and is involved in the biosynthesis of paeoniflorin. Two new terpene synthase genes were isolated from the transcriptome data of Peaonia lactiflora. Phylogenetic analysis and sequence characterization revealed that one gene, named PlPIN, encoded a monoterpene synthase that might be involved in the biosynthesis of paeoniflorin. In vitro enzyme assay showed that, in contrast to most monoterpene synthases, PlPIN encoded an α-pinene synthase which converted GPP into α-pinene as a single product. This newly identified α-pinene synthase could be used for improving paeoniflorin accumulation by metabolic engineering or for producing α-pinene via synthetic biology.

SbnG, a Citrate Synthase in Staphylococcus aureus

PubMed Central

Kobylarz, Marek J.; Grigg, Jason C.; Sheldon, Jessica R.; Heinrichs, David E.; Murphy, Michael E. P.

2014-01-01

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. We present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic gene clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. A structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production. PMID:25336653

Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants.

PubMed

Degenhardt, Jörg; Köllner, Tobias G; Gershenzon, Jonathan

2009-01-01

The multitude of terpene carbon skeletons in plants is formed by enzymes known as terpene synthases. This review covers the monoterpene and sesquiterpene synthases presenting an up-to-date list of enzymes reported and evidence for their ability to form multiple products. The reaction mechanisms of these enzyme classes are described, and information on how terpene synthase proteins mediate catalysis is summarized. Correlations between specific amino acid motifs and terpene synthase function are described, including an analysis of the relationships between active site sequence and cyclization type and a discussion of whether specific protein features might facilitate multiple product formation.

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

PubMed

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

2016-12-01

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

Mitochondrial Genome Integrity Mutations Uncouple the Yeast Saccharomyces cerevisiae ATP Synthase*║

PubMed Central

Wang, Yamin; Singh, Usha; Mueller, David M.

2013-01-01

The mitochondrial ATP synthase is a molecular motor, which couples the flow of rotons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the α-, β-, and γ-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the γ-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk. PMID:17244612

Purification and Characterization of 1-Aminocyclopropane-1-Carboxylate Synthase from Apple Fruits 1

PubMed Central

Yip, Wing-Kin; Dong, Jian-Guo; Yang, Shang Fa

1991-01-01

1-Aminocyclopropane-1-carboxylate (ACC) synthase, a key enzyme in ethylene biosynthesis, was isolated and partially purified from apple (Malus sylvestris Mill.) fruits. Unlike ACC synthase isolated from other sources, apple ACC synthase is associated with the pellet fraction and can be solubilized in active form with Triton X-100. Following five purification steps, the solubilized enzyme was purified over 5000-fold to a specific activity of 100 micromoles per milligram protein per hour, and its purity was estimated to be 20 to 30%. Using this preparation, specific monoclonal antibodies were raised. Monoclonal antibodies against ACC synthase immunoglobulin were coupled to protein-A agarose to make an immunoaffinity column, which effectively purified the enzyme from a relatively crude enzyme preparation (100 units per milligram protein). As with the tomato enzyme, apple ACC synthase was inactivated and radiolabeled by its substrate S-adenosyl-l-methionine. Apple ACC synthase was identified to be a 48-kilodalton protein based on the observation that it was specifically bound to immunoaffinity column and it was specifically radiolabeled by its substrate S-adenosyl-l-methionine. Images Figure 4 Figure 6 PMID:16667960

Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, K.K.; Sanduja, R.; Tsai, A.L.

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. Similarlymore » 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.« less

Cloning and kinetic characterization of Arabidopsis thaliana solanesyl diphosphate synthase.

PubMed

Hirooka, Kazutake; Bamba, Takeshi; Fukusaki, Ei-ichiro; Kobayashi, Akio

2003-03-01

trans -Long-chain prenyl diphosphate synthases catalyse the sequential condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate to produce the C(30)-C(50) prenyl diphosphates, which are precursors of the side chains of prenylquinones. Based on the relationship between product specificity and the region around the first aspartate-rich motif in trans -prenyl diphosphate synthases characterized so far, we have isolated the cDNA for a member of trans -long-chain prenyl diphosphate synthases from Arabidopsis thaliana. The cDNA was heterologously expressed in Escherichia coli, and the recombinant His(6)-tagged protein was purified and characterized. Product analysis revealed that the cDNA encodes solanesyl diphosphate (C(45)) synthase (At-SPS). At-SPS utilized farnesyl diphosphate (FPP; C(15)) and geranylgeranyl diphosphate (GGPP; C(20)), but did not accept either the C(5) or the C(10) allylic diphosphate as a primer substrate. The Michaelis constants for FPP and GGPP were 5.73 microM and 1.61 microM respectively. We also performed an analysis of the side chains of prenylquinones extracted from the A. thaliana plant, and showed that its major prenylquinones, i.e. plastoquinone and ubiquinone, contain the C(45) prenyl moiety. This suggests that At-SPS might be devoted to the biosynthesis of either or both of the prenylquinone side chains. This is the first established trans -long-chain prenyl diphosphate synthase from a multicellular organism.

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*

PubMed Central

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

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.

PubMed

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.

Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

PubMed Central

Ludtmann, Marthe H.R.; Angelova, Plamena R.; Ninkina, Natalia N.; Gandhi, Sonia

2016-01-01

Misfolded α-synuclein is a key factor in the pathogenesis of Parkinson's disease (PD). However, knowledge about a physiological role for the native, unfolded α-synuclein is limited. Using brains of mice lacking α-, β-, and γ-synuclein, we report that extracellular monomeric α-synuclein enters neurons and localizes to mitochondria, interacts with ATP synthase subunit α, and modulates ATP synthase function. Using a combination of biochemical, live-cell imaging and mitochondrial respiration analysis, we found that brain mitochondria of α-, β-, and γ-synuclein knock-out mice are uncoupled, as characterized by increased mitochondrial respiration and reduced mitochondrial membrane potential. Furthermore, synuclein deficiency results in reduced ATP synthase efficiency and lower ATP levels. Exogenous application of low unfolded α-synuclein concentrations is able to increase the ATP synthase activity that rescues the mitochondrial phenotypes observed in synuclein deficiency. Overall, the data suggest that α-synuclein is a previously unrecognized physiological regulator of mitochondrial bioenergetics through its ability to interact with ATP synthase and increase its efficiency. This may be of particular importance in times of stress or PD mutations leading to energy depletion and neuronal cell toxicity. SIGNIFICANCE STATEMENT Misfolded α-synuclein aggregations in the form of Lewy bodies have been shown to be a pathological hallmark in histological staining of Parkinson's disease (PD) patient brains. It is known that misfolded α-synuclein is a key driver in PD pathogenesis, but the physiological role of unfolded monomeric α-synuclein remains unclear. Using neuronal cocultures and isolated brain mitochondria of α-, β-, and γ-synuclein knock-out mice and monomeric α-synuclein, this current study shows that α-synuclein in its unfolded monomeric form improves ATP synthase efficiency and mitochondrial function. The ability of monomeric α-synuclein to enhance

Fo-driven Rotation in the ATP Synthase Direction against the Force of F1 ATPase in the FoF1 ATP Synthase*

PubMed Central

Martin, James; Hudson, Jennifer; Hornung, Tassilo; Frasch, Wayne D.

2015-01-01

Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the Fo motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F1 ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single FoF1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of Fo-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with Fo stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation. PMID:25713065

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

PubMed

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.

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.

PubMed

Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

2015-10-06

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases.

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM

PubMed Central

Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

2015-01-01

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001 PMID:26439008

Oligosaccharide Binding in Escherichia coli Glycogen Synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheng, Fang; Yep, Alejandra; Feng, Lei

2010-11-17

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

The Role of Light-Dark Regulation of the Chloroplast ATP Synthase.

PubMed

Kohzuma, Kaori; Froehlich, John E; Davis, Geoffry A; Temple, Joshua A; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A; Kramer, David M

2017-01-01

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se . Instead

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

PubMed Central

Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; Temple, Joshua A.; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A.; Kramer, David M.

2017-01-01

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead

Identification and Characterization of Daurichromenic Acid Synthase Active in Anti-HIV Biosynthesis.

PubMed

Iijima, Miu; Munakata, Ryosuke; Takahashi, Hironobu; Kenmoku, Hiromichi; Nakagawa, Ryuichi; Kodama, Takeshi; Asakawa, Yoshinori; Abe, Ikuro; Yazaki, Kazufumi; Kurosaki, Fumiya; Taura, Futoshi

2017-08-01

Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from Rhododendron dauricum We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of R. dauricum The gene coded for a 533-amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino-terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine-112 and cysteine-175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic Pichia pastoris , exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from Cannabis sativa , whereas DCA synthase catalyzes a novel cyclization reaction of the farnesyl moiety of a meroterpenoid natural product of plant origin. Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of R. dauricum plants, based on various analytical studies at the chemical, biochemical, and molecular levels. The extracellular localization of DCA also was confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA: the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells. © 2017 American Society of Plant Biologists. All Rights Reserved.

Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia).

PubMed

Landmann, Christian; Fink, Barbara; Festner, Maria; Dregus, Márta; Engel, Karl-Heinz; Schwab, Wilfried

2007-09-15

The essential oil of lavender (Lavandula angustifolia) is mainly composed of mono- and sesquiterpenes. Using a homology-based PCR strategy, two monoterpene synthases (LaLIMS and LaLINS) and one sesquiterpene synthase (LaBERS) were cloned from lavender leaves and flowers. LaLIMS catalyzed the formation of (R)-(+)-limonene, terpinolene, (1R,5S)-(+)-camphene, (1R,5R)-(+)-alpha-pinene, beta-myrcene and traces of alpha-phellandrene. The proportions of these products changed significantly when Mn(2+) was supplied as the cofactor instead of Mg(2+). The second enzyme LaLINS produced exclusively (R)-(-)-linalool, the main component of lavender essential oil. LaBERS transformed farnesyl diphosphate and represents the first reported trans-alpha-bergamotene synthase. It accepted geranyl diphosphate with higher affinity than farnesyl diphosphate and also produced monoterpenes, albeit at low rates. LaBERS is probably derived from a parental monoterpene synthase by the loss of the plastidial signal peptide and by broadening its substrate acceptance spectrum. The identification and description of the first terpene synthases from L. angustifolia forms the basis for the biotechnological modification of essential oil composition in lavender.

Producing alpha-olefins using polyketide synthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fortman, Jeffrey L.; Katz, Leonard; Steen, Eric J.

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lei, Lei; Singh, Abhishek; Bashline, Logan

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 plasmamore » 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.« less

The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

PubMed

Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

2015-05-22

HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genetics Home Reference: N-acetylglutamate synthase deficiency

MedlinePlus

... Hyperammonemia due to N-acetylglutamate synthase deficiency Screening, Technology and Research in Genetics Patient Support and Advocacy Resources (4 links) Children Living with Inherited Metabolic Diseases National Organization for ...

The Sucrose Synthase Gene Family in Chinese Pear (Pyrus bretschneideri Rehd.): Structure, Expression, and Evolution.

PubMed

Abdullah, Muhammad; Cao, Yungpeng; Cheng, Xi; Meng, Dandan; Chen, Yu; Shakoor, Awais; Gao, Junshan; Cai, Yongping

2018-05-11

Sucrose synthase (SS) is a key enzyme involved in sucrose metabolism that is critical in plant growth and development, and particularly quality of the fruit. Sucrose synthase gene families have been identified and characterized in plants various plants such as tobacco, grape, rice, and Arabidopsis . However, there is still lack of detailed information about sucrose synthase gene in pear. In the present study, we performed a systematic analysis of the pear ( Pyrus bretschneideri Rehd.) genome and reported 30 sucrose synthase genes. Subsequently, gene structure, phylogenetic relationship, chromosomal localization, gene duplications, promoter regions, collinearity, RNA-Seq data and qRT-PCR were conducted on these sucrose synthase genes. The transcript analysis revealed that 10 PbSSs genes (30%) were especially expressed in pear fruit development. Additionally, qRT-PCR analysis verified the RNA-seq data and shown that PbSS30 , PbSS24 , and PbSS15 have a potential role in the pear fruit development stages. This study provides important insights into the evolution of sucrose synthase gene family in pear and will provide assistance for further investigation of sucrose synthase genes functions in the process of fruit development, fruit quality and resistance to environmental stresses.

S-Sulfhydration of ATP synthase by hydrogen sulfide stimulates mitochondrial bioenergetics.

PubMed

Módis, Katalin; Ju, YoungJun; Ahmad, Akbar; Untereiner, Ashley A; Altaany, Zaid; Wu, Lingyun; Szabo, Csaba; Wang, Rui

2016-11-01

Mammalian cells can utilize hydrogen sulfide (H 2 S) to support mitochondrial respiration. The aim of our study was to explore the potential role of S-sulfhydration (a H 2 S-induced posttranslational modification, also known as S-persulfidation) of the mitochondrial inner membrane protein ATP synthase (F1F0 ATP synthase/Complex V) in the regulation of mitochondrial bioenergetics. Using a biotin switch assay, we have detected S-sulfhydration of the α subunit (ATP5A1) of ATP synthase in response to exposure to H 2 S in vitro. The H 2 S generator compound NaHS induced S-sulfhydration of ATP5A1 in HepG2 and HEK293 cell lysates in a concentration-dependent manner (50-300μM). The activity of immunocaptured mitochondrial ATP synthase enzyme isolated from HepG2 and HEK293 cells was stimulated by NaHS at low concentrations (10-100nM). Site-directed mutagenesis of ATP5A1 in HEK293 cells demonstrated that cysteine residues at positions 244 and 294 are subject to S-sulfhydration. The double mutant ATP synthase protein (C244S/C294S) showed a significantly reduced enzyme activity compared to control and the single-cysteine-mutated recombinant proteins (C244S or C294S). To determine whether endogenous H 2 S plays a role in the basal S-sulfhydration of ATP synthase in vivo, we compared liver tissues harvested from wild-type mice and mice deficient in cystathionine-gamma-lyase (CSE, one of the three principal mammalian H 2 S-producing enzymes). Significantly reduced S-sulfhydration of ATP5A1 was observed in liver homogenates of CSE -/- mice, compared to wild-type mice, suggesting a physiological role for CSE-derived endogenous H 2 S production in the S-sulfhydration of ATP synthase. Various forms of critical illness (including burn injury) upregulate H 2 S-producing enzymes and stimulate H 2 S biosynthesis. In liver tissues collected from mice subjected to burn injury, we detected an increased S-sulfhydration of ATP5A1 at the early time points post-burn. At later time points

ESR studies on reactivity of protein-derived tyrosyl radicals formed by prostaglandin H synthase and ribonucleotide reductase.

PubMed

Lassmann, G; Curtis, J; Liermann, B; Mason, R P; Eling, T E

1993-01-01

Using ESR spectroscopy, the ability of enzyme inhibitors to quench protein-derived tyrosyl radicals was studied in two different enzymes, prostaglandin H synthase and ribonucleotide reductase. The prostaglandin H synthase inhibitors indomethacin, eugenol, and MK-410 effectively prevent the formation of tyrosyl radicals during the oxidation of arachidonic acid by prostaglandin H synthase from ram seminal vesicles. A direct reaction with preformed tyrosyl radicals was observed only with eugenol. The other prostaglandin H synthase inhibitors were ineffective. The ribonucleotide reductase inhibitors hydroxyurea and 4-hydroxyanisole, which effectively inactivate the tyrosyl radical in the active site of ribonucleotide reductase present in tumor cells, exhibit a different reactivity with tyrosyl radicals formed by prostaglandin H synthase. Hydroxyurea quenches preformed tyrosyl radicals in prostaglandin H synthase weakly, whereas 4-hydroxyanisole does not quench tyrosyl radicals in prostaglandin H synthase at all. Eugenol, which quenches preformed prostaglandin H synthase-derived tyrosyl radicals, also quenches the tyrosyl radical in ribonucleotide reductase. The results suggest that the reactivity of protein-linked tyrosyl radicals in ribonucleotide reductase and those formed during prostaglandin H synthase catalysis are very different and have unrelated roles in enzyme catalysis.

The Maize Gene terpene synthase 1 Encodes a Sesquiterpene Synthase Catalyzing the Formation of (E)-β-Farnesene, (E)-Nerolidol, and (E,E)-Farnesol after Herbivore Damage1

PubMed Central

Schnee, Christiane; Köllner, Tobias G.; Gershenzon, Jonathan; Degenhardt, Jörg

2002-01-01

Maize (Zea mays) emits a mixture of volatile compounds upon attack by the Egyptian cotton leafworm (Spodoptera littoralis). These substances, primarily mono- and sesquiterpenes, are used by parasitic wasps to locate the lepidopteran larvae, which are their natural hosts. This interaction among plant, lepidopteran larvae, and hymenopteran parasitoids benefits the plant and has been termed indirect defense. The committed step in the biosynthesis of the different skeletal types of mono- and sesquiterpenes is catalyzed by terpene synthases, a class of enzymes that forms a large variety of mono- and sesquiterpene products from prenyl diphosphate precursors. We isolated a terpene synthase gene, terpene synthase 1 (tps1), from maize that exhibits only a low degree of sequence identity to previously identified terpene synthases. Upon expression in a bacterial system, the encoded enzyme produced the acyclic sesquiterpenes, (E)-β-farnesene, (E,E)-farnesol, and (3R)-(E)-nerolidol, the last an intermediate in the formation of (3E)-4,8-dimethyl-1,3,7-nonatriene. Both (E)-β-farnesene and (3E)-4,8-dimethyl-1,3,7-nonatriene are prominent compounds of the maize volatile blend that is emitted after herbivore damage. The biochemical characteristics of the encoded enzyme are similar to those of terpene synthases from both gymnosperms and dicotyledonous angiosperms, suggesting that catalysis involves a similar electrophilic reaction mechanism. The transcript level of tps1 in the maize cv B73 was elevated after herbivory, mechanical damage, and treatment with elicitors. In contrast, the increase in the transcript level of the tps1 gene or gene homolog in the maize cv Delprim after herbivory was less pronounced, suggesting that the regulation of terpene synthase expression may vary among maize varieties. PMID:12481088

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

DOE PAGES

Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; ...

2017-07-24

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas thosemore » expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Secdependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. But, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Thus, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas thosemore » expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Secdependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. But, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Thus, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead

Interactions of citrate synthases from osmoconforming and osmoregulating animals with salt: possible signs of molecular eco-adaptation?

PubMed

Sarkissian, I V

1977-01-01

This study considers differential sensitivity of citrate synthase (citrate oxaloacetatelyase [CoA acetylating]) EC 4.1.3.7. from an osmoconforming animal (sea anemone) and an osmoregulating animal (the pig) to salt. Attention is drawn to the fact that the osmoconforming sea anemone is in essence a sessile creature while the pig is readily mobile and able to change its ionic environment at will. It had been shown earlier that citrate synthase from another osmoconformer (oyster) is also not sensitive to ionic strength while citrate synthase from osmoregulating white shrimp is sensitive to increasing levels of salt. However, these enzymes are characteristically regulated by ATP and alpha-ketoglutarate. Both forms of citrate synthase are denatured by 6 M guanidine hydrochloride and are aided by salt levels in their refolding but the rate and extent of refolding of the osmoconformer citrate synthase are greater than those of the osmoregulator citrate synthase. Catalytic activity of both forms of citrate synthase is inhibited by incubation in distilled water; osmoconformer citrate synthase was inhibited completely in 7 h while osmoregulator citrate synthase was inhibited only 60% in this time and 80% after 22 h in distilled water. The eco-adaptive and evolutionary implications of these findings are discussed.

Insights into the surface topology of polyhydroxyalkanoate synthase: self-assembly of functionalized inclusions.

PubMed

Hooks, David O; Rehm, Bernd H A

2015-10-01

The polyhydroxyalkanoate (PHA) synthase catalyzes the synthesis of PHA and remains attached to the hydrophobic PHA inclusions it creates. Although this feature is actively exploited to generate functionalized biobeads via protein engineering, little is known about the structure of the PHA synthase. Here, the surface topology of Ralstonia eutropha PHA synthase was probed to inform rational protein engineering toward the production of functionalized PHA beads. Surface-exposed residues were detected by conjugating biotin to inclusion-bound PHA synthase and identifying the biotin-conjugated lysine and cysteine residues using peptide fingerprinting analysis. The identified sites (K77, K90, K139, C382, C459, and K518) were investigated as insertion sites for the generation of new protein fusions. Insertions of FLAG epitopes into exposed sites K77, K90, K139, and K518 were tolerated, retaining >65 % of in vivo activity. Sites K90, K139, and K518 were also tested by insertion of the immunoglobulin G (IgG)-binding domain (ZZ), successfully producing PHA inclusions able to bind human IgG in vitro. Although simultaneous insertions of the ZZ domain into two sites was permissive, insertion at all three lysine sites inactivated the synthase. The K90/K139 double ZZ insertion had the optimum IgG-binding capacity of 16 mg IgG/g wet PHA beads and could selectively purify the IgG fraction from human serum. Overall, this study identified surface-exposed flexible regions of the PHA synthase which either tolerate protein/peptide insertions or are critical for protein function. This further elucidates the structure and function of PHA synthase and provides new opportunities for generating functionalized PHA biobeads.

Exploring the Influence of Domain Architecture on the Catalytic Function of Diterpene Synthases.

PubMed

Pemberton, Travis A; Chen, Mengbin; Harris, Golda G; Chou, Wayne K W; Duan, Lian; Köksal, Mustafa; Genshaft, Alex S; Cane, David E; Christianson, David W

2017-04-11

Terpenoid synthases catalyze isoprenoid cyclization reactions underlying the generation of more than 80,000 natural products. Such dramatic chemodiversity belies the fact that these enzymes generally consist of only three domain folds designated as α, β, and γ. Catalysis by class I terpenoid synthases occurs exclusively in the α domain, which is found with α, αα, αβ, and αβγ domain architectures. Here, we explore the influence of domain architecture on catalysis by taxadiene synthase from Taxus brevifolia (TbTS, αβγ), fusicoccadiene synthase from Phomopsis amygdali (PaFS, (αα) 6 ), and ophiobolin F synthase from Aspergillus clavatus (AcOS, αα). We show that the cyclization fidelity and catalytic efficiency of the α domain of TbTS are severely compromised by deletion of the βγ domains; however, retention of the β domain preserves significant cyclization fidelity. In PaFS, we previously demonstrated that one α domain similarly influences catalysis by the other α domain [ Chen , M. , Chou , W. K. W. , Toyomasu , T. , Cane , D. E. , and Christianson , D. W. ( 2016 ) ACS Chem. Biol. 11 , 889 - 899 ]. Here, we show that the hexameric quaternary structure of PaFS enables cluster channeling. We also show that the α domains of PaFS and AcOS can be swapped so as to make functional chimeric αα synthases. Notably, both cyclization fidelity and catalytic efficiency are altered in all chimeric synthases. Twelve newly formed and uncharacterized C 20 diterpene products and three C 25 sesterterpene products are generated by these chimeras. Thus, engineered αβγ and αα terpenoid cyclases promise to generate chemodiversity in the greater family of terpenoid natural products.

Lessons from 455 Fusarium polyketide synthases

USDA-ARS?s Scientific Manuscript database

In fungi, polyketide synthases (PKSs) synthesize a structurally diverse array of secondary metabolites (SMs) with a range of biological activities. The most studied SMs are toxic to animals and/or plants, alter plant growth, have beneficial pharmaceutical activities, and/or are brightly colored pigm...

Bisabosquals, novel squalene synthase inhibitors. I. Taxonomy, fermentation, isolation and biological activities.

PubMed

Minagawa, K; Kouzuki, S; Nomura, K; Yamaguchi, T; Kawamura, Y; Matsushima, K; Tani, H; Ishii, K; Tanimoto, T; Kamigauchi, T

2001-11-01

In the course of screening for yeast squalene synthase inhibitors, bisabosqual A was isolated from the culture broth of Stachybotrys sp. RF-7260. The related compounds bisabosquals B, C and D were also isolated from Stachybotrys ruwenzoriensis RF-6853. Bisabosquals inhibited squalene synthases. IC50 values of bisabosqual A against the microsomal squalene synthases from Saccharomyces cerevisiae, Candida albicans, HepG2 cell and rat liver were 0.43, 0.25, 0.95 and 2.5 microg/ml, respectively. Bisabosqual C exhibited inhibitory activities similar to bisabosqual A. Bisabosqual A showed broad spectrum antifungal activity in vitro.

Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

2014-08-01

Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. Copyright © 2014 Elsevier Inc. All rights reserved.

5-Fluoroindole Resistance Identifies Tryptophan Synthase Beta Subunit Mutants in Arabidopsis Thaliana

PubMed Central

Barczak, A. J.; Zhao, J.; Pruitt, K. D.; Last, R. L.

1995-01-01

A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation. PMID:7635295

Resistance Phenotypes Mediated by Aminoacyl-Phosphatidylglycerol Synthases

PubMed Central

Arendt, Wiebke; Hebecker, Stefanie; Jäger, Sonja; Nimtz, Manfred

2012-01-01

The specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine or with lysine catalyzed by aminoacyl-phosphatidylglycerol synthases (aaPGS) was shown to render various organisms less susceptible to antibacterial agents. This study makes use of Pseudomonas aeruginosa chimeric mutant strains producing lysyl-phosphatidylglycerol (L-PG) instead of the naturally occurring alanyl-phosphatidylglycerol (A-PG) to study the resulting impact on bacterial resistance. Consequences of such artificial phospholipid composition were studied in the presence of an overall of seven antimicrobials (β-lactams, a lipopeptide antibiotic, cationic antimicrobial peptides [CAMPs]) to quantitatively assess the effect of A-PG substitution (with L-PG, L-PG and A-PG, increased A-PG levels). For the employed Gram-negative P. aeruginosa model system, an exclusive charge repulsion mechanism does not explain the attenuated antimicrobial susceptibility due to PG modification. Additionally, the specificity of nine orthologous aaPGS enzymes was experimentally determined. The newly characterized protein sequences allowed for the establishment of a significant group of A-PG synthase sequences which were bioinformatically compared to the related group of L-PG synthesizing enzymes. The analysis revealed a diverse origin for the evolution of A-PG and L-PG synthases, as the specificity of an individual enzyme is not reflected in terms of a characteristic sequence motif. This finding is relevant for future development of potential aaPGS inhibitors. PMID:22267511

Isolation and Characterization of Three New Monoterpene Synthases from Artemisia annua

PubMed Central

Ruan, Ju-Xin; Li, Jian-Xu; Fang, Xin; Wang, Ling-Jian; Hu, Wen-Li; Chen, Xiao-Ya; Yang, Chang-Qing

2016-01-01

Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5, and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, β-myrcene. Although both Mg2+ and Mn2+ were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn2+ for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography–mass spectrometry detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate, salicylic acid, and gibberellin, suggesting a role of these monoterpene synthases in plant–environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant. PMID:27242840

Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis).

PubMed

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.

Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

PubMed Central

Hyakutake, Manami; Tomizawa, Satoshi; Mizuno, Kouhei; Abe, Hideki

2014-01-01

Polyhydroxyalkanoate (PHA)-producing Bacillus strains express class IV PHA synthase, which is composed of the subunits PhaR and PhaC. Recombinant Escherichia coli expressing PHA synthase from Bacillus cereus strain YB-4 (PhaRCYB-4) showed an unusual reduction of the molecular weight of PHA produced during the stationary phase of growth. Nuclear magnetic resonance analysis of the low-molecular-weight PHA revealed that its carboxy end structure was capped by ethanol, suggesting that the molecular weight reduction was the result of alcoholytic cleavage of PHA chains by PhaRCYB-4 induced by endogenous ethanol. This scission reaction was also induced by exogenous ethanol in both in vivo and in vitro assays. In addition, PhaRCYB-4 was observed to have alcoholysis activity for PHA chains synthesized by other synthases. The PHA synthase from Bacillus megaterium (PhaRCBm) from another subgroup of class IV synthases was also assayed and was shown to have weak alcoholysis activity for PHA chains. These results suggest that class IV synthases may commonly share alcoholysis activity as an inherent feature. PMID:24334666

Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in Drosophila.

PubMed

Sawyer, Eric M; Brunner, Elizabeth C; Hwang, Yihharn; Ivey, Lauren E; Brown, Olivia; Bannon, Megan; Akrobetu, Dennis; Sheaffer, Kelsey E; Morgan, Oshauna; Field, Conroy O; Suresh, Nishita; Gordon, M Grace; Gunnell, E Taylor; Regruto, Lindsay A; Wood, Cricket G; Fuller, Margaret T; Hales, Karen G

2017-03-23

In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure. The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits. We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.

Torque generation mechanism of ATP synthase

NASA Astrophysics Data System (ADS)

Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

2010-03-01

ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

Genetics Home Reference: GM3 synthase deficiency

MedlinePlus

... GM3 synthase deficiency is characterized by recurrent seizures (epilepsy) and problems with brain development. Within the first ... Testing (1 link) Genetic Testing Registry: Amish infantile epilepsy syndrome Other Diagnosis and Management Resources (2 links) ...

Producing dicarboxylic acids using polyketide synthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

PubMed

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

2016-02-23

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

Medicinal Chemistry of ATP Synthase: A Potential Drug Target of Dietary Polyphenols and Amphibian Antimicrobial Peptides

PubMed Central

Ahmad, Zulfiqar; Laughlin, Thomas F.

2015-01-01

In this review we discuss the inhibitory effects of dietary polyphenols and amphibian antimicrobial/antitumor peptides on ATP synthase. In the beginning general structural features highlighting catalytic and motor functions of ATP synthase will be described. Some details on the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it an interesting drug target with respect to dietary polyphenols and amphibian antimicrobial peptides will also be reviewed. ATP synthase is known to have distinct polyphenol and peptide binding sites at the interface of α/β subunits. Molecular interaction of polyphenols and peptides with ATP synthase at their respective binding sites will be discussed. Binding and inhibition of other proteins or enzymes will also be covered so as to understand the therapeutic roles of both types of molecules. Lastly, the effects of polyphenols and peptides on the inhibition of Escherichia coli cell growth through their action on ATP synthase will also be presented. PMID:20586714

ATP Synthase, a Target for Dementia and Aging?

PubMed

Larrick, James W; Larrick, Jasmine W; Mendelsohn, Andrew R

2018-02-01

Advancing age is the biggest risk factor for development for the major life-threatening diseases in industrialized nations accounting for >90% of deaths. Alzheimer's dementia (AD) is among the most devastating. Currently approved therapies fail to slow progression of the disease, providing only modest improvements in memory. Recently reported work describes mechanistic studies of J147, a promising therapeutic molecule previously shown to rescue the severe cognitive deficits exhibited by aged, transgenic AD mice. Apparently, J147 targets the mitochondrial alpha-F1-ATP synthase (ATP5A). Modest inhibition of the ATP synthase modulates intracellular calcium to activate AMP-activated protein kinase to inhibit mammalian target of rapamycin, a known mechanism of lifespan extension from worms to mammals.

Interaction of Constitutive Nitric Oxide Synthases with Cyclooxygenases in Regulation of Bicarbonate Secretion in the Gastric Mucosa.

PubMed

Zolotarev, V A; Andreeva, Yu V; Vershinina, E; Khropycheva, R P

2017-05-01

Neuronal NO synthase blocker 7-nitroindazole suppressed bicarbonate secretion in rat gastric mucosa induced by mild local irritation with 1 M NaCl (pH 2.0). Non-selective blocker of neuronal and endothelial synthases, Nω-nitro-L-arginine (L-NNA), did not affect HCO 3 - production, but inhibited secretion after pretreatment with omeprazole. Non-selective cyclooxygenase blocker indomethacin inhibited HCO 3 - production under conditions of normal synthase activity and in the presence of L-NNA, but was ineffective when co-administered with 7-nitroindazole. It was concluded that neuronal and endothelial synthases are involved in different mechanisms of regulation of HCO 3 - secretion in the gastric mucosa induced by mild irritation. Activation of neuronal synthase stimulated HCO 3 - production, which is mediated mainly through activation of cyclooxygenase. Theoretically, activation of endothelial synthase should suppress HCO 3 - production. The effect of endothelial synthase depends on acid secretion in the stomach and bicarbonate concentration in the submucosa, as it was demonstrated in experiments with intravenous NaHCO 3 infusion.

Reduced methylation of the thromboxane synthase gene is correlated with its increased vascular expression in preeclampsia.

PubMed

Mousa, Ahmad A; Strauss, Jerome F; Walsh, Scott W

2012-06-01

Preeclampsia is characterized by increased thromboxane and decreased prostacyclin levels, which predate symptoms, and can explain some of the clinical manifestations of preeclampsia, including hypertension and thrombosis. In this study, we examined DNA methylation of the promoter region of the thromboxane synthase gene (TBXAS1) and the expression of thromboxane synthase in systemic blood vessels of normal pregnant and preeclamptic women. Thromboxane synthase is responsible for the synthesis of thromboxane A(2), a potent vasoconstrictor and activator of platelets. We also examined the effect of experimentally induced DNA hypomethylation on the expression of thromboxane synthase in a neutrophil-like cell line (HL-60 cells) and in cultured vascular smooth muscle and endothelial cells. We found that DNA methylation of the TBXAS1 promoter was decreased and thromboxane synthase expression was increased in omental arteries of preeclamptic women as compared with normal pregnant women. Increased thromboxane synthase expression was observed in vascular smooth muscles cells, endothelial cells, and infiltrating neutrophils. Experimentally induced DNA hypomethylation only increased expression of thromboxane synthase in the neutrophil-like cell line, whereas tumor necrosis factor-α, a neutrophil product, increased its expression in cultured vascular smooth muscle cells. Our study suggests that epigenetic mechanisms and release of tumor necrosis factor-α by infiltrating neutrophils could contribute to the increased expression of thromboxane synthase in maternal systemic blood vessels, contributing to the hypertension and coagulation abnormalities associated with preeclampsia.

The Polyketide Components of Waxes and the Cer-cqu Gene Cluster Encoding a Novel Polyketide Synthase, the β-Diketone Synthase, DKS.

PubMed

von Wettstein-Knowles, Penny

2017-07-10

The primary function of the outermost, lipophilic layer of plant aerial surfaces, called the cuticle, is preventing non-stomatal water loss. Its exterior surface is often decorated with wax crystals, imparting a blue-grey color. Identification of the barley Cer-c , -q and -u genes forming the 101 kb Cer-cqu gene cluster encoding a novel polyketide synthase-the β-diketone synthase (DKS), a lipase/carboxyl transferase, and a P450 hydroxylase, respectively, establishes a new, major pathway for the synthesis of plant waxes. The major product is a β-diketone (14,16-hentriacontane) aliphatic that forms long, thin crystalline tubes. A pathway branch leads to the formation of esterified alkan-2-ols.

Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot1[OPEN

PubMed Central

Yahyaa, Mosaab; Matsuba, Yuki; Brandt, Wolfgang; Doron-Faigenboim, Adi; Bar, Einat; McClain, Alan; Davidovich-Rikanati, Rachel; Lewinsohn, Efraim; Pichersky, Eran; Ibdah, Mwafaq

2015-01-01

Bay laurel (Laurus nobilis) is an agriculturally and economically important dioecious tree in the basal dicot family Lauraceae used in food and drugs and in the cosmetics industry. Bay leaves, with their abundant monoterpenes and sesquiterpenes, are used to impart flavor and aroma to food, and have also drawn attention in recent years because of their potential pharmaceutical applications. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, we performed RNA sequencing to profile the transcriptome of L. nobilis leaves. Bioinformatic analysis led to the identification of eight TPS complementary DNAs. We characterized the enzymes encoded by three of these complementary DNAs: a monoterpene synthase that belongs to the TPS-b clade catalyzes the formation of mostly 1,8-cineole; a sesquiterpene synthase belonging to the TPS-a clade catalyzes the formation of mainly cadinenes; and a diterpene synthase of the TPS-e/f clade catalyzes the formation of geranyllinalool. Comparison of the sequences of these three TPSs indicated that the TPS-a and TPS-b clades of the TPS gene family evolved early in the evolution of the angiosperm lineage, and that geranyllinalool synthase activity is the likely ancestral function in angiosperms of genes belonging to an ancient TPS-e/f subclade that diverged from the kaurene synthase gene lineages before the split of angiosperms and gymnosperms. PMID:26157114

Cyclophilin D Promotes Brain Mitochondrial F1FO ATP Synthase Dysfunction in Aging Mice

PubMed Central

Gauba, Esha; Guo, Lan; Du, Heng

2017-01-01

Brain aging is the known strongest risk factor for Alzheimer’s disease (AD). In recent years, mitochondrial deficits have been proposed to be a common mechanism linking brain aging to AD. Therefore, to elucidate the causative mechanisms of mitochondrial dysfunction in aging brains is of paramount importance for our understanding of the pathogenesis of AD, in particular its sporadic form. Cyclophilin D (CypD) is a specific mitochondrial protein. Recent studies have shown that F1FO ATP synthase oligomycin sensitivity conferring protein (OSCP) is a binding partner of CypD. The interaction of CypD with OSCP modulates F1FO ATP synthase function and mediates mitochondrial permeability transition pore (mPTP) opening. Here, we have found that increased CypD expression, enhanced CypD/OSCP interaction, and selective loss of OSCP are prominent brain mitochondrial changes in aging mice. Along with these changes, brain mitochondria from the aging mice demonstrated decreased F1FO ATP synthase activity and defective F1FO complex coupling. In contrast, CypD deficient mice exhibited substantially mitigated brain mitochondrial F1FO ATP synthase dysfunction with relatively preserved mitochondrial function during aging. Interestingly, the aging-related OSCP loss was also dramatically attenuated by CypD depletion. Therefore, the simplest interpretation of this study is that CypD promotes F1FO ATP synthase dysfunction and the resultant mitochondrial deficits in aging brains. In addition, in view of CypD and F1FO ATP synthase alterations seen in AD brains, the results further suggest that CypD-mediated F1FO ATP synthase deregulation is a shared mechanism linking mitochondrial deficits in brain aging and AD. PMID:27834780

Cyclophilin D Promotes Brain Mitochondrial F1FO ATP Synthase Dysfunction in Aging Mice.

PubMed

Gauba, Esha; Guo, Lan; Du, Heng

2017-01-01

Brain aging is the known strongest risk factor for Alzheimer's disease (AD). In recent years, mitochondrial deficits have been proposed to be a common mechanism linking brain aging to AD. Therefore, to elucidate the causative mechanisms of mitochondrial dysfunction in aging brains is of paramount importance for our understanding of the pathogenesis of AD, in particular its sporadic form. Cyclophilin D (CypD) is a specific mitochondrial protein. Recent studies have shown that F1FO ATP synthase oligomycin sensitivity conferring protein (OSCP) is a binding partner of CypD. The interaction of CypD with OSCP modulates F1FO ATP synthase function and mediates mitochondrial permeability transition pore (mPTP) opening. Here, we have found that increased CypD expression, enhanced CypD/OSCP interaction, and selective loss of OSCP are prominent brain mitochondrial changes in aging mice. Along with these changes, brain mitochondria from the aging mice demonstrated decreased F1FO ATP synthase activity and defective F1FO complex coupling. In contrast, CypD deficient mice exhibited substantially mitigated brain mitochondrial F1FO ATP synthase dysfunction with relatively preserved mitochondrial function during aging. Interestingly, the aging-related OSCP loss was also dramatically attenuated by CypD depletion. Therefore, the simplest interpretation of this study is that CypD promotes F1FO ATP synthase dysfunction and the resultant mitochondrial deficits in aging brains. In addition, in view of CypD and F1FO ATP synthase alterations seen in AD brains, the results further suggest that CypD-mediated F1FO ATP synthase deregulation is a shared mechanism linking mitochondrial deficits in brain aging and AD.

Expression and Activity of Nitric Oxide Synthase Isoforms in Methamphetamine-Induced Striatal Dopamine Toxicity

PubMed Central

Friend, Danielle M.; Son, Jong H.; Keefe, Kristen A.

2013-01-01

Nitric oxide is implicated in methamphetamine (METH)-induced neurotoxicity; however, the source of the nitric oxide has not been identified. Previous work has also revealed that animals with partial dopamine loss induced by a neurotoxic regimen of methamphetamine fail to exhibit further decreases in striatal dopamine when re-exposed to methamphetamine 7–30 days later. The current study examined nitric oxide synthase expression and activity and protein nitration in striata of animals administered saline or neurotoxic regimens of methamphetamine at postnatal days 60 and/or 90, resulting in four treatment groups: Saline:Saline, METH:Saline, Saline:METH, and METH:METH. Acute administration of methamphetamine on postnatal day 90 (Saline:METH and METH:METH) increased nitric oxide production, as evidenced by increased protein nitration. Methamphetamine did not, however, change the expression of endothelial or inducible isoforms of nitric oxide synthase, nor did it change the number of cells positive for neuronal nitric oxide synthase mRNA expression or the amount of neuronal nitric oxide synthase mRNA per cell. However, nitric oxide synthase activity in striatal interneurons was increased in the Saline:METH and METH:METH animals. These data suggest that increased nitric oxide production after a neurotoxic regimen of methamphetamine results from increased nitric oxide synthase activity, rather than an induction of mRNA, and that constitutively expressed neuronal nitric oxide synthase is the most likely source of nitric oxide after methamphetamine administration. Of interest, animals rendered resistant to further methamphetamine-induced dopamine depletions still show equivalent degrees of methamphetamine-induced nitric oxide production, suggesting that nitric oxide production alone in response to methamphetamine is not sufficient to induce acute neurotoxic injury. PMID:23230214

14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase.

PubMed

Bunney, T D; van Walraven, H S; de Boer, A H

2001-03-27

Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our understanding of regulatory mechanisms is still rather preliminary. Here we report a role for 14-3-3 proteins in the regulation of ATP synthases. These 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate a wide range of enzymes in plants, animals, and yeast. Recently, the presence of 14-3-3 proteins in chloroplasts was illustrated, and we show here that plant mitochondria harbor 14-3-3s within the inner mitochondrial-membrane compartment. There, the 14-3-3 proteins were found to be associated with the ATP synthases, in a phosphorylation-dependent manner, through direct interaction with the F(1) beta-subunit. The activity of the ATP synthases in both organelles is drastically reduced by recombinant 14-3-3. The rapid reduction in chloroplast ATPase activity during dark adaptation was prevented by a phosphopeptide containing the 14-3-3 interaction motif, demonstrating a role for endogenous 14-3-3 in the down-regulation of the CF(o)F(1) activity. We conclude that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply.

RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula).

PubMed

Adal, Ayelign M; Sarker, Lukman S; Malli, Radesh P N; Liang, Ping; Mahmoud, Soheil S

2018-06-09

Using RNA-Seq, we cloned and characterized a unique monoterpene synthase responsible for the formation of a scent-determining S-linalool constituent of lavender oils from Lavandula × intermedia. Several species of Lavandula produce essential oils (EOs) consisting mainly of monoterpenes including linalool, one of the most abundant and scent-determining oil constituents. Although R-linalool dominates the EOs of lavenders, varying amounts (depending on the species) of the S-linalool enantiomer can also be found in these plants. Despite its relatively low abundance, S-linalool contributes a sweet, pleasant scent and is an important constituent of lavender EOs. While several terpene synthase genes including R-linalool synthase have been cloned from lavenders many important terpene synthases including S-linalool synthase have not been described from these plants. In this study, we employed RNA-Seq and other complementary sequencing data to clone and functionally characterize the sparsely expressed S-linalool synthase cDNA (LiS-LINS) from Lavandula × intermedia. Recombinant LiS-LINS catalyzed the conversion of the universal monoterpene precursor geranyl diphosphate to S-linalool as the sole product. Intriguingly, LiS-LINS exhibited very low (~ 30%) sequence similarity to other Lavandula terpene synthases, including R-linalool synthase. However, the predicted 3D structure of this protein, including the composition and arrangement of amino acids at the active site, is highly homologous to known terpene synthase proteins. LiS-LINS transcripts were detected in flowers, but were much less abundant than those corresponding to LiR-LINS, paralleling enantiomeric composition of linalool in L. × intermedia oils. These data indicate that production of S-linalool is at least partially controlled at the level of transcription from LiS-LINS. The cloned LiS-LINS cDNA may be used to enhance oil composition in lavenders and other plants through metabolic engineering.

The Structure of Sucrose Synthase-1 from Arabidopsis thaliana and Its Functional Implications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Yi; Anderson, Spencer; Zhang, Yanfeng

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) hasmore » 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.« less

Identification and characterization of two bisabolene synthases from linear glandular trichomes of sunflower (Helianthus annuus L., Asteraceae).

PubMed

Aschenbrenner, Anna-Katharina; Kwon, Moonhyuk; Conrad, Jürgen; Ro, Dae-Kyun; Spring, Otmar

2016-04-01

Sunflower is known to produce a variety of bisabolene-type sesquiterpenes and accumulates these substances in trichomes of leaves, stems and flowering parts. A bioinformatics approach was used to identify the enzyme responsible for the initial step in the biosynthesis of these compounds from its precursor farnesyl pyrophosphate. Based on sequence similarity with a known bisabolene synthases from Arabidopsis thaliana AtTPS12, candidate genes of Helianthus were searched in EST-database and used to design specific primers. PCR experiments identified two candidates in the RNA pool of linear glandular trichomes of sunflower. Their sequences contained the typical motifs of sesquiterpene synthases and their expression in yeast functionally characterized them as bisabolene synthases. Spectroscopic analysis identified the stereochemistry of the product of both enzymes as (Z)-γ-bisabolene. The origin of the two sunflower bisabolene synthase genes from the transcripts of linear trichomes indicates that they may be involved in the synthesis of sesquiterpenes produced in these trichomes. Comparison of the amino acid sequences of the sunflower bisabolene synthases showed high similarity with sesquiterpene synthases from other Asteracean species and indicated putative evolutionary origin from a β-farnesene synthase. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of neuronal nitric oxide synthase in ovine model of acute lung injury*

PubMed Central

Enkhbaatar, Perenlei; Connelly, Rhykka; Wang, Jianpu; Nakano, Yoshimitsu; Lange, Matthias; Hamahata, Atsumori; Horvath, Eszter; Szabo, Csaba; Jaroch, Stefan; Hölscher, Peter; Hillmann, Margrit; Traber, Lillian D.; Schmalstieg, Frank C.; Herndon, David N.; Traber, Daniel L.

2013-01-01

Objective Acute respiratory distress syndrome/acute lung injury is a serious complication of burn patients with concomitant smoke inhalation injury. Nitric oxide has been shown to play a major role in pulmonary dysfunction from thermal damage. In this study, we have tested the hypothesis that inhibition of neuronal nitric oxide synthase could ameliorate the severity of acute lung injury using our well-established ovine model of cutaneous burn and smoke inhalation. Design Prospective, randomized, controlled, experimental animals study. Setting Investigational intensive care unit at university hospital. Subjects Adult female sheep Interventions Female sheep (n = 16) were surgically prepared for the study. Seven days after surgery, all sheep were randomly allocated into three study groups: sham (noninjured, nontreated, n = 6); control (injured, treated with saline, n = 6); and neuronal nitric oxide synthase (injured, treated with specific neuronal nitric oxide synthase inhibitor, ZK 234238 (n = 4). Control and neuronal nitric oxide synthase groups were given a cutaneous burn (40% of total body surface, third degree) and insufflated with cotton smoke (48 breaths, <40°C) under halothane anesthesia. Animals in sham group received fake injury also under halothane anesthesia. After injury or fake injury procedure, all sheep were placed on ventilators and resuscitated with lactated Ringer's solution. Neuronal nitric oxide synthase group was administered with continuous infusion of ZK 234238 started 1 hr postinjury with a dose of 100 μg/kg/hr. Sham and control groups received same amount of saline. Measurements and Main Results Cardiopulmonary hemodynamics monitored during the 24-hr experimental time period was stable in the sham group. Control sheep developed multiple signs of acute lung injury. This pathophysiology included decreased pulmonary gas exchange and lung compliance, increased pulmonary edema, and inflammatory indices, such as interleukin-8. Treatment of

Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica.

PubMed

Maluf, Mirian Perez; da Silva, Carla Cristina; de Oliveira, Michelle de Paula Abreu; Tavares, Aline Gomes; Silvarolla, Maria Bernadete; Guerreiro, Oliveiro

2009-10-01

In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

PubMed Central

2009-01-01

In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence. PMID:21637458

Isolation and characterization of beta-glucan synthase: A potential biochemical regulator of gravistimulated differential cell wall loosening

NASA Technical Reports Server (NTRS)

Kuzmanoff, K. M.

1984-01-01

In plants, gravity stimulates differential growth in the upper and lower halves of horizontally oriented organs. Auxin regulation of cell wall loosening and elongation is the basis for most models of this phenomenon. Auxin treatment of pea stem tissue rapidly increases the activity of Golgi-localized Beta-1,4-glucan synthase, an enzyme involved in biosynthesis of wall xyloglucan which apparently constitutes the substrate for the wall loosening process. The primary objective is to determine if auxin induces de novo formation of Golgi glucan synthase and increases the level of this glucan synthase mRNA. This shall be accomplished by (a) preparation of a monoclonal antibody to the synthase, (b) isolation, and characterization of the glucan synthase, and (c) examination for cross reactivity between the antibody and translation products of auxin induced mRNAs in pea tissue. The antibody will also be used to localize the glucan synthase in upper and lower halves of pea stem tissue before, during and after the response to gravity.

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

PubMed

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.

Twisting and subunit rotation in single FOF1-ATP synthase

PubMed Central

Sielaff, Hendrik; Börsch, Michael

2013-01-01

FOF1-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single FOF1-ATP synthases. PMID:23267178

Multiple defects in muscle glycogen synthase activity contribute to reduced glycogen synthesis in non-insulin dependent diabetes mellitus.

PubMed Central

Thorburn, A W; Gumbiner, B; Bulacan, F; Brechtel, G; Henry, R R

1991-01-01

To define the mechanisms of impaired muscle glycogen synthase and reduced glycogen formation in non-insulin dependent diabetes mellitus (NIDDM), glycogen synthase activity was kinetically analyzed during the basal state and three glucose clamp studies (insulin approximately equal to 300, 700, and 33,400 pmol/liter) in eight matched nonobese NIDDM and eight control subjects. Muscle glycogen content was measured in the basal state and following clamps at insulin levels of 33,400 pmol/liter. NIDDM subjects had glucose uptake matched to controls in each clamp by raising serum glucose to 15-20 mmol/liter. The insulin concentration required to half-maximally activate glycogen synthase (ED50) was approximately fourfold greater for NIDDM than control subjects (1,004 +/- 264 vs. 257 +/- 110 pmol/liter, P less than 0.02) but the maximal insulin effect was similar. Total glycogen synthase activity was reduced approximately 38% and glycogen content was approximately 30% lower in NIDDM. A positive correlation was present between glycogen content and glycogen synthase activity (r = 0.51, P less than 0.01). In summary, defects in muscle glycogen synthase activity and reduced glycogen content are present in NIDDM. NIDDM subjects also have less total glycogen synthase activity consistent with reduced functional mass of the enzyme. These findings and the correlation between glycogen synthase activity and glycogen content support the theory that multiple defects in glycogen synthase activity combine to cause reduced glycogen formation in NIDDM. PMID:1899428

Crystallization of Δ{sup 1}-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shoyama, Yoshinari; Takeuchi, Ayako; Taura, Futoshi

Δ{sup 1}-Tetrahydrocannabinolic acid (THCA) synthase from C. sativa was crystallized. The crystal diffracted to 2.7 Å resolution with sufficient quality for further structure determination. Δ{sup 1}-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 Mmore » sodium citrate. The crystal diffracted to 2.7 Å resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 Å. The calculated Matthews coefficient was approximately 4.1 or 2.0 Å{sup 3} Da{sup −1} assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.« less

Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase

PubMed Central

2015-01-01

The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity. PMID:24517311

A domain swapping approach to elucidate differential regiospecific hydroxylation by geraniol and linalool synthases from perilla.

PubMed

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.

Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

PubMed

Cheng, Jiujun; Charles, Trevor C

2016-09-01

Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

Gene cloning and overexpression of a geranylgeranyl diphosphate synthase of an extremely thermophilic bacterium, Thermus thermophilus.

PubMed

Ohto, C; Ishida, C; Koike-Takeshita, A; Yokoyama, K; Muramatsu, M; Nishino, T; Obata, S

1999-02-01

A geranylgeranyl diphosphate (GGPP) synthase gene of an extremely thermophilic bacterium, Thermus thermophilus, was cloned and sequenced. T. thermophilus GGPP synthase, overexpressed in Escherichia coli cells as a glutathione S-transferase fusion protein, was purified and characterized. The fusion protein, retaining thermostability, formed a homodimer, and showed higher specific activity than did a partially purified thermostable enzyme previously reported. Optimal reaction conditions and kinetic parameters were also examined. The deduced amino acid sequence indicated that T. thermophilus GGPP synthase was excluded from the group of bacterial type GGPP synthases and lacked the insertion amino acid residues in the first aspartate-rich motif as do archaeal and eukaryotic short-chain prenyltransferases.

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

USDA-ARS?s Scientific Manuscript database

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

Sesquiterpene furan compound CJ-01, a novel chitin synthase 2 inhibitor from Chloranthus japonicus SIEB.

PubMed

Yim, Nam Hui; Hwang, Eui Il; Yun, Bong Sik; Park, Ki Duk; Moon, Jae Sun; Lee, Sang Han; Sung, Nack Do; Kim, Sung Uk

2008-05-01

A novel sesquiterpene furan compound CJ-01 was isolated from the methanol extract of the whole plant of Chloranthus japonicus SIEB. by monitoring the inhibitory activity of chitin synthase 2 from Saccharomyces cerevisiae. Based on spectroscopic analysis, the structure of compound CJ-01 was determined as 3,4,8a-trimethyl-4a,7,8,8a-tetrahydro-4a-naphto[2,3-b]furan-9-one. The compound inhibited chitin synthase 2 of Saccharomyces cerevisiae in a dose-dependent manner with an IC50 of 39.6 microg/ml, whereas it exhibited no inhibitory activities against chitin synthase 1 and 3 of S. cerevisiae up to 280 microg/ml. CJ-01 has 1.7-fold stronger inhibitory activity than polyoxin D (IC50=70 microg/ml), a well-known chitin synthase inhibitor. These results indicate that the compound is a specific inhibitor of chitin synthase 2 from S. cerevisiae. In addition, CJ-01 showed antifungal activities against various human and phytopathogenic fungi. Therefore, the compound might be an interesting lead to develop effective antifungal agents.

Energy transduction in the F1 motor of ATP synthase.

PubMed

Wang, H; Oster, G

1998-11-19

ATP synthase is the universal enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. It can also reverse itself and hydrolyse ATP to pump protons against an electrochemical gradient. ATP synthase carries out both its synthetic and hydrolytic cycles by a rotary mechanism. This has been confirmed in the direction of hydrolysis after isolation of the soluble F1 portion of the protein and visualization of the actual rotation of the central 'shaft' of the enzyme with respect to the rest of the molecule, making ATP synthase the world's smallest rotary engine. Here we present a model for this engine that accounts for its mechanochemical behaviour in both the hydrolysing and synthesizing directions. We conclude that the F1 motor achieves its high mechanical torque and almost 100% efficiency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque.

Energy transduction in the F1 motor of ATP synthase

NASA Astrophysics Data System (ADS)

Wang, Hongyun; Oster, George

1998-11-01

ATP synthase is the universal enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. It can also reverse itself and hydrolyse ATP to pump protons against an electrochemical gradient. ATP synthase carries out both its synthetic and hydrolytic cycles by a rotary mechanism. This has been confirmed in the direction of hydrolysis, after isolation of the soluble F1 portion of the protein and visualization of the actual rotation of the central `shaft' of the enzyme with respect to the rest of the molecule, making ATP synthase the world's smallest rotary engine. Here we present a model for this engine that accounts for its mechanochemical behaviour in both the hydrolysing and synthesizing directions. We conclude that the F1 motor achieves its high mechanical torque and almost 100% efficiency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque.

Regulation of expression, activity and localization of fungal chitin synthases

PubMed Central

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

Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii

PubMed Central

Sutton, Kristin A.; Breen, Jennifer; Russo, Thomas A.; Schultz, L. Wayne; Umland, Timothy C.

2016-01-01

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301–Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate. PMID:26919521

Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii.

PubMed

Sutton, Kristin A; Breen, Jennifer; Russo, Thomas A; Schultz, L Wayne; Umland, Timothy C

2016-03-01

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301-Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartwig, S.; Frister, T.; Alemdar, S.

2015-03-20

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

Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis.

PubMed

Larbat, Romain; Kellner, Sandra; Specker, Silvia; Hehn, Alain; Gontier, Eric; Hans, Joachim; Bourgaud, Frederic; Matern, Ulrich

2007-01-05

Ammi majus L. accumulates linear furanocoumarins by cytochrome P450 (CYP)-dependent conversion of 6-prenylumbelliferone via (+)-marmesin to psoralen. Relevant activities, i.e. psoralen synthase, are induced rapidly from negligible background levels upon elicitation of A. majus cultures with transient maxima at 9-10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocoumarin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for (+)-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via (+)-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, (+)-columbianetin failed as substrate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate (+)-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution.

Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

PubMed Central

Balakrishna, Asha Manikkoth; Seelert, Holger; Marx, Sven-Hendric; Dencher, Norbert A.; Grüber, Gerhard

2014-01-01

In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF1FO-ATP synthase) of plants is integrated into the thylakoid membrane via its FO-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H+-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F1 sector. Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF1FO-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å. PMID:27919036

Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis

PubMed Central

Begley, Darren W.; Edwards, Thomas E.; Raymond, Amy C.; Smith, Eric R.; Hartley, Robert C.; Abendroth, Jan; Sankaran, Banumathi; Lorimer, Donald D.; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J.

2011-01-01

Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research. PMID:21904052

Native granule associated short chain length polyhydroxyalkanoate synthase from a marine derived Bacillus sp. NQ-11/A2.

PubMed

Prabhu, Nimali N; Santimano, Maria Celisa; Mavinkurve, Suneela; Bhosle, Saroj N; Garg, Sandeep

2010-01-01

A rapidly growing marine derived Bacillus sp. strain NQ-11/A2, identified as Bacillus megaterium, accumulated 61% polyhydroxyalkanoate by weight. Diverse carbon sources served as substrates for the accumulation of short chain length polyhydroxyalkanoate. Three to nine granules either single or attached as buds could be isolated intact from each cell. Maximum activity of polyhydroxyalkanoate synthase was associated with the granules. Granule-bound polyhydroxyalkanoate synthase had a K(m) of 7.1 x 10(-5) M for DL-beta-hydroxybutyryl-CoA. Temperature and pH optima for maximum activity were 30 degrees C and 7.0, respectively. Sodium ions were required for granule-bound polyhydroxyalkanoate synthase activity and inhibited by potassium. Granule-bound polyhydroxyalkanoate synthase was apparently covalently bound to the polyhydroxyalkanoate-core of the granules and affected by the chaotropic reagent urea. Detergents inhibited the granule-bound polyhydroxyalkanoate synthase drastically whilst glycerol and bovine serum albumin stabilized the synthase.

Acetohydroxy acid synthase is a target for leucine containing peptide toxicity in Escherichia coli.

PubMed Central

Gollop, N; Tavori, H; Barak, Z

1982-01-01

Acetohydroxy acid synthase from a mutant resistant to leucine-containing peptides was insensitive to leucine inhibition. It is concluded that acetohydroxy acid synthase is a target for the toxicity of the high concentrations of leucine brought into Escherichia coli K-12 by leucine-containing peptides. PMID:7033214

Expression, crystallization and preliminary crystallographic studies of a novel bifunctional N-­acetylglutamate synthase/kinase from Xanthomonas campestris homologous to vertebrate N-acetylglutamate synthase

PubMed Central

Shi, Dashuang; Caldovic, Ljubica; Jin, Zhongmin; Yu, Xiaolin; Qu, Qiuhao; Roth, Lauren; Morizono, Hiroki; Hathout, Yetrib; Allewell, Norma M.; Tuchman, Mendel

2006-01-01

A novel N-acetylglutamate synthase/kinase bifunctional enzyme of arginine biosynthesis that was homologous to vertebrate N-acetylglutamate synthases was identified in Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the hexagonal space group P6222, with unit-cell parameters a = b = 134.60, c = 192.11 Å, and diffract to about 3.0 Å resolution. Selenomethionine-substituted recombinant protein was produced and selenomethionine substitution was verified by mass spectroscopy. Multiple anomalous dispersion (MAD) data were collected at three wavelengths at SER-CAT, Advanced Photon Source, Argonne National Laboratory. Structure determination is under way using the MAD phasing method. PMID:17142901

In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

PubMed

Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M

2014-04-01

A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

Acute electroacupuncture inhibits nitric oxide synthase expression in the spinal cord of neuropathic rats.

PubMed

Cha, Myeoung Hoon; Bai, Sun Joon; Lee, Kyung Hee; Cho, Zang Hee; Kim, Young-Bo; Lee, Hye-Jung; Lee, Bae Hwan

2010-02-01

To examine the effects of electroacupuncture stimulation on behavioral changes and neuronal nitric oxide synthase expression in the rat spinal cord after nerve injury. Under pentobarbital anesthesia, male Sprague-Dawley rats were subjected to neuropathic surgery by tightly ligating and cutting the left tibial and sural nerves. Behavioral responses to mechanical stimulation were tested for 2 weeks post-operatively. At the end of behavioral testing, electroacupuncture stimulation was applied to ST36 (Choksamni) and SP9 (Eumleungcheon) acupoints. Immunocytochemical staining was performed to investigate changes in the expression of neuronal nitric oxide synthase-immunoreactive neurons in the L4-5 spinal cord. Mechanical allodynia was observed by nerve injury. The mechanical allodynia was decreased after electroacupuncture stimulation. Neuronal nitric oxide synthase expression was also decreased in L4-5 spinal cord by electroacupuncture treatment. These results suggest that electroacupuncture relieves mechanical allodynia in the neuropathic rats possibly by the inhibition of neuronal nitric oxide synthase expression in the spinal cord.

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

PubMed

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

2013-03-01

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

Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium.

PubMed

d'Emmanuele di Villa Bianca, Roberta; Mitidieri, Emma; Esposito, Davide; Donnarumma, Erminia; Donnarumm, Erminia; Russo, Annapina; Fusco, Ferdinando; Ianaro, Angela; Mirone, Vincenzo; Cirino, Giuseppe; Russo, Giulia; Sorrentino, Raffaella

2015-01-01

Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP) but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP) causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity.

Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium

PubMed Central

d’Emmanuele di Villa Bianca, Roberta; Donnarumm, Erminia; Russo, Annapina; Fusco, Ferdinando; Ianaro, Angela; Mirone, Vincenzo; Cirino, Giuseppe; Russo, Giulia; Sorrentino, Raffaella

2015-01-01

Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP) but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP) causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity. PMID:26368121

Genetic-deletion of Cyclooxygenase-2 Downstream Prostacyclin Synthase Suppresses Inflammatory Reactions but Facilitates Carcinogenesis, unlike Deletion of Microsomal Prostaglandin E Synthase-1.

PubMed

Sasaki, Yuka; Kamiyama, Shuhei; Kamiyama, Azusa; Matsumoto, Konomi; Akatsu, Moe; Nakatani, Yoshihito; Kuwata, Hiroshi; Ishikawa, Yukio; Ishii, Toshiharu; Yokoyama, Chieko; Hara, Shuntaro

2015-11-27

Prostacyclin synthase (PGIS) and microsomal prostaglandin E synthase-1 (mPGES-1) are prostaglandin (PG) terminal synthases that function downstream of inducible cyclooxygenase (COX)-2 in the PGI2 and PGE2 biosynthetic pathways, respectively. mPGES-1 has been shown to be involved in various COX-2-related diseases such as inflammatory diseases and cancers, but it is not yet known how PGIS is involved in these COX-2-related diseases. Here, to clarify the pathophysiological role of PGIS, we investigated the phenotypes of PGIS and mPGES-1 individual knockout (KO) or double KO (DKO) mice. The results indicate that a thioglycollate-induced exudation of leukocytes into the peritoneal cavity was suppressed by the genetic-deletion of PGIS. In the PGIS KO mice, lipopolysaccharide-primed pain nociception (as assessed by the acetic acid-induced writhing reaction) was also reduced. Both of these reactions were suppressed more effectively in the PGIS/mPGES-1 DKO mice than in the PGIS KO mice. On the other hand, unlike mPGES-1 deficiency (which suppressed azoxymethane-induced colon carcinogenesis), PGIS deficiency up-regulated both aberrant crypt foci formation at the early stage of carcinogenesis and polyp formation at the late stage. These results indicate that PGIS and mPGES-1 cooperatively exacerbate inflammatory reactions but have opposing effects on carcinogenesis, and that PGIS-derived PGI2 has anti-carcinogenic effects.

(-)-Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase.

PubMed

Ramírez-Sánchez, Israel; Rodríguez, Alonso; Moreno-Ulloa, Aldo; Ceballos, Guillermo; Villarreal, Francisco

2016-05-01

(-)-Epicatechin increases indicators associated with mitochondrial biogenesis in endothelial cells and myocardium. We investigated endothelial nitric oxide synthase involvement on (-)-epicatechin-induced increases in indicators associated with mitochondrial biogenesis in human coronary artery endothelial cells cultured in normal-glucose and high-glucose media, as well as to restore indicators of cardiac mitochondria from the effects of simulated diabetes. Here, we demonstrate the role of endothelial nitric oxide synthase on (-)-epicatechin-induced increases in mitochondrial proteins, transcription factors and sirtuin 1 under normal-glucose conditions. In simulated diabetes endothelial nitric oxide synthase function, mitochondrial function-associated and biogenesis-associated indicators were adversely impacted by high glucose, effects that were reverted by (-)-epicatechin. As an animal model of type 2 diabetes, 2-month old C57BL/6 mice were fed a high-fat diet for 16 weeks. Fasting and fed blood glucose levels were increased and NO plasma levels decreased. High-fat-diet-fed mice myocardium revealed endothelial nitric oxide synthase dysfunction, reduced mitochondrial activity and markers of mitochondrial biogenesis. The administration of 1 mg/kg (-)-epicatechin for 15 days by oral gavage shifted these endpoints towards control mice values. Results suggest that endothelial nitric oxide synthase mediates (-)-epicatechin-induced increases of indicators associated with mitochondrial biogenesis in endothelial cells. (-)-Epicatechin also counteracts the negative effects that high glucose or simulated type 2 diabetes has on endothelial nitric oxide synthase function. © The Author(s) 2016.

Protein modelling of triterpene synthase genes from mangrove plants using Phyre2 and Swiss-model

NASA Astrophysics Data System (ADS)

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.

The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

NASA Astrophysics Data System (ADS)

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.

Homocysteine threshold value based on cystathionine beta synthase and paraoxonase 1 activities in mice.

PubMed

Hamelet, J; Aït-Yahya-Graison, E; Matulewicz, E; Noll, C; Badel-Chagnon, A; Camproux, A-C; Demuth, K; Paul, J-L; Delabar, J M; Janel, N

2007-12-01

Hyperhomocysteinaemia is a metabolic disorder associated with the development of premature atherosclerosis. Among the determinants which predispose to premature thromboembolic and atherothrombotic events, serum activity of paraoxonase 1, mainly synthesized in the liver, has been shown to be a predictor of cardiovascular disease and to be negatively correlated with serum homocysteine levels in human. Even though treatments of hyperhomocysteinaemic patients ongoing cardiovascular complications are commonly used, it still remains unclear above which homocysteine level a preventive therapy should be started. In order to establish a threshold of plasma homocysteine concentration we have analyzed the hepatic cystathionine beta synthase and paraoxonase 1 activities in a moderate to intermediate murine model of hyperhomocysteinaemia. Using wild type and heterozygous cystathionine beta synthase deficient mice fed a methionine enriched diet or a control diet, we first studied the link between cystathionine beta synthase and paraoxonase 1 activities and plasma homocysteine concentration. Among the animals used in this study, we observed a negative correlation between plasma homocysteine level and cystathionine beta synthase activity (rho=-0.52, P=0.0008) or paraoxonase 1 activity (rho=-0.49, P=0.002). Starting from these results, a homocysteine cut-off value of 15 microm has been found for both cystathionine beta synthase (P=0.0003) and paraoxonase 1 (P=0.0007) activities. Our results suggest that both cystathionine beta synthase and paraoxonase 1 activities are significantly decreased in mice with a plasma homocysteine value greater than 15 microm. In an attempt to set up preventive treatment for cardiovascular disease our results indicate that treatments should be started from 15 microm of plasma homocysteine.

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

PubMed Central

Khosla, C; McDaniel, R; Ebert-Khosla, S; Torres, R; Sherman, D H; Bibb, M J; Hopwood, D A

1993-01-01

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases. Images PMID:8468280

Biochemical identification of residues that discriminate between 3,4-dihydroxyphenylalanine decarboxylase and 3,4-dihydroxyphenylacetaldehyde synthase-mediated reactions.

PubMed

Liang, Jing; Han, Qian; Ding, Haizhen; Li, Jianyong

2017-12-01

In available insect genomes, there are several L-3,4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC)-like or aromatic amino acid decarboxylase (AAAD) sequences. This contrasts to those of mammals whose genomes contain only one DDC. Our previous experiments established that two DDC-like proteins from Drosophila actually mediate a complicated decarboxylation-oxidative deamination process of dopa in the presence of oxygen, leading to the formation of 3,4-dihydroxyphenylacetaldehyde (DHPA), CO 2 , NH 3, and H 2 O 2 . This contrasts to the typical DDC-catalyzed reaction, which produces CO 2 and dopamine. These DDC-like proteins were arbitrarily named DHPA synthases based on their critical role in insect soft cuticle formation. Establishment of reactions catalyzed by these AAAD-like proteins solved a puzzle that perplexed researchers for years, but to tell a true DHPA synthase from a DDC in the insect AAAD family remains problematic due to high sequence similarity. In this study, we performed extensive structural and biochemical comparisons between DHPA synthase and DDC. These comparisons identified several target residues potentially dictating DDC-catalyzed and DHPA synthase-catalyzed reactions, respectively. Comparison of DHPA synthase homology models with crystal structures of typical DDC proteins, particularly residues in the active sites, provided further insights for the roles these identified target residues play. Subsequent site-directed mutagenesis of the tentative target residues and activity evaluations of their corresponding mutants determined that active site His192 and Asn192 are essential signature residues for DDC- and DHPA synthase-catalyzed reactions, respectively. Oxygen is required in DHPA synthase-mediated process and this oxidizing agent is reduced to H 2 O 2 in the process. Biochemical assessment established that H 2 O 2 , formed in DHPA synthase-mediated process, can be reused as oxidizing agent and this active oxygen species is reduced to H 2

Binding Isotope Effects for para-Aminobenzoic Acid with Dihydropteroate Synthase from Staphylococcus aureus and Plasmodium falciparum.

PubMed

Stratton, Christopher F; Namanja-Magliano, Hilda A; Cameron, Scott A; Schramm, Vern L

2015-10-16

Dihydropteroate synthase is a key enzyme in folate biosynthesis and is the target of the sulfonamide class of antimicrobials. Equilibrium binding isotope effects and density functional theory calculations indicate that the substrate binding sites for para-aminobenzoic acid on the dihydropteroate synthase enzymes from Staphylococcus aureus and Plasmodium falciparum present distinct chemical environments. Specifically, we show that para-aminobenzoic acid occupies a more sterically constrained vibrational environment when bound to dihydropteroate synthase from P. falciparum relative to that of S. aureus. Deletion of a nonhomologous, parasite-specific insert from the plasmodial dihydropteroate synthase abrogated the binding of para-aminobenzoic acid. The loop specific to P. falciparum is important for effective substrate binding and therefore plays a role in modulating the chemical environment at the substrate binding site.

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

PubMed

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.

Impact of drought stress on specialised metabolism: Biosynthesis and the expression of monoterpene synthases in sage (Salvia officinalis).

PubMed

Radwan, Alzahraa; Kleinwächter, Maik; Selmar, Dirk

2017-09-01

In previous experiments, we demonstrated that the amount of monoterpenes in sage is increased massively by drought stress. Our current study is aimed to elucidate whether this increase is due, at least in part, to elevated activity of the monoterpene synthases responsible for the biosynthesis of essential oils in sage. Accordingly, the transcription rates of the monoterpene synthases were analyzed. Salvia officinalis plants were cultivated under moderate drought stress. The concentrations of monoterpenes as well as the expression of the monoterpene synthases were analyzed. The amount of monoterpenes massively increased in response to drought stress; it doubled after just two days of drought stress. The observed changes in monoterpene content mostly match with the patterns of monoterpene synthase expressions. The expression of bornyl diphosphate synthase was strongly up-regulated; its maximum level was reached after two days. Sabinene synthase increased gradually and reached a maximum after two weeks. In contrast, the transcript level of cineole synthase continuously declined. This study revealed that the stress related increase of biosynthesis is not only due to a "passive" shift caused by the stress related over-reduced status, but also is due - at least in part-to an "active" up-regulation of the enzymes involved. Copyright © 2017 Elsevier Ltd. All rights reserved.

Creation of a high-amylose durum wheat through mutagenesis of starch synthase II (SSIIa)

USDA-ARS?s Scientific Manuscript database

In cereal seeds mutations in one or more starch synthases lead to decreased amylopectin and increased amylose content. Here, the impact of starch synthase IIa (SSIIa or SGP-1) mutations upon durum starch was investigated. A screen of durum accessions identified two lines lacking SGP-A1, the A geno...

Synergy as design principle for metabolic engineering of 1-propanol production in Escherichia coli.

PubMed

Shen, Claire R; Liao, James C

2013-05-01

Synthesis of a desired product can often be achieved via more than one metabolic pathway. Whether naturally evolved or synthetically engineered, these pathways often exhibit specific properties that are suitable for production under distinct conditions and host organisms. Synergy between pathways arises when the underlying pathway characteristics, such as reducing equivalent demand, ATP requirement, intermediate utilization, and cofactor preferences, are complementary to each other. Utilization of such pathways in combination leads to an increased metabolite productivity and/or yield compared to using each pathway alone. This work illustrates the principle of synergy between two different pathways for 1-propanol production in Escherichia coli. A model-guided design based on maximum theoretical yield calculations identified synergy of the native threonine pathway and the heterologous citramalate pathway in terms of production yield across all flux ratios between the two pathways. Characterization of the individual pathways by host gene deletions demonstrates their distinct metabolic characteristics: the necessity of TCA cycle for threonine pathway and the independence of TCA cycle for the citramalate pathway. The two pathways are also complementary in driving force demands. Production experiments verified the synergistic effects predicted by the yield model, in which the platform with dual pathway for 2-ketobutyrate synthesis achieved higher yield (0.15g/g of glucose) and productivity (0.12g/L/h) of 1-propanol than individual ones alone: the threonine pathway (0.09g/g; 0.04g/L/h) or the citramalate pathway (0.11g/g; 0.04g/L/h). Thus, incorporation of synergy into the design principle of metabolic engineering may improve the production yield and rate of the desired compound. Copyright © 2013 Elsevier Inc. All rights reserved.

Squalene synthase inhibition: a novel target for the management of dyslipidemia.

PubMed

Davidson, Michael H

2007-01-01

A new class of compounds, known as squalene synthase inhibitors, has recently reached phase III clinical trials and may provide another therapeutic option for clinicians to improve risk management of low-density lipoprotein cholesterol (LDL-C). The clinical need for another LDL-C-lowering therapy is evident by the inability to achieve an LDL-C target of less than 70 mg/dL in the majority of very high-risk patients on statin monotherapy. Human clinical trial data with TAK-475, a novel and potent inhibitor of squalene synthase, have not yet been published.

Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemazanyy, Ivan; Panasyuk, Ganna; Breus, Oksana

2006-03-24

CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy {beta} and originally identified CoA synthase, CoASy {alpha}. The transcript specific for CoASy {beta} was identified by electronic screening and by RT-PCR analysismore » of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy {beta}. In contrast to CoASy {alpha}, which shows ubiquitous expression, CoASy {beta} is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation.« less

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schoenitzer, Veronika; Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg; Eichner, Norbert

Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report themore » heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.« less

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite.

PubMed

Masetti, Massimo; Locci, Teresa; Cecchettini, Antonella; Lucchesi, Paolo; Magi, Marta; Malvaldi, Gino; Bruschi, Fabrizio

2004-05-01

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.

Development of intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase for discriminating Curcuma species.

PubMed

Kita, Tomoko; Komatsu, Katsuko; Zhu, Shu; Iida, Osamu; Sugimura, Koji; Kawahara, Nobuo; Taguchi, Hiromu; Masamura, Noriya; Cai, Shao-Qing

2016-03-01

Various Curcuma rhizomes have been used as medicines or spices in Asia since ancient times. It is very difficult to distinguish them morphologically, especially when they are boiled and dried, which causes misidentification leading to a loss of efficacy. We developed a method for discriminating Curcuma species by intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase. This method could apply to identification of not only fresh plants but also samples of crude drugs or edible spices. By applying this method to Curcuma specimens and samples, and constructing a dendrogram based on these markers, seven Curcuma species were clearly distinguishable. Moreover, Curcuma longa specimens were geographically distinguishable. On the other hand, Curcuma kwangsiensis (gl type) specimens also showed intraspecies polymorphism, which may have occurred as a result of hybridization with other Curcuma species. The molecular method we developed is a potential tool for global classification of the genus Curcuma. Copyright © 2015 Elsevier Ltd. All rights reserved.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

PubMed

Chin, Randall M; Fu, Xudong; Pai, Melody Y; Vergnes, Laurent; Hwang, Heejun; Deng, Gang; Diep, Simon; Lomenick, Brett; Meli, Vijaykumar S; Monsalve, Gabriela C; Hu, Eileen; Whelan, Stephen A; Wang, Jennifer X; Jung, Gwanghyun; Solis, Gregory M; Fazlollahi, Farbod; Kaweeteerawat, Chitrada; Quach, Austin; Nili, Mahta; Krall, Abby S; Godwin, Hilary A; Chang, Helena R; Faull, Kym F; Guo, Feng; Jiang, Meisheng; Trauger, Sunia A; Saghatelian, Alan; Braas, Daniel; Christofk, Heather R; Clarke, Catherine F; Teitell, Michael A; Petrascheck, Michael; Reue, Karen; Jung, Michael E; Frand, Alison R; Huang, Jing

2014-06-19

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing

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

PubMed

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

2015-03-01

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

Studies on the Expression of Sesquiterpene Synthases Using Promoter-β-Glucuronidase Fusions in Transgenic Artemisia annua L

PubMed Central

Wang, Hongzhen; Han, Junli; Kanagarajan, Selvaraju; Lundgren, Anneli; Brodelius, Peter E.

2013-01-01

In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production. PMID:24278301

Orotic aciduria and uridine monophosphate synthase: a reappraisal.

PubMed

Bailey, C J

2009-12-01

Three subtypes of hereditary orotic aciduria are described in the literature, all related to deficiencies in uridine monophosphate synthase, the multifunctional enzyme that contains both orotate: pyrophosphoryl transferase and orotidine monophosphate decarboxylase activities. The type of enzyme defect present in the subtypes has been re-examined by steady-state modelling of the relative outputs of the three enzymic products, uridine monophosphate, urinary orotic acid and urinary orotidine. It is shown that the ratio of urinary outputs of orotidine to orotate provides a means of testing for particular forms of enzyme defect. It is confirmed that the type I defect is caused by loss of uridine monophosphate synthase activity. Cells and tissue of type I cases have a residual amount of activity that is qualitatively unchanged: the relative rates of the transferase and decarboxylase do not differ from those of wild-type enzyme. The single claimed case of type II, thought to be due to specific inactivation of orotidine monophosphate decarboxylase, is shown to have a product spectrum inconsistent with that claim. It is proposed that this type II form does not differ sufficiently to be accepted as separate from type I. The third subtype, hereditary orotic aciduria without megaloblastic anaemia, occurs in two cases. It has the product spectrum expected of a defect in orotidine monophosphate decarboxylase. This form is the only one that appears to have a qualitatively different uridine monophosphate synthase. The possibility that orotidine monophosphate may control flux through the pyrimidine biosynthesis pathway in hereditary orotic aciduria is discussed.

Crystallization of Δ1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

PubMed Central

Shoyama, Yoshinari; Takeuchi, Ayako; Taura, Futoshi; Tamada, Taro; Adachi, Motoyasu; Kuroki, Ryota; Shoyama, Yukihiro; Morimoto, Satoshi

2005-01-01

Δ1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 Å resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 Å. The calculated Matthews coefficient was approximately 4.1 or 2.0 Å3 Da−1 assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively. PMID:16511162

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

PubMed Central

Souza-Moreira, Tatiana M.; Alves, Thaís B.; Pinheiro, Karina A.; Felippe, Lidiane G.; De Lima, Gustavo M. A.; Watanabe, Tatiana F.; Barbosa, Cristina C.; Santos, Vânia A. F. F. M.; Lopes, Norberto P.; Valentini, Sandro R.; Guido, Rafael V. C.; Furlan, Maysa; Zanelli, Cleslei F.

2016-01-01

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65–74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase. PMID:27874020

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

NASA Astrophysics Data System (ADS)

Souza-Moreira, Tatiana M.; Alves, Thaís B.; Pinheiro, Karina A.; Felippe, Lidiane G.; de Lima, Gustavo M. A.; Watanabe, Tatiana F.; Barbosa, Cristina C.; Santos, Vânia A. F. F. M.; Lopes, Norberto P.; Valentini, Sandro R.; Guido, Rafael V. C.; Furlan, Maysa; Zanelli, Cleslei F.

2016-11-01

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

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

PubMed Central

2016-01-01

Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three α-helical domains (αβγ), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (α) and type II TSs (βγ). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtmT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 Å, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg2+-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs. PMID:27490479

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

PubMed

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

2016-08-31

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

Sucrose Synthase Expression during Cold Acclimation in Wheat 1

PubMed Central

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

1991-01-01

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

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

PubMed

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.

Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

PubMed

Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J; Schaller, Hubert

2014-01-01

The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

Hypotensive effect of agmatine, arginine metabolite, is affected by NO synthase.

PubMed

Gerová, M; Török, J

2004-01-01

The metabolites of arginine were recently shown to be involved in cardiovascular control. The study addresses the general cardiovascular response of anaesthetized rats to agmatine, a decarboxylated arginine. The relation between two arginine metabolic pathways governed by arginine decarboxylase and nitric oxide synthase was investigated. Intravenous administration of agmatine 30 and 60 microM/0.1 ml saline elicited remarkable hypotension of 42.6+/-4.6 and 70.9+/-6.5 mm Hg, respectively. The hypotension was characterized by long duration with half-time of return 171.6+/-2.9 and 229.2+/-3.8 s, respectively. The time of total blood pressure BP recovery was about 10 min. Dose-dependent relaxation to agmatine was also found in aorta rings in vitro. Both doses of agmatine administered 60-180 min after NO synthase inhibition L-NAME 40 mg/kg i.v. caused greater hypotension 59.0+/-7.6 and 95.8 8.8 mm Hg P<0.01 both compared to animals with intact NO synthase, but this was accompanied by a significant shortening of the half-time of BP return. If agmatine was administered to hypertensive NO-deficient rats treated with 40 mg/kg/day L-NAME for 4 weeks, similar significant enhancement of hypotension was observed at both agmatine doses, again with a significant shortening of half-time of BP return. It can be summarized that the long-lasting hypotension elicited by agmatine was amplified after acute or chronic NO synthase inhibition, indicating a feedback relation between the two metabolic pathways of arginine.

Structural Characteristics and Function of a New Kind of Thermostable Trehalose Synthase from Thermobaculum terrenum.

PubMed

Wang, Junqing; Ren, Xudong; Wang, Ruiming; Su, Jing; Wang, Feng

2017-09-06

Trehalose has important applications in the food industry and pharmaceutical manufacturing. The thermostable enzyme trehalose synthase from Thermobaculum terrenum (TtTS) catalyzes the reversible interconversion of maltose and trehalose. Here, we investigated the structural characteristics of TtTS in complex with the inhibitor TriS. TtTS exhibits the typical three domain glycoside hydrolase family 13 structure. The catalytic cleft consists of Asp202-Glu244-Asp310 and various conserved substrate-binding residues. However, among trehalose synthases, TtTS demonstrates obvious thermal stability. TtTS has more polar (charged) amino acids distributed on its protein structure surface and more aromatic amino acids buried within than other mesophilic trehalose synthases. Furthermore, TtTS structural analysis revealed four potential metal ion-binding sites rather than the two in a homologous structure. These factors may render TtTS relatively more thermostable among mesophilic trehalose synthases. The detailed thermophilic enzyme structure provided herein may provide guidance for further protein engineering in the design of stabilized enzymes.

Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae.

PubMed

Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

2010-07-01

An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions.

Producing biofuels using polyketide synthases

DOEpatents

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.

Modified cellulose synthase gene from Arabidopsis thaliana confers herbicide resistance to plants

DOEpatents

Somerville, Chris R [Portola Valley, CA; Scheible, Wolf [Golm, DE

2007-07-10

Cellulose synthase ("CS"), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl)phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

Modified cellulose synthase gene from 'Arabidopsis thaliana' confers herbicide resistance to plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Somerville, Chris R.; Scieble, Wolf

Cellulose synthase ('CS'), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS genemore » can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.« less

Co-expression of peppermint geranyl diphosphate synthase small subunit enhances monoterpene production in transgenic tobacco plants.

PubMed

Yin, Jun-Lin; Wong, Woon-Seng; Jang, In-Cheol; Chua, Nam-Hai

2017-02-01

Monoterpenes are important for plant survival and useful to humans. In addition to their function in plant defense, monoterpenes are also used as flavors, fragrances and medicines. Several metabolic engineering strategies have been explored to produce monoterpene in tobacco but only trace amounts of monoterpenes have been detected. We investigated the effects of Solanum lycopersicum 1-deoxy-d-xylulose-5-phosphate synthase (SlDXS), Arabidopsis thaliana geranyl diphosphate synthase 1 (AtGPS) and Mentha × piperita geranyl diphosphate synthase small subunit (MpGPS.SSU) on production of monoterpene and geranylgeranyl diphosphate (GGPP) diversities, and plant morphology by transient expression in Nicotiana benthamiana and overexpression in transgenic Nicotiana tabacum. We showed that MpGPS.SSU could enhance the production of various monoterpenes such as (-)-limonene, (-)-linalool, (-)-α-pinene/β-pinene or myrcene, in transgenic tobacco by elevating geranyl diphosphate synthase (GPS) activity. In addition, overexpression of MpGPS.SSU in tobacco caused early flowering phenotype and increased shoot branching by elevating contents of GA 3 and cytokinins due to upregulated transcript levels of several plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway genes, geranylgeranyl diphosphate synthases 3 (GGPPS3) and GGPPS4. Our method would allow the identification of new monoterpene synthase genes using transient expression in N. benthamiana and the improvement of monoterpene production in transgenic tobacco plants. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Overexpression of an archaeal geranylgeranyl diphosphate synthase in Escherichia coli cells.

PubMed

Ohto, C; Nakane, H; Hemmi, H; Ohnuma, S; Obata, S; Nishino, T

1998-06-01

An archaeal geranylgeranyl diphosphate synthase was overexpressed in Escherichia coli cells as fusion proteins. These fusion proteins retained their thermostability and had higher specific activity than did a partially purified native enzyme Previously reported. We purified 24.3 mg of MBP (maltose-binding protein)-fusion protein and 5.4 mg of GST (glutathione S-transferase)-fusion protein from a one-liter culture of E. coli. The MBP-fusion proteins existed in dimer, tetramer, octamer, or dodecamer form, and their product specificities were altered according to the oligomerization. The MBP-fusion protein has protease-sensitive sites in the portion corresponding to geranylgeranyl diphosphate synthase.

Biosynthesis of Lipoic Acid in Arabidopsis: Cloning and Characterization of the cDNA for Lipoic Acid Synthase1

PubMed Central

Yasuno, Rie; Wada, Hajime

1998-01-01

Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southern-hybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide. PMID:9808738

Effect of inhibition of glycogen synthase kinase-3 on cardiac hypertrophy during acute pressure overload.

PubMed

Tateishi, Atsushi; Matsushita, Masayuki; Asai, Tomohiro; Masuda, Zenichi; Kuriyama, Mitsuhito; Kanki, Kazushige; Ishino, Kozo; Kawada, Masaaki; Sano, Shunji; Matsui, Hideki

2010-06-01

A large number of diverse signaling molecules in cell and animal models participate in the stimulus-response pathway through which the hypertrophic growth of the myocardium is controlled. However, the mechanisms of signaling pathway including the influence of lithium, which is known as an inhibitor of glycogen synthase kinase-3beta, in pressure overload hypertrophy remain unclear. The aim of our study was to determine whether glycogen synthase kinase-3beta inhibition by lithium has acute effects on the myocyte growth mechanism in a pressure overload rat model. First, we created a rat model of acute pressure overload cardiac hypertrophy by abdominal aortic banding. Protein expression time courses for beta-catenin, glycogen synthase kinase-3beta, and phosphoserine9-glycogen synthase kinase-3beta were then examined. The rats were divided into four groups: normal rats with or without lithium administration and pressure-overloaded rats with or without lithium administration. Two days after surgery, Western blot analysis of beta-catenin, echo-cardiographic evaluation, left ventricular (LV) weight, and LV atrial natriuretic peptide mRNA levels were evaluated. We observed an increase in the level of glycogen synthase kinase-3beta phosphorylation on Ser 9. A significant enhancement of LV heart weight (P < 0.05) and interventricular septum and posterior wall thickness (P < 0.05) with pressure-overloaded hypertrophy in animals treated with lithium were also observed. Atrial natriuretic peptide mRNA levels were significantly increased with pressure overload hypertrophy in animals treated with lithium. We have shown in an animal model that inhibition of glycogen synthase kinase-3beta by lithium has an additive effect on pressure overload cardiac hypertrophy.

Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation

NASA Technical Reports Server (NTRS)

Paliyath, G.; Poovaiah, B. W.

1988-01-01

Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris.

PubMed

Rubin, J L; Gaines, C G; Jensen, R A

1984-07-01

Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg. et Comes with glyphosate (N-[phosphonomethyl]glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK(a) values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO(-)CH(2)NH(2) (+)CH(2)PO(3) (2-), and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K(i) = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K(i)' = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an [enzyme:shikimate-3-P] complex and ultimately forms the dead-end complex of [enzyme:shikimate-3-P:glyphosate].

Cloning and characterization of chsD, a chitin synthase-like gene of Aspergillus fumigatus.

PubMed

Mellado, E; Specht, C A; Robbins, P W; Holden, D W

1996-09-15

A chitin synthase-like gene (chsD) was isolated from an Aspergillus fumigatus genomic DNA library. Comparisons with the predicted amino acid sequence from chsD reveals low but significant similarity to chitin synthases, to other N-acetylglucosaminyltransferases (NodC from Rhizopus spp., HasA from Streptococcus spp. and DG42 from vertebrates. A chsD- mutant strain constructed by gene disruption has a 20% reduction in total mycelial chitin content; however, no differences between the wild-type strain and the chsD- strain were found with respect to morphology, chitin synthase activity or virulence in a neutropenic murine model of aspergillosis. The results show that the chsD product has an important but inessential role in the synthesis of chitin in A. fumigatus.

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

PubMed Central

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

2013-01-01

The mountain pine beetle (Dendroctonus ponderosae Hopkins) is the most destructive pest of western North American pine forests. Adult males produce frontalin, an eight-carbon antiaggregation pheromone, via the mevalonate pathway, as part of several pheromones that initiate and modulate the mass attack of host trees. Frontalin acts as a pheromone, attractant, or kairomone in most Dendroctonus species, other insects, and even elephants. 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to originate from 10-carbon geranyl diphosphate (GPP), 15-carbon farnesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate (GGPP) via a dioxygenase- or cytochrome P450-mediated carbon–carbon bond cleavage. To investigate the role of isoprenyl diphosphate synthases in pheromone biosynthesis, we characterized a bifunctional GPP/FPP synthase and a GGPP synthase in the mountain pine beetle. The ratio of GPP to FPP produced by the GPP/FPP synthase was highly dependent on the ratio of the substrates isopentenyl diphosphate and dimethylallyl diphosphate used in the assay. Transcript levels in various tissues and life stages suggested that GGPP rather than GPP or FPP is used as a precursor to frontalin. Reduction of transcript levels by RNA interference of the isoprenyl diphosphate synthases identified GGPP synthase as having the largest effect on frontalin production, suggesting that frontalin is derived from a 20-carbon isoprenoid precursor rather than from the 10- or 15-carbon precursors. PMID:24167290

Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment.

PubMed

Ma, Zhan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Wang, Yingzhi; Yang, Cuixia; Wang, Wenjuan; Du, Yan; Zhou, Muqing; Gao, Feng

2010-08-01

F1Fo-ATP synthase was originally thought to exclusively locate in the inner membrane of the mitochondria. However, recent studies prove the existence of ectopic F1Fo-ATP synthase on the outside of the cell membrane. Ectopic ATP synthase was proposed as a marker for tumor target therapy. Nevertheless, the protein transport mechanism of the ectopic ATP synthase is still unclear. The specificity of the ectopic ATP synthase, with regard to tumors, is questioned because of its widespread expression. In the current study, we constructed green fluorescent protein-ATP5B fusion protein and introduced it into HepG2 cells to study the localization of the ATP synthase. The expression of ATP5B was analyzed in six cell lines with different 'malignancies'. These cells were cultured in both normal and tumor-like acidic and hypoxic conditions. The results suggested that the ectopic expression of ATP synthase is a consequence of translocation from the mitochondria. The expression and catalytic activity of ectopic ATP synthase were similar on the surface of malignant cells as on the surface of less malignant cells. Interestingly, the expression of ectopic ATP synthase was not up-regulated in tumor-like acidic and hypoxic microenvironments. However, the catalytic activity of ectopic ATP synthase was up-regulated in tumor-like microenvironments. Therefore, the specificity of ectopic ATP synthase for tumor target therapy relies on the high level of catalytic activity that is observed in acidic and hypoxic microenvironments in tumor tissues.

Umchs5, a gene coding for a class IV chitin synthase in Ustilago maydis.

PubMed

Xoconostle-Cázares, B; Specht, C A; Robbins, P W; Liu, Y; León, C; Ruiz-Herrera, J

1997-12-01

A fragment corresponding to a conserved region of a fifth gene coding for chitin synthase in the plant pathogenic fungus Ustilago maydis was amplified by means of the polymerase chain reaction (PCR). The amplified fragment was utilized as a probe for the identification of the whole gene in a genomic library of the fungus. The predicted gene product of Umchs5 has highest similarity with class IV chitin synthases encoded by the CHS3 genes from Saccharomyces cerevisiae and Candida albicans, chs-4 from Neurospora crassa, and chsE from Aspergillus nidulans. Umchs5 null mutants were constructed by substitution of most of the coding sequence with the hygromycin B resistance cassette. Mutants displayed significant reduction in growth rate, chitin content, and chitin synthase activity, specially in the mycelial form. Virulence to corn plantules was also reduced in the mutants. PCR was also used to obtain a fragment of a sixth chitin synthase, Umchs6. It is suggested that multigenic control of chitin synthesis in U. maydis operates as a protection mechanism for fungal viability in which the loss of one activity is partially compensated by the remaining enzymes. Copyright 1997 Academic Press.

The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex

PubMed Central

Gay, Darren C.; Wagner, Drew T.; Meinke, Jessica L.; Zogzas, Charles E.; Gay, Glen R.; Keatinge-Clay, Adrian T.

2016-01-01

Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line. PMID:26724270

Intrathecal oxotremorine affects formalin-induced behavior and spinal nitric oxide synthase immunoreactivity in rats.

PubMed

Przewlocka, B; Mika, J; Capone, F; Machelska, H; Pavone, F

1999-03-01

The present research was undertaken to investigate, by behavioral and immunohistochemical methods, the effects of intrathecal (i.th.) injection of the muscarinic agonist oxotremorine on the response to the long-lasting nociceptive stimulus induced by injection of formalin into the rat hind paw. Formalin injection induced a biphasic, pain-induced behavioral response (paw jerks), as well as an increase in the number of nitric oxide (NO) synthase-labeled neurons in laminae I-III, IV, and X, but not in laminae V-VI. Oxotremorine (0.1-10 ng, i.th.) inhibited paw-jerk frequency in both phases of formalin-induced behavior. The immunohistochemical results showed that i.th.-injected oxotremorine differently affected the level of NO synthase in lumbar part of the spinal cord: no change or increase after the dose of 1 ng, and a significant reduction of nitric oxide synthase neurons after the higher dose (10 ng). These results evidenced a role of cholinergic system in the modulation of tonic pain and in nitric oxide synthase expression at the spinal cord level, which further suggests that these two systems could be involved in phenomena induced by long-lasting nociceptive stimulation.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rudolf, Jeffrey D.; Dong, Liao-Bin; Cao, Hongnan

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

Macromolecular organization of ATP synthase and complex I in whole mitochondria

PubMed Central

Davies, Karen M.; Strauss, Mike; Daum, Bertram; Kief, Jan H.; Osiewacz, Heinz D.; Rycovska, Adriana; Zickermann, Volker; Kühlbrandt, Werner

2011-01-01

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F1 heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis. PMID:21836051

40 CFR 174.533 - Glycine max Herbicide-Resistant Acetolactate Synthase (GM-HRA) inert ingredient; exemption from...

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Glycine max Herbicide-Resistant... Glycine max Herbicide-Resistant Acetolactate Synthase (GM-HRA) inert ingredient; exemption from the requirement of a tolerance. Residues of Glycine max herbicide-resistant acetolactate synthase (GM-HRA) enzyme...

40 CFR 174.533 - Glycine max Herbicide-Resistant Acetolactate Synthase (GM-HRA) inert ingredient; exemption from...

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Glycine max Herbicide-Resistant... Glycine max Herbicide-Resistant Acetolactate Synthase (GM-HRA) inert ingredient; exemption from the requirement of a tolerance. Residues of Glycine max herbicide-resistant acetolactate synthase (GM-HRA) enzyme...

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

PubMed

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

2015-01-01

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

Contribution of extracellular ATP on the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation.

PubMed

Kita, Toshiyuki; Arakaki, Naokatu

2015-01-01

Cell-surface F1F0-ATP synthase was involved in the cell signaling mediating various biological functions. Recently, we found that cell-surface F1F0-ATP synthase plays a role on intracellular triacylglycerol accumulation in adipocytes, and yet, the underlying mechanisms remained largely unknown. In this study, we investigated the role of extracellular ATP on the intracellular triacylglycerol accumulation. We demonstrated that significant amounts of ATP were produced extracellularly by cultured 3T3-L1 adipocytes and that the antibodies against α and β subunits of F1F0-ATP synthase inhibited the extracellular ATP production. Piceatannol, a F1F0-ATP synthase inhibitor, and apyrase, an enzyme which degrades extracellular ATP, suppressed triacylglycerol accumulation. The selective P2Y1 receptor antagonist MRS2500 significantly inhibited triacylglycerol accumulation, whereas the selective P2X receptor antagonist NF279 has less effect. The present results indicate that cell-surface F1F0-ATP synthase on adipocytes is functional in extracellular ATP production and that the extracellular ATP produced contributes, at least in part, to the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation in adipocytes through P2Y1 receptor.

An unusual plant triterpene synthase with predominant α-amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus × domestica.

PubMed

Brendolise, Cyril; Yauk, Yar-Khing; Eberhard, Ellen D; Wang, Mindy; Chagne, David; Andre, Christelle; Greenwood, David R; Beuning, Lesley L

2011-07-01

The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti-inflammatory, antitumour and antimicrobial properties. α-Amyrin and β-amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full-length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus × domestica 'Royal Gala') that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC-MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of α-amyrin to β-amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of α-amyrin produced is > 80% of the total product and is, therefore, primarily an α-amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC-MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either �

Adaptive responses of GLUT-4 and citrate synthase in fast-twitch muscle of voluntary running rats

NASA Technical Reports Server (NTRS)

Henriksen, E. J.; Halseth, A. E.

1995-01-01

Glucose transporter (GLUT-4) protein, hexokinase, and citrate synthase (proteins involved in oxidative energy production from blood glucose catabolism) increase in response to chronically elevated neuromuscular activity. It is currently unclear whether these proteins increase in a coordinated manner in response to this stimulus. Therefore, voluntary wheel running (WR) was used to chronically overload the fast-twitch rat plantaris muscle and the myocardium, and the early time courses of adaptative responses of GLUT-4 protein and the activities of hexokinase and citrate synthase were characterized and compared. Plantaris hexokinase activity increased 51% after just 1 wk of WR, whereas GLUT-4 and citrate synthase were increased by 51 and 40%, respectively, only after 2 wk of WR. All three variables remained comparably elevated (+50-64%) through 4 wk of WR. Despite the overload of the myocardium with this protocol, no substantial elevations in these variables were observed. These findings are consistent with a coordinated upregulation of GLUT-4 and citrate synthase in the fast-twitch plantaris, but not in the myocardium, in response to this increased neuromuscular activity. Regulation of hexokinase in fast-twitch muscle appears to be uncoupled from regulation of GLUT-4 and citrate synthase, as increases in the former are detectable well before increases in the latter.

The anisotropy1 D604N mutation in the Arabidopsis cellulose synthase1 catalytic domain reduces cell wall crystallinity and the velocity of cellulose synthase complexes.

PubMed

Fujita, Miki; Himmelspach, Regina; Ward, Juliet; Whittington, Angela; Hasenbein, Nortrud; Liu, Christine; Truong, Thy T; Galway, Moira E; Mansfield, Shawn D; Hocart, Charles H; Wasteneys, Geoffrey O

2013-05-01

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1's permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature.

Stilbene synthase gene transfer caused alterations in the phenylpropanoid metabolism of transgenic strawberry (Fragaria×ananassa)

PubMed Central

Hanhineva, Kati; Kokko, Harri; Siljanen, Henri; Rogachev, Ilana; Aharoni, Asaph; Kärenlampi, Sirpa O.

2009-01-01

The gene encoding stilbene synthase is frequently used to modify plant secondary metabolism with the aim of producing the self-defence phytoalexin resveratrol. In this study, strawberry (Fragaria×ananassa) was transformed with the NS-Vitis3 gene encoding stilbene synthase from frost grape (Vitis riparia) under the control of the cauliflower mosaic virus 35S and the floral filament-specific fil1 promoters. Changes in leaf metabolites were investigated with UPLC-qTOF-MS (ultra performance liquid chromatography-quadrupole time of flight mass spectrometry) profiling, and increased accumulation of cinnamate, coumarate, and ferulate derivatives concomitantly with a decrease in the levels of flavonols was observed, while the anticipated resveratrol or its derivatives were not detected. The changed metabolite profile suggested that chalcone synthase was down-regulated by the genetic modification; this was verified by decreased chalcone synthase transcript levels. Changes in the levels of phenolic compounds led to increased susceptibility of the transgenic strawberry to grey mould fungus. PMID:19443619

Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori.

PubMed

Webby, Celia J; Patchett, Mark L; Parker, Emily J

2005-08-15

DAH7P (3-Deoxy-D-arabino-heptulosonate 7-phosphate) synthase catalyses the condensation reaction between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) as the first committed step in the biosynthesis of aromatic compounds in plants and micro-organisms. Previous work has identified two families of DAH7P synthases based on sequence similarity and molecular mass, with the majority of the mechanistic and structural studies being carried out on the type I paralogues from Escherichia coli. Whereas a number of organisms possess genes encoding both type I and type II DAH7P synthases, the pathogen Helicobacter pylori has only a single, type II, enzyme. Recombinant DAH7P synthase from H. pylori was partially solubilized by co-expression with chaperonins GroEL/GroES in E. coli, and purified to homogeneity. The enzyme reaction follows an ordered sequential mechanism with the following kinetic parameters: K(m) (PEP), 3 microM; K(m) (E4P), 6 microM; and kcat, 3.3 s(-1). The enzyme reaction involves interaction of the si face of PEP with the re face of E4P. H. pylori DAH7P synthase is not inhibited by phenylalanine, tyrosine, tryptophan or chorismate. EDTA inactivates the enzyme, and activity is restored by a range of bivalent metal ions, including (in order of decreasing effectiveness) Co2+, Mn2+, Ca2+, Mg2+, Cu2+ and Zn2+. Analysis of type II DAH7P synthase sequences reveals several highly conserved motifs, and comparison with the type I enzymes suggests that catalysis by these two enzyme types occurs on a similar active-site scaffold and that the two DAH7P synthase families may indeed be distantly related.

Molecular cloning and functional characterization of three terpene synthases from unripe fruit of black pepper (Piper nigrum).

PubMed

Jin, Zhehao; Kwon, Moonhyuk; Lee, Ah-Reum; Ro, Dae-Kyun; Wungsintaweekul, Juraithip; Kim, Soo-Un

2018-01-15

To identify terpene synthases (TPS) responsible for the biosynthesis of the sesquiterpenes that contribute to the characteristic flavors of black pepper (Piper nigrum), unripe peppercorn was subjected to the Illumina transcriptome sequencing. The BLAST analysis using amorpha-4,11-diene synthase as a query identified 19 sesquiterpene synthases (sesqui-TPSs), of which three full-length cDNAs (PnTPS1 through 3) were cloned. These sesqui-TPS cDNAs were expressed in E. coli to produce recombinant enzymes for in vitro assays, and also expressed in the engineered yeast strain to assess their catalytic activities in vivo. PnTPS1 produced β-caryophyllene as a main product and humulene as a minor compound, and thus was named caryophyllene synthase (PnCPS). Likewise, PnTPS2 and PnTPS3 were, respectively, named cadinol/cadinene synthase (PnCO/CDS) and germacrene D synthase (PnGDS). PnGDS expression in yeast yielded β-cadinene and α-copaene, the rearrangement products of germacrene D. Their k cat /K m values (20-37.7 s -1  mM -1 ) were comparable to those of other sesqui-TPSs. Among three PnTPSs, the transcript level of PnCPS was the highest, correlating with the predominant β-caryophyllene biosynthesis in the peppercorn. The products and rearranged products of three PnTPSs could account for about a half of the sesquiterpenes in number found in unripe peppercorn. Copyright © 2017 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maley, F.; Maley, G.F.

1983-01-01

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

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

PubMed

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.

Dynamics of meso and thermo citrate synthases with implicit solvation

NASA Astrophysics Data System (ADS)

Cordeiro, J. M. M.

The dynamics of hydration of meso and thermo citrate synthases has been investigated using the EEF1 methodology implemented with the CHARMM program. The native enzymes are composed of two identical subunits, each divided into a small and large domain. The dynamics behavior of both enzymes at 30°C and 60°C has been compared. The results of simulations show that during the hydration process, each subunit follows a different pathway of hydration, in spite of the identical sequence. The hydrated structures were compared with the crystalline structure, and the root mean square deviation (RMSD) of each residue along the trajectory was calculated. The regions with larger and smaller mobility were identified. In particular, helices belonging to the small domain are more mobile than those of the large domain. In contrast, the residues that constitute the active site show a much lower displacement compared with the crystalline structure. Hydration free energy calculations point out that Thermoplasma acidophilum citrate synthase (TCS) is more stable than chicken citrate synthase (CCS), at high temperatures. Such result has been ascribed to the higher number of superficial charges in the thermophilic homologue, which stabilizes the enzyme, while the mesophilic homologue denatures. These results are in accord with the experimental found that TCS keeps activity at temperatures farther apart from the catalysis regular temperature than the CCS.

Incorporation of phosphate into glycogen by glycogen synthase.

PubMed

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.

INCORPORATION OF PHOSPHATE INTO GLYCOGEN BY GLYCOGEN SYNTHASE

PubMed Central

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

2016-01-01

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 Metab 13, 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 Metab 17, 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 32P from [β-32P]UDP-glucose to glycogen by glycogen synthase. The 32P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The 32P-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-32P]UDP generated in the reaction. Furthermore, [32P]UDP did not bind non-covalently to glycogen. The 32P 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, perhaps C3, phosphorylation. PMID:27036853

Piceid (resveratrol glucoside) synthesis in stilbene synthase transgenic apple fruit.

PubMed

Rühmann, Susanne; Treutter, Dieter; Fritsche, Steffi; Briviba, Karlis; Szankowski, Iris

2006-06-28

A stilbene synthase gene along with the selectable marker gene bar for herbicide resistance was transferred via Agrobacterium tumefaciens mediated transformation into apple (Malus domesticaBorkh.) cvs. 'Elstar' and 'Holsteiner Cox'. The stilbene synthase catalyzes the conversion of 1 molecule of p-coumaroyl-CoA and 3 molecules of malonyl-CoA into 3,4',5-trihydroxystilbene, commonly known as resveratrol. This phytoalexin has implications in both phytopathology and human health. Greenhouse-grown transgenic and nontransformed control plants were grafted onto dwarfing rootstock M27. Flowering and fruiting occurred within the following years, offering the opportunity to analyze transgenic apple fruit and fertility of transgenic plants as well as inheritance of the transgenes into the seedling progeny. Molecular analysis revealed that the stilbene synthase is expressed in transgenic plants and in the skin and flesh of transgenic apple fruit. After formation, resveratrol is modified by the addition of a hexose sugar. The resulting component was characterized as piceid. With the aim of characterizing the influence of the novel biosynthetic pathway on the accumulation of other phenolic compounds naturally present in apple fruit, the amounts of flavanols, flavonols, phloretin derivatives and hydroxycinnamic acids in wild type and transgenic fruit were determined by HPLC. In all investigated transformed lines that accumulated piceid, no negative correlation between levels of piceid and the above-mentioned compounds was observed, except for the flavonol contents, which slightly decreased. Inheritance of the transgenes was confirmed in the seedling progeny, which were obtained after pollination of transgenic plants with nontransgenic pollen and vice versa after pollination of nontransgenic plants with pollen obtained from transgenic plants. The fertility of stilbene synthase transgenic plants was demonstrated. To the authors' knowledge this is the first time that data are

Identification of novel isoprene synthases through genome mining and expression in Escherichia coli.

PubMed

Ilmén, Marja; Oja, Merja; Huuskonen, Anne; Lee, Sangmin; Ruohonen, Laura; Jung, Simon

2015-09-01

Isoprene is a naturally produced hydrocarbon emitted into the atmosphere by green plants. It is also a constituent of synthetic rubber and a potential biofuel. Microbial production of isoprene can become a sustainable alternative to the prevailing chemical production of isoprene from petroleum. In this work, sequence homology searches were conducted to find novel isoprene synthases. Candidate sequences were functionally expressed in Escherichia coli and the desired enzymes were identified based on an isoprene production assay. The activity of three enzymes was shown for the first time: expression of the candidate genes from Ipomoea batatas, Mangifera indica, and Elaeocarpus photiniifolius resulted in isoprene formation. The Ipomoea batatas isoprene synthase produced the highest amounts of isoprene in all experiments, exceeding the isoprene levels obtained by the previously known Populus alba and Pueraria montana isoprene synthases that were studied in parallel as controls. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Glucose induces the translocation and the aggregation of glycogen synthase in rat hepatocytes.

PubMed Central

Fernández-Novell, J M; Ariño, J; Vilaró, S; Guinovart, J J

1992-01-01

Incubation of rat hepatocytes with glucose results in a decrease in the amount of glycogen synthase activity found in supernatants obtained after centrifugation of cell homogenates at 9200 g. The enzymic activity was quantitatively recovered in the sediments. This effect of translocation was dose- and time-dependent and correlated with the amount of immunoreactive enzyme determined by immunoblotting in both fractions. Hydrolysis by alpha-amylase of glycogen accumulated upon incubation with the sugar did not affect the translocation pattern. Translocation was also observed when cells were incubated with 2-deoxyglucose, which did not result in accumulation of glycogen. Immunocytochemical evidence indicates that glucose induces the aggregation of glycogen synthase molecules into clusters which are recovered in the sediments. These results indicate that glucose, in addition to activating glycogen synthase, may trigger changes in the localization of the enzyme in the cell. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:1736893

Molecular biological effects of selective neuronal nitric oxide synthase inhibition in ovine lung injury

PubMed Central

Westphal, Martin; Enkhbaatar, Perenlei; Wang, Jianpu; Pazdrak, Konrad; Nakano, Yoshimitsu; Hamahata, Atsumori; Jonkam, Collette C.; Lange, Matthias; Connelly, Rhykka L.; Kulp, Gabriela A.; Cox, Robert A.; Hawkins, Hal K.; Schmalstieg, Frank C.; Horvath, Eszter; Szabo, Csaba; Traber, Lillian D.; Whorton, Elbert; Herndon, David N.; Traber, Daniel L.

2010-01-01

Neuronal nitric oxide synthase is critically involved in the pathogenesis of acute lung injury resulting from combined burn and smoke inhalation injury. We hypothesized that 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, blocks central molecular mechanisms involved in the pathophysiology of this double-hit insult. Twenty-five adult ewes were surgically prepared and randomly allocated to 1) an uninjured, untreated sham group (n = 7), 2) an injured control group with no treatment (n = 7), 3) an injury group treated with 7-nitroindazole from 1-h postinjury to the remainder of the 24-h study period (n = 7), or 4) a sham-operated group subjected only to 7-nitroindazole to judge the effects in health. The combination injury was associated with twofold increased activity of neuronal nitric oxide synthase and oxidative/nitrosative stress, as indicated by significant increases in plasma nitrate/nitrite concentrations, 3-nitrotyrosine (an indicator of peroxynitrite formation), and malondialdehyde lung tissue content. The presence of systemic inflammation was evidenced by twofold, sixfold, and threefold increases in poly(ADP-ribose) polymerase, IL-8, and myeloperoxidase lung tissue concentrations, respectively (each P < 0.05 vs. sham). These molecular changes were linked to tissue damage, airway obstruction, and pulmonary shunting with deteriorated gas exchange. 7-Nitroindazole blocked, or at least attenuated, all these pathological changes. Our findings suggest 1) that nitric oxide formation derived from increased neuronal nitric oxide synthase activity represents a pivotal reactive agent in the patho-physiology of combined burn and smoke inhalation injury and 2) that selective neuronal nitric oxide synthase inhibition represents a goal-directed approach to attenuate the degree of injury. PMID:19965980

Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

PubMed

Schimpl, Flávia Camila; Kiyota, Eduardo; Mayer, Juliana Lischka Sampaio; Gonçalves, José Francisco de Carvalho; da Silva, José Ferreira; Mazzafera, Paulo

2014-09-01

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of Combined Stress on Morphological Changes and Expression of NO Synthases in Rat Ventral Hippocampus.

PubMed

Smirnov, A V; Tyurenkov, I N; Shmidt, M V; Ekova, M R; Mednikov, D S; Borodin, D D

2015-11-01

Adult rats were subjected to 7-day combined stress with stochastic changes of stressors of different modalities (noise, vibration, pulsating bright light) along with mobility restriction and elevated temperature in the chamber during stress exposures (daily 30-min sessions). Circulatory disorders, inhibition of endothelial NO-synthase expression in endothelial cells of the microcirculatory bed, perivascular edema, pronounced degenerative changes, and enhanced expression of inducible NO synthase in CA3 pyramidal neurons in the ventral hippocampus of stressed 12-month-old rats were observed. These findings can attest to the involvement NOdependent mechanisms and different contribution of NO synthase isoforms into the formation of hippocampal neuronal damage.

The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex.

PubMed

Gay, Darren C; Wagner, Drew T; Meinke, Jessica L; Zogzas, Charles E; Gay, Glen R; Keatinge-Clay, Adrian T

2016-03-01

Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line. Copyright © 2015 Elsevier Inc. All rights reserved.

Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its Implications for Diterpenoid Fragrance Production*

PubMed Central

Zerbe, Philipp; Chiang, Angela; Yuen, Macaire; Hamberger, Björn; Hamberger, Britta; Draper, Jason A.; Britton, Robert; Bohlmann, Jörg

2012-01-01

The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production. PMID:22337889

NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with μ-Opioid Agonist Activity.

PubMed

Renton, Paul; Green, Brenda; Maddaford, Shawn; Rakhit, Suman; Andrews, John S

2012-03-08

A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the μ-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the μ-opioid GPCR was predicated on the modulatory role of nitric oxide on μ-opioid receptor function. Structure-activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 μM), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent μ-opioid binding affinity, K i = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 μM). This work represents a novel approach in the development of new analgesics for the treatment of pain.

Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe.

PubMed

Jeangkhwoa, Pattraporn; Bandhaya, Achirapa; Umpunjun, Puangpaka; Chuenboonngarm, Ngarmnij; Panvisavas, Nathinee

2017-03-01

This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace. Copyright © 2016. Published by Elsevier B.V.

Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP.

PubMed

Cirovic, Natasa; Kecmanovic, Miljana; Keckarevic, Dusan; Keckarevic Markovic, Milica

2017-10-01

Cannabis sativa subspecies, known as industrial hemp (C. sativa sativa) and marijuana (C. sativa indica) show no evident morphological distinctions, but they contain different levels of psychoactive Δ-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp. C. sativa subspecies differ in sequence of tetrahydrocannabinolic acid (THCA) synthase gene, responsible for THC production, and only one active copy of the gene, distinctive for marijuana, is capable of producing THC in concentration more then 0,3% in dried plants, usually punishable by the law. Twenty different samples of marijuana that contain THC in concentration more then 0,3% and three varieties of industrial hemp were analyzed for presence of an active copy of THCA synthase gene using in-house developed restriction fragment length polymorphism (RFLP) method All twenty samples of marijuana were positive for the active copy of THCA synthase gene, 16 of them heterozygous. All three varieties of industrial hemp were homozygous for inactive copy. An algorithm for the fast and accurate forensic analysis of samples suspected to be marijuana was constructed, answering the question if an analyzed sample is capable of producing THC in concentrations higher than 0.3%. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Analysis of GD2/GM2 synthase mRNA as a biomarker for small cell lung cancer.

PubMed

Chen, Lin-Chi; Brown, Andrew B; Cheung, Irene Y; Cheung, Nai-Kong V; Kris, Mark G; Krug, Lee M

2010-02-01

GD2/GM2 synthase is a key enzyme in the synthesis of GD2 and GM2 gangliosides found on the surface of neuroblastoma and small cell lung carcinoma (SCLC) cells. In neuroblastoma, persistent levels of GD2/GM2 synthase RNA in bone marrow (BM) following therapy portend poorer progression-free and overall survival. We conducted this study to determine if GD2/GM2 synthase RNA could be detected in SCLC cell lines and human tissues, and whether mRNA transcript levels corresponded with disease status. Initially, a pilot study enrolled patients with SCLC to determine the rate of GD2 expression at various points in the patients' disease course. Peripheral blood (PB), bone marrow and tumor tissues were used to measure GD2/GM2 synthase levels. In addition, SCLC cell lines were analyzed for GD2/GM2 synthase expression. Based on data from that initial analysis, a prospective trial was developed enrolling patients with newly diagnosed SCLC and following them serially. GD2/GM2 synthase transcript was determined by a sensitive quantitative reverse transcription-PCR (qRT-PCR) assay and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Six SCLC cell lines were assayed for expression of GD2/GM2 synthase, and high expression was detected in all. GD2/GM2 synthase transcript levels were obtained from tumor tissue, BM, or PB of 29 patients in the pilot study. 6/10 (60%) tumor tissues or BM samples were positive (median 332.7 units; range 13-2323 units); 8/19 (42%) untreated patients were GD2/GM2 synthase positive in their PB prior to beginning therapy (median 10.2; range 5.1-32.2); 3/4 (75%) patients who were first tested when they developed recurrent disease were positive in their PB (median 16.1; range 8.5-19.9). The fourth patient had an initial value of 2.0 (negative), which increased to 8.4 (positive) within 1 month without treatment. Seven of 12 patients with baseline positive GD2/GM2 synthase values had post-treatment levels measured, all of which were

SIRT3 Deacetylates ATP Synthase F1 Complex Proteins in Response to Nutrient- and Exercise-Induced Stress

PubMed Central

Vassilopoulos, Athanassios; Pennington, J. Daniel; Andresson, Thorkell; Rees, David M.; Bosley, Allen D.; Fearnley, Ian M.; Ham, Amy; Flynn, Charles Robb; Hill, Salisha; Rose, Kristie Lindsey; Kim, Hyun-Seok; Walker, John E.

2014-01-01

Abstract Aims: Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. Results: By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCPK139 directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. Innovation: This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. Conclusion: Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins. Antioxid. Redox Signal. 21, 551–564. PMID:24252090

Stachyose synthesis in seeds of adzuki bean (Vigna angularis): molecular cloning and functional expression of stachyose synthase.

PubMed

Peterbauer, T; Mucha, J; Mayer, U; Popp, M; Glössl, J; Richter, A

1999-12-01

Stachyose is the major soluble carbohydrate in seeds of a number of important crop species. It is synthesized from raffinose and galactinol by the action of stachyose synthase (EC 2.4.1.67). We report here on the identification of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). Based on internal amino acid sequences of the enzyme purified from adzuki bean, oligonucleotides were designed and used to amplify corresponding sequences from adzuki bean cDNA by RT-PCR, followed by rapid amplification of cDNA ends (RACE-PCR). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was amplified by PCR, engineered into the baculovirus expression vector pVL1393 and introduced into Spodoptera frugiperda (Sf21) insect cells for heterologous expression. The recombinant protein was immunologically reactive with polyclonal antibodies raised against stachyose synthase purified from adzuki bean and was shown to be a functional stachyose synthase with the same catalytic properties as its native counterpart. High levels of stachyose synthase mRNA were transiently accumulated midway through seed development, and the enzyme was also present in mature seeds and during germination.

Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species

NASA Technical Reports Server (NTRS)

Haney, P. J.; Badger, J. H.; Buldak, G. L.; Reich, C. I.; Woese, C. R.; Olsen, G. J.

1999-01-01

The genome sequence of the extremely thermophilic archaeon Methanococcus jannaschii provides a wealth of data on proteins from a thermophile. In this paper, sequences of 115 proteins from M. jannaschii are compared with their homologs from mesophilic Methanococcus species. Although the growth temperatures of the mesophiles are about 50 degrees C below that of M. jannaschii, their genomic G+C contents are nearly identical. The properties most correlated with the proteins of the thermophile include higher residue volume, higher residue hydrophobicity, more charged amino acids (especially Glu, Arg, and Lys), and fewer uncharged polar residues (Ser, Thr, Asn, and Gln). These are recurring themes, with all trends applying to 83-92% of the proteins for which complete sequences were available. Nearly all of the amino acid replacements most significantly correlated with the temperature change are the same relatively conservative changes observed in all proteins, but in the case of the mesophile/thermophile comparison there is a directional bias. We identify 26 specific pairs of amino acids with a statistically significant (P < 0.01) preferred direction of replacement.

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

PubMed

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

2017-05-01

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

[Role of NO-synthase in stimulation of opiate receptors and kidney oxidative stress resistance].

PubMed

Orlova, E A; Komarevtseva, I A

2004-01-01

It was established that dalarginum injection before ARI (acute renal insufficiency) formation prevented an increases of proteolysis, decrease of SOD (superoxide dismutase), increase of NO2-/NO3- content in kidney tissue. Antioxidant effect of opiate receptor agonist was completely abolished by preliminary injection of OR antagonist--naloxone. Aminoguanidine nitrate (inducible NO-synthase inhibitor) injection removed positive effect of OR stimulation too. Thus OR stimulation increases kidney oxidative stress resistance due to NO-synthase and SOD activation.

Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones

PubMed Central

Thomson, Nicholas M.; Saika, Azusa; Ushimaru, Kazunori; Sangiambut, Smith; Tsuge, Takeharu; Summers, David K.

2013-01-01

The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter−1), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter−1) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer. PMID:23335776

NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with μ-Opioid Agonist Activity

PubMed Central

2012-01-01

A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the μ-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the μ-opioid GPCR was predicated on the modulatory role of nitric oxide on μ-opioid receptor function. Structure–activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 μM), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent μ-opioid binding affinity, Ki = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 μM). This work represents a novel approach in the development of new analgesics for the treatment of pain. PMID:24900459

Chitin synthases are required for survival, fecundity and egg-hatch in the red flour beetle, Tribolium castaneum

USDA-ARS?s Scientific Manuscript database

The synthesis of chitin, the Beta-1,4-linked polymer of N-acetylglucosamine, is catalyzed by chitin synthase (CHS). Chitin is essential for the structural integrity of the exoskeletal cuticle and midgut peritrophic membrane (PM) of insects. To study the functions of the two chitin synthase genes, ...

Discovery of a new polyhydroxyalkanoate synthase from limestone soil through metagenomic approach.

PubMed

Tai, Yen Teng; Foong, Choon Pin; Najimudin, Nazalan; Sudesh, Kumar

2016-04-01

PHA synthase (PhaC) is the key enzyme in the production of biodegradable plastics known as polyhydroxyalkanoate (PHA). Nevertheless, most of these enzymes are isolated from cultivable bacteria using traditional isolation method. Most of the microorganisms found in nature could not be successfully cultivated due to the lack of knowledge on their growth conditions. In this study, a culture-independent approach was applied. The presence of phaC genes in limestone soil was screened using primers targeting the class I and II PHA synthases. Based on the partial gene sequences, a total of 19 gene clusters have been identified and 7 clones were selected for full length amplification through genome walking. The complete phaC gene sequence of one of the clones (SC8) was obtained and it revealed 81% nucleotide identity to the PHA synthase gene of Chromobacterium violaceum ATCC 12472. This gene obtained from uncultured bacterium was successfully cloned and expressed in a Cupriavidus necator PHB(-)4 PHA-negative mutant resulting in the accumulation of significant amount of PHA. The PHA synthase activity of this transformant was 64 ± 12 U/g proteins. This paper presents a pioneering study on the discovery of phaC in a limestone area using metagenomic approach. Through this study, a new functional phaC was discovered from uncultured bacterium. Phylogenetic classification for all the phaCs isolated from this study has revealed that limestone hill harbors a great diversity of PhaCs with activities that have not yet been investigated. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

PubMed Central

Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H.; Blondel, Marc; di Rago, Jean-Paul

2010-01-01

Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

PubMed Central

Hong, Sangjin; Pedersen, Peter L.

2008-01-01

Summary: ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and Pi, the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology. PMID:19052322

TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells.

PubMed

Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

2015-03-12

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics.

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

PubMed Central

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

2012-01-01

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

Modulation of inherent dynamical tendencies of the bisabolyl cation via preorganization in epi-isozizaene synthase.

PubMed

Pemberton, Ryan P; Ho, Krystina C; Tantillo, Dean J

2015-04-01

The relative importance of preorganization, selective transition state stabilization and inherent reactivity are assessed through quantum chemical and docking calculations for a sesquiterpene synthase ( epi -isozizaene synthase, EIZS). Inherent reactivity of the bisabolyl cation, both static and dynamic, appears to determine the pathway to product, although preorganization and selective binding of the final transition state structure in the multi-step carbocation cascade that forms epi -isozizaene appear to play important roles.

ATP synthase--a marvellous rotary engine of the cell.

PubMed

Yoshida, M; Muneyuki, E; Hisabori, T

2001-09-01

ATP synthase can be thought of as a complex of two motors--the ATP-driven F1 motor and the proton-driven Fo motor--that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.

Cloning and characterization of the Schizosaccharomyces pombe tRNA:pseudouridine synthase Pus1p

PubMed Central

Hellmuth, Klaus; Grosjean, Henri; Motorin, Yuri; Deinert, Karina; Hurt, Ed; Simos, George

2000-01-01

Saccharomyces cerevisiae cells that carry deletions in both the LOS1 (a tRNA export receptor) and the PUS1 (a tRNA:pseudouridine synthase) genes exhibit a thermosensitive growth defect. A Schizosaccharomyces pombe gene, named spPUS1, was cloned from a cDNA library by complementation of this conditional lethal phenotype. The corresponding protein, spPus1p, shows sequence similarity to S.cerevisiae and murine Pus1p as well as other known members of the pseudouridine synthase family. Accordingly, recombinant spPus1p can catalyze in vitro the formation of pseudouridines at positions 27, 28, 34, 35 and 36 of yeast tRNA transcripts. The sequence and functional conservation of the Pus1p proteins in fungi and mammalian species and their notable absence from prokaryotes suggest that this family of pseudouridine synthases is required for a eukaryote-specific step of tRNA biogenesis, such as nuclear export. PMID:11095668

Cloning and characterization of the Schizosaccharomyces pombe tRNA:pseudouridine synthase Pus1p.

PubMed

Hellmuth, K; Grosjean, H; Motorin, Y; Deinert, K; Hurt, E; Simos, G

2000-12-01

Saccharomyces cerevisiae cells that carry deletions in both the LOS1 (a tRNA export receptor) and the PUS1 (a tRNA:pseudouridine synthase) genes exhibit a thermosensitive growth defect. A Schizosaccharomyces pombe gene, named spPUS1, was cloned from a cDNA library by complementation of this conditional lethal phenotype. The corresponding protein, spPus1p, shows sequence similarity to S. cerevisiae and murine Pus1p as well as other known members of the pseudouridine synthase family. Accordingly, recombinant spPus1p can catalyze in vitro the formation of pseudouridines at positions 27, 28, 34, 35 and 36 of yeast tRNA transcripts. The sequence and functional conservation of the Pus1p proteins in fungi and mammalian species and their notable absence from prokaryotes suggest that this family of pseudouridine synthases is required for a eukaryote-specific step of tRNA biogenesis, such as nuclear export.

Cardioprotective Effect of Danshensu against Ischemic/Reperfusion Injury via c-Subunit of ATP Synthase Inhibition

PubMed Central

Zhao, JingYi; Fan, Zixuan; Bao, Jiadi; Sun, Dawei; Sun, Chun

2017-01-01

Mitochondrial permeability transition pore (MPTP) opening is the main culprit of ischemic/reperfusion (IR) injury. It is reported that c-subunit of ATP synthase is the core component of MPTP. Danshensu (DSS), a monomer isolated from the traditional Chinese herb Danshen, has showed cardioprotective effect against IR injury through unknown mechanism. In this study, rat hearts were suspended in Langendorff instrument and perfused with Krebs-Henseleit (KH) buffer containing DSS for 60 minutes, followed by 30 minutes of global ischemia. Parameters including heart rate, left ventricular developed pressure, and the rate of left ventricle diastolic pressure change were recorded to assess their cardiac function. All these indexes were improved in DSS group. The rate of cardiomyocytes apoptosis and MPTP opening were both inhibited in DSS group. In addition, DSS administration leads to downregulation of c-subunit of ATP synthase in both mRNA and protein levels. Consistently, when c-subunit of ATP synthase was overexpressed in H9C2 cells through pcDNA3/5G1 plasmid transfection, MPTP opening was enhanced when the cardioprotective effect of DSS also tapers. In conclusion, DSS could alleviate cardiac IR injury via inhibiting c-subunit of ATP synthase expression. PMID:29250127

Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Hongmei; Ma, Leyuan; Zhao, Cong

To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leavesmore » and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.« less

The First Prokaryotic Trehalose Synthase Complex Identified in the Hyperthermophilic Crenarchaeon Thermoproteus tenax

PubMed Central

Bräsen, Christopher; Hensel, Reinhard; Lupas, Andrei N.; Brinkmann, Henner; Siebers, Bettina

2013-01-01

The role of the disaccharide trehalose, its biosynthesis pathways and their regulation in Archaea are still ambiguous. In Thermoproteus tenax a fused trehalose-6-phosphate synthase/phosphatase (TPSP), consisting of an N-terminal trehalose-6-phosphate synthase (TPS) and a C-terminal trehalose-6-phosphate phosphatase (TPP) domain, was identified. The tpsp gene is organized in an operon with a putative glycosyltransferase (GT) and a putative mechanosensitive channel (MSC). The T. tenax TPSP exhibits high phosphatase activity, but requires activation by the co-expressed GT for bifunctional synthase-phosphatase activity. The GT mediated activation of TPS activity relies on the fusion of both, TPS and TPP domain, in the TPSP enzyme. Activation is mediated by complex-formation in vivo as indicated by yeast two-hybrid and crude extract analysis. In combination with first evidence for MSC activity the results suggest a sophisticated stress response involving TPSP, GT and MSC in T. tenax and probably in other Thermoproteales species. The monophyletic prokaryotic TPSP proteins likely originated via a single fusion event in the Bacteroidetes with subsequent horizontal gene transfers to other Bacteria and Archaea. Furthermore, evidence for the origin of eukaryotic TPSP fusions via HGT from prokaryotes and therefore a monophyletic origin of eukaryotic and prokaryotic fused TPSPs is presented. This is the first report of a prokaryotic, archaeal trehalose synthase complex exhibiting a much more simple composition than the eukaryotic complex described in yeast. Thus, complex formation and a complex-associated regulatory potential might represent a more general feature of trehalose synthesizing proteins. PMID:23626675

chs-4, a class IV chitin synthase gene from Neurospora crassa.

PubMed

Din, A B; Specht, C A; Robbins, P W; Yarden, O

1996-02-05

In Saccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by the CSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized as CSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomycete Neurospora crassa and have used a "reverse genetics" approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of a N. crassa gene(designated chs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those of S. cerevisiae and Candida albicans. N. crassa strains in which chs-4 had been inactivated by the Repeat-Induced point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in the chs-4RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme in N. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.

Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase.

PubMed

Michel, J B; Feron, O; Sase, K; Prabhakar, P; Michel, T

1997-10-10

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.

Isolation and bacterial expression of a sesquiterpene synthase CDNA clone from peppermint(mentha .chi. piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Crock, John E.

1999-01-01

A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) 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 (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene 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 (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-farnesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

Three 1-Aminocyclopropane-1-Carboxylate Synthase Genes Regulated by Primary and Secondary Pollination Signals in Orchid Flowers1

PubMed Central

Bui, Anhthu Q.; Neill, Sharman D. O'

1998-01-01

The temporal and spatial expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes were investigated in pollinated orchid (Phalaenopsis spp.) flowers. Pollination signals initiate a cascade of development events in multiple floral organs, including the induction of ethylene biosynthesis, which coordinates several postpollination developmental responses. The initiation and propagation of ethylene biosynthesis is regulated by the coordinated expression of three distinct ACC synthase genes in orchid flowers. One ACC synthase gene (Phal-ACS1) is regulated by ethylene and participates in amplification and interorgan transmission of the pollination signal, as we have previously described in a related orchid genus. Two additional ACC synthase genes (Phal-ACS2 and Phal-ACS3) are expressed primarily in the stigma and ovary of pollinated orchid flowers. Phal-ACS2 mRNA accumulated in the stigma within 1 h after pollination, whereas Phal-ACS1 mRNA was not detected until 6 h after pollination. Similar to the expression of Phal-ACS2, the Phal-ACS3 gene was expressed within 2 h after pollination in the ovary. Exogenous application of auxin, but not ACC, mimicked pollination by stimulating a rapid increase in ACC synthase activity in the stigma and ovary and inducing Phal-ACS2 and Phal-ACS3 mRNA accumulation in the stigma and ovary, respectively. These results provide the basis for an expanded model of interorgan regulation of three ACC synthase genes that respond to both primary (Phal-ACS2 and Phal-ACS3) and secondary (Phal-ACS1) pollination signals. PMID:9449850

TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells

PubMed Central

Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

2015-01-01

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics. PMID:25762467

Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction

PubMed Central

2012-01-01

Background Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined. Method We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability. Results In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W. Conclusion Hypercapnia with and without acidosis increased HPV during

Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction.

PubMed

Ketabchi, Farzaneh; Ghofrani, Hossein A; Schermuly, Ralph T; Seeger, Werner; Grimminger, Friedrich; Egemnazarov, Bakytbek; Shid-Moosavi, S Mostafa; Dehghani, Gholam A; Weissmann, Norbert; Sommer, Natascha

2012-01-31

Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined. We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability. In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W. Hypercapnia with and without acidosis increased HPV during conditions of sustained hypoxia. The

UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.

Prostaglandins belong to a class of cyclic lipid-derived mediators synthesized from arachidonic acid via COX-1, COX-2 and various prostaglandin synthases. Members of this family include prostaglandins such as PGE{sub 2}, PGF{sub 2{alpha}}, PGD{sub 2} and PGI{sub 2} (prostacyclin) as well as thromboxane. In the present studies we analyzed the effects of UVB on prostaglandin production and prostaglandin synthase expression in primary cultures of undifferentiated and calcium-differentiated mouse keratinocytes. Both cell types were found to constitutively synthesize PGE{sub 2}, PGD{sub 2} and the PGD{sub 2} metabolite PGJ{sub 2}. Twenty-four hours after treatment with UVB (25 mJ/cm{sup 2}), production of PGE{sub 2}more » and PGJ{sub 2} increased, while PGD{sub 2} production decreased. This was associated with increased expression of COX-2 mRNA and protein. UVB (2.5-25 mJ/cm{sup 2}) also caused marked increases in mRNA expression for the prostanoid synthases PGDS, mPGES-1, mPGES-2, PGFS and PGIS, as well as expression of receptors for PGE{sub 2} (EP1 and EP2), PGD{sub 2} (DP and CRTH2) and prostacyclin (IP). UVB was more effective in inducing COX-2 and DP in differentiated cells and EP1 and IP in undifferentiated cells. UVB readily activated keratinocyte PI-3-kinase (PI3K)/Akt, JNK and p38 MAP signaling pathways which are known to regulate COX-2 expression. While inhibition of PI3K suppressed UVB-induced mPGES-1 and CRTH2 expression, JNK inhibition suppressed mPGES-1, PGIS, EP2 and CRTH2, and p38 kinase inhibition only suppressed EP1 and EP2. These data indicate that UVB modulates expression of prostaglandin synthases and receptors by distinct mechanisms. Moreover, both the capacity of keratinocytes to generate prostaglandins and their ability to respond to these lipid mediators are stimulated by exposure to UVB.« less

Two Bacterial Diterpene Synthases from Allokutzneria albata for Bonnadiene and for Phomopsene and Allokutznerene.

PubMed

Lauterbach, Lukas; Rinkel, Jan; Dickschat, Jeroen Sidney

2018-05-14

Two diterpene synthases from Allokutzneria albata were studied for their products, resulting in the identification of the new compound bonnadiene from the first enzyme. Although phylogenetically unrelated to fungal phomopsene synthase, the second enzyme produced a mixture of phomopsene and a biosynthetically linked new compound, allokutznerene, besides spiroviolene. Both enzymes were deeply studied for their mechanisms by isotopic labelling experiments, metal cofactor variation and site-directed mutagenesis. Oxidation products of phomopsene and allokutznerene are also discussed. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Crystallization of the c14-rotor of the chloroplast ATP synthase reveals that it contains pigments

PubMed Central

Varco-Merth, Benjamin; Fromme, Raimund; Wang, Meitian; Fromme, Petra

2012-01-01

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 phosphate, 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 (c10–14γε) relative to stator moiety (α3β3δab2). Here we report the isolation and crystallization of the c14-ring of subunit c from the spinach chloroplast enzyme diffracting as far as 2.8 Å. Though ATP synthase was not previously known to contain any pigments, the crystals of the c-subunit possessed a strong yellow color. The pigment analysis revealed 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. PMID:18515064

Phylogenomic and Domain Analysis of Iterative Polyketide Synthases in Aspergillus Species

PubMed Central

Lin, Shu-Hsi; Yoshimoto, Miwa; Lyu, Ping-Chiang; Tang, Chuan-Yi; Arita, Masanori

2012-01-01

Aspergillus species are industrially and agriculturally important as fermentors and as producers of various secondary metabolites. Among them, fungal polyketides such as lovastatin and melanin are considered a gold mine for bioactive compounds. We used a phylogenomic approach to investigate the distribution of iterative polyketide synthases (PKS) in eight sequenced Aspergilli and classified over 250 fungal genes. Their genealogy by the conserved ketosynthase (KS) domain revealed three large groups of nonreducing PKS, one group inside bacterial PKS, and more than 9 small groups of reducing PKS. Polyphyly of nonribosomal peptide synthase (NRPS)-PKS genes raised questions regarding the recruitment of the elegant conjugation machinery. High rates of gene duplication and divergence were frequent. All data are accessible through our web database at http://metabolomics.jp/wiki/Category:PK. PMID:22844193

The anisotropy1 D604N Mutation in the Arabidopsis Cellulose Synthase1 Catalytic Domain Reduces Cell Wall Crystallinity and the Velocity of Cellulose Synthase Complexes1[W][OA

PubMed Central

Fujita, Miki; Himmelspach, Regina; Ward, Juliet; Whittington, Angela; Hasenbein, Nortrud; Liu, Christine; Truong, Thy T.; Galway, Moira E.; Mansfield, Shawn D.; Hocart, Charles H.; Wasteneys, Geoffrey O.

2013-01-01

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1’s permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature. PMID:23532584

Genetic Analysis of Comamonas acidovorans Polyhydroxyalkanoate Synthase and Factors Affecting the Incorporation of 4-Hydroxybutyrate Monomer

PubMed Central

Sudesh, Kumar; Fukui, Toshiaki; Doi, Yoshiharu

1998-01-01

The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaCCa) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp β-ketothiolase gene (phaACa). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A. eutrophus. The arrangement of PHA biosynthesis genes in C. acidovorans was also similar to that in A. eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaACa. The cloned fragment complemented a PHA-negative mutant of A. eutrophus, PHB−4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of C. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in C. acidovorans, it is the metabolic pathway, and not the specificity of the organism’s PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content. PMID:9726894

Muscle-Specific Deletion of Rictor Impairs Insulin-Stimulated Glucose Transport and Enhances Basal Glycogen Synthase Activity▿

PubMed Central

Kumar, Anil; Harris, Thurl E.; Keller, Susanna R.; Choi, Kin M.; Magnuson, Mark A.; Lawrence, John C.

2008-01-01

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity. PMID:17967879

Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis.

PubMed

Sitthithaworn, W; Kojima, N; Viroonchatapan, E; Suh, D Y; Iwanami, N; Hayashi, T; Noji, M; Saito, K; Niwa, Y; Sankawa, U

2001-02-01

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene-producing plants, Scoparia dulcis and Croton sublyratus, have been isolated using the homology-based polymerase chain reaction (PCR) method. Both clones contained highly conserved aspartate-rich motifs (DDXX(XX)D) and their N-terminal residues exhibited the characteristics of chloroplast targeting sequence. When expressed in Escherichia coli, both the full-length and truncated proteins in which the putative targeting sequence was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to produce geranylgeranyl diphosphate (GGPP). The structural factors determining the product length in plant GGPPSs were investigated by constructing S. dulcis GGPPS mutants on the basis of sequence comparison with the first aspartate-rich motif (FARM) of plant farnesyl diphosphate synthase. The result indicated that in plant GGPPSs small amino acids, Met and Ser, at the fourth and fifth positions before FARM and Pro and Cys insertion in FARM play essential roles in determination of product length. Further, when a chimeric gene comprised of the putative transit peptide of the S. dulcis GGPPS gene and a green fluorescent protein was introduced into Arabidopsis leaves by particle gun bombardment, the chimeric protein was localized in chloroplasts, indicating that the cloned S. dulcis GGPPS is a chloroplast protein.

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

PubMed

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

2010-07-01

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

The role of inducible nitric oxide synthase in vascular hyporeactivity of endotoxin-treated and portal hypertensive rats.

PubMed

Heinemann, A; Stauber, R E

1995-05-04

The involvement of the inducible nitric oxide (NO) synthase in the vascular hyporeactivity in portal vein-ligated rats was assessed in isolated perfused mesenteric arterial beds. Aminoguanidine, a selective inhibitor of the inducible NO synthase, restored the pressor responses to methoxamine in arteries of endotoxin-treated rats, but was ineffective in hyporeactive portal vein-ligated vessels. NG-Nitro-L-arginine methyl ester enhanced the responsiveness both in portal vein-ligated and sham-operated rats, without changing the difference between the two groups. These results not only indicate that the inducible NO synthase is not involved in the hyporeactivity to methoxamine in mesenteric arteries of portal hypertensive rats, but also suggest a role for factors other than NO.

Polyketide synthases from poison hemlock (Conium maculatum L.).

PubMed

Hotti, Hannu; Seppänen-Laakso, Tuulikki; Arvas, Mikko; Teeri, Teemu H; Rischer, Heiko

2015-11-01

Coniine is a toxic alkaloid, the biosynthesis of which is not well understood. A possible route, supported by evidence from labelling experiments, involves a polyketide formed by the condensation of one acetyl-CoA and three malonyl-CoAs catalysed by a polyketide synthase (PKS). We isolated PKS genes or their fragments from poison hemlock (Conium maculatum L.) by using random amplification of cDNA ends (RACE) and transcriptome analysis, and characterized three full-length enzymes by feeding different starter-CoAs in vitro. On the basis of our in vitro experiments, two of the three characterized PKS genes in poison hemlock encode chalcone synthases (CPKS1 and CPKS2), and one encodes a novel type of PKS (CPKS5). We show that CPKS5 kinetically favours butyryl-CoA as a starter-CoA in vitro. Our results suggest that CPKS5 is responsible for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoAs. © 2015 FEBS.

Methionine biosynthesis in higher plants. I. Purification and characterization of cystathionine gamma-synthase from spinach chloroplasts.

PubMed

Ravanel, S; Droux, M; Douce, R

1995-01-10

Cystathionine gamma-synthase, the first enzyme specific for the methionine biosynthetic pathway, was purified to apparent homogeneity from spinach leaf chloroplasts. A nonradioactive assay based on O-phthaldialdehyde derivatization of L-cystathionine and fluorescence detection was developed to determine the cystathionine gamma-synthase activity. A unique cystathionine gamma-synthase activity was located in the stromal fraction of chloroplasts while cystathionine beta-lyase, the second enzyme of the transsulfuration pathway, was associated with both the chloroplastic and cytosolic compartments (see companion manuscript). The purified enzyme exhibited a specific activity of 13 U mg-1. As estimated by gel filtration and polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions followed by activity staining, the native enzyme had an apparent M(r) of 215,000. On the basis of sodium dodecyl sulfate-PAGE, purified cystathionine gamma-synthase migrated as two molecular species of M(r) 53,000 and 50,000 that are identical in their N-termini. The absorption spectrum obtained at pH 7.5 exhibited a peak at 425 nm due to pyridoxal 5'-phosphate (PLP). The purified enzyme catalyzed the formation of L-cystathionine or L-homocysteine depending on the sulfur-containing substrate, L-cysteine or sulfide. Maximal cystathionine gamma-synthase activity was found at pH 7.4. The apparent Km values for O-phospho-L-homoserine (the unique homoserine ester synthesized in the chloroplast), L-cysteine, and sulfide were 1.4, 0.18, and 0.6 mM, respectively. Inactivation of cystathionine gamma-synthase by DL-propargylglycine (PAG) showed pseudo-first-order kinetics and data were consistent with the existence of an intermediate reversible enzyme-inhibitor complex (Kappi = 140 microM) preceding the formation of a final enzyme-inhibitor complex (kd = 24 x 10(-3) s-1). The irreversibility of the inhibition and the partial restoration of the activity by pyridoxal-phosphate suggest that

Tailoring lumazine synthase assemblies for bionanotechnology.

PubMed

Azuma, Yusuke; Edwardson, Thomas G W; Hilvert, Donald

2018-05-21

Nanoscale compartments formed by hierarchical protein self-assembly are valuable platforms for nanotechnology development. The well-defined structure and broad chemical functionality of protein cages, as well as their amenability to genetic and chemical modification, have enabled their repurposing for diverse applications. In this review, we summarize progress in the engineering of the cage-forming enzyme lumazine synthase. This bacterial nanocompartment has proven to be a malleable scaffold. The natural protein has been diversified to afford a family of unique proteinaceous capsules that have been modified, evolved and assembled with other components to produce nanoreactors, artificial organelles, delivery vehicles and virus mimics.

Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and α-bisabolene synthases.

PubMed

Parveen, Iffat; Wang, Mei; Zhao, Jianping; Chittiboyina, Amar G; Tabanca, Nurhayat; Ali, Abbas; Baerson, Scott R; Techen, Natascha; Chappell, Joe; Khan, Ikhlas A; Pan, Zhiqiang

2015-11-01

Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including bioactive flavonoids, diterpene lactones, terpenoids and polysaccharides which accumulate in foliar tissues. Despite this chemical diversity, relatively few enzymes associated with any biosynthetic pathway from ginkgo have been characterized to date. In the present work, predicted transcripts potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoid compounds, including putative terpene synthases, were first identified by mining publicly-available G. biloba RNA-seq data sets. Recombinant enzyme studies with two of the TPS-like sequences led to the identification of GbTPS1 and GbTPS2, encoding farnesol and bisabolene synthases, respectively. Additionally, the phylogenetic analysis revealed the two terpene synthase genes as primitive genes that might have evolved from an ancestral diterpene synthase.

Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase.

PubMed

Lozovaya, Vera V; Lygin, Anatoliy V; Zernova, Olga V; Ulanov, Alexander V; Li, Shuxian; Hartman, Glen L; Widholm, Jack M

2007-02-01

Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen.

Sulfonamide Resistance in Clinical Isolates of Campylobacter jejuni: Mutational Changes in the Chromosomal Dihydropteroate Synthase

PubMed Central

Gibreel, Amera; Sköld, Ola

1999-01-01

The characterization of the genetic basis of sulfonamide resistance in Campylobacter jejuni was attempted. The resistance determinant from a sulfonamide-resistant strain of C. jejuni was cloned and was found to show 42% identity with the folP gene (which codes for dihydropteroate synthase, the target of sulfonamides) of the related bacterium Helicobacter pylori. The sequences of the areas surrounding the folP gene in C. jejuni showed similarity to those of the areas surrounding the corresponding gene in H. pylori. The folP gene of C. jejuni, which mediates the resistance, was observed to show particular features when it was compared to other known folP genes. One of these features is the presence of two pairs of direct repeats (15 and 27 bp) within the coding sequence of the gene. Comparison of the C. jejuni folP genes that mediate susceptibility and resistance revealed the occurrence of mutations that changed four amino acid residues. Resistance of C. jejuni to sulfonamides could be associated with one or several of these four mutational substitutions, which all occurred in the five different resistant isolates studied. The codon for one of these changed amino acids was found to be located in the second direct repeat within the coding sequence of the gene. The change made the repeat perfect. The transformation of both the resistance and the susceptibility variants of the gene into an Escherichia coli folP knockout mutant was found to complement the dihydropteroate synthase deficiency, confirming that the characterized sulfonamide resistance determinant codes for the C. jejuni dihydropteroate synthase enzyme. Kinetic measurements established different affinities of sulfonamide for the dihydropteroate synthase enzyme isolated from the resistant and susceptible strains. In conclusion, sulfonamide resistance in C. jejuni was shown to be associated with mutational changes in the chromosomally located gene for dihydropteroate synthase, the target of sulfonamides. PMID

Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

PubMed Central

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

2014-01-01

The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KM DXP = 252 µM and KM NADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development. PMID:25171339

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

DOEpatents

Croteau, Rodney Bruce; Crock, John E.

2005-01-25

A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) 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 (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene 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 (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-famesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

GMP Synthase Is Required for Virulence Factor Production and Infection by Cryptococcus neoformans*

PubMed Central

Chitty, Jessica L.; Tatzenko, Tayla L.; Williams, Simon J.; Koh, Y. Q. Andre E.; Corfield, Elizabeth C.; Butler, Mark S.; Robertson, Avril A. B.; Cooper, Matthew A.; Kappler, Ulrike; Kobe, Bostjan; Fraser, James A.

2017-01-01

Over the last four decades the HIV pandemic and advances in medical treatments that also cause immunosuppression have produced an ever-growing cohort of individuals susceptible to opportunistic pathogens. Of these, AIDS patients are particularly vulnerable to infection by the encapsulated yeast Cryptococcus neoformans. Most commonly found in the environment in purine-rich bird guano, C. neoformans experiences a drastic change in nutrient availability during host infection, ultimately disseminating to colonize the purine-poor central nervous system. Investigating the consequences of this challenge, we have characterized C. neoformans GMP synthase, the second enzyme in the guanylate branch of de novo purine biosynthesis. We show that in the absence of GMP synthase, C. neoformans becomes a guanine auxotroph, the production of key virulence factors is compromised, and the ability to infect nematodes and mice is abolished. Activity assays performed using recombinant protein unveiled differences in substrate binding between the C. neoformans and human enzymes, with structural insights into these kinetic differences acquired via homology modeling. Collectively, these data highlight the potential of GMP synthase to be exploited in the development of new therapeutic agents for the treatment of disseminated, life-threatening fungal infections. PMID:28062578

Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity.

PubMed Central

Gojković, Z; Sandrini, M P; Piskur, J

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-carbamyl-beta-alanine as the sole nitrogen source and exhibits diminished beta-alanine synthase activity was used to clone analogous genes from different eukaryotes. Putative PYD3 sequences from the yeast S. kluyveri, the slime mold Dictyostelium discoideum, and the fruit fly Drosophila melanogaster complemented the pyd3 defect. When the S. kluyveri PYD3 gene was expressed in S. cerevisiae, which has 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-carbamyl-beta-alanine, but not by uracil. This work establishes S. kluyveri as a model organism for studying pyrimidine degradation and beta-alanine production in eukaryotes. PMID:11454750

Cooxidation of 13-cis-retinoic acid by prostaglandin H synthase.

PubMed

Samokyszyn, V M; Sloane, B F; Honn, K V; Marnett, L J

1984-10-30

Cooxidative metabolism of 13-cis-retinoic acid (13-CIS) via prostaglandin H synthase was investigated employing ram seminal vesicle microsomes. Oxidation of 13-CIS utilizing H2O2, 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-OOH-18:2), or 1-hydroperoxy-5-phenyl-4-pentene was detected by measurement of O2 incorporation. UV spectroscopy and HPLC of extracted incubation mixtures demonstrated that 13-CIS was metabolized to oxidized derivatives. Similar spectral changes and HPLC profiles were obtained with H2O2, 13-OOH-18:2, or arachidonic acid as substrates. 4-Hydroxy-13-cis-retinoic acid and all trans-retinoic acid were products of cooxidation as well as other polar metabolites. Oxidation was inhibited by the antioxidant butylated hydroxyanisole and the spin trap, nitrosobenzene. These results indicate that 13-cis-retinoic acid is cooxidized by prostaglandin H synthase and suggest a free radical mechanism resembling that of lipid peroxidation.

A non-synonymous nucleotide substitution can account for one evolutionary route to sesquiterpene synthase activity in the TPS-b subgroup.

PubMed

Green, Sol; Baker, Edward N; Laing, William

2011-06-23

Plant sesquiterpene and hemiterpene synthases in the monoterpene synthase dominated TPS-b subgroup are thought to have evolved independently from a monoterpene synthase ancestor. A TPS-b sesquiterpene synthase from apple (MdAFS1), which predominantly produces α-farnesene, can also synthesize the monoterpene (E)-β-ocimene. The dual activity offered a functional link to an ancestral MdAFS1 enzyme and a rational basis for investigation of the evolution of TPS-b sesquiterpene enzymes. Protein modelling and mutagenesis analysis of the MdAFS1 active site identified a non-synonymous nucleotide substitution that could account for the requisite shift in substrate specificity necessary for the emergence of its sesquiterpene activity during the evolution of the TPS-b enzymes. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents.

PubMed

Richie, Daryl L; Thompson, Katherine V; Studer, Christian; Prindle, Vivian C; Aust, Thomas; Riedl, Ralph; Estoppey, David; Tao, Jianshi; Sexton, Jessica A; Zabawa, Thomas; Drumm, Joseph; Cotesta, Simona; Eichenberger, Jürg; Schuierer, Sven; Hartmann, Nicole; Movva, N Rao; Tallarico, John A; Ryder, Neil S; Hoepfner, Dominic

2013-05-01

High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo.

Identification and Evaluation of Novel Acetolactate Synthase Inhibitors as Antifungal Agents

PubMed Central

Richie, Daryl L.; Thompson, Katherine V.; Studer, Christian; Prindle, Vivian C.; Aust, Thomas; Riedl, Ralph; Estoppey, David; Tao, Jianshi; Sexton, Jessica A.; Zabawa, Thomas; Drumm, Joseph; Cotesta, Simona; Eichenberger, Jürg; Schuierer, Sven; Hartmann, Nicole; Movva, N. Rao; Tallarico, John A.

2013-01-01

High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo. PMID:23478965

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

PubMed

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.

UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyata, Maiko; Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065; Ichihara, Masatoshi

Highlights: • Melanocytes showed low ST8SIA1 and high B3GALT4 levels in contrast with melanomas. • Direct UVB irradiation of melanocytes did not induce ganglioside synthase genes. • Culture supernatants of UVB-irradiated keratinocytes induced ST8SIA1 in melanocytes. • TNFα and IL-6 secreted from keratinocytes enhanced ST8SIA1 expression in melanocytes. • Inflammatory cytokines induced melanoma-related ST8SIA1 in melanocytes. - Abstract: Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas frommore » melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.« less

β-Glucoside Activators of Mung Bean UDP-Glucose: β-Glucan Synthase 1

PubMed Central

Callaghan, Theresa; Ross, Peter; Weinberger-Ohana, Patricia; Benziman, Moshe

1988-01-01

n-Alkyl (C6-C12) β-d-monoglucopyranosides have been found to be highly potent activators of mung bean β-glucan synthase in vitro, increasing the Vmax of the enzyme as much as 60-fold and with Ka values as low as 10 micromolar. Activation is highly specific for the β-linked terminal glucose residue; other alkyl glycosides such as, octyl-α-glucoside, dodecyl β-maltoside, 6-lauryl sucrose, 6-lauryl glucose, which lack this structure, are ineffective as activators. Based on the similarities in their structure and effects on β-glucan synthesis under a variety of conditions, it is proposed that the alkyl β-glucosides are structural analogs of the native glucolipid activator of β-glucan synthase isolated from mung bean extracts. PMID:16666039

Glycogen synthase kinase 3: more than a namesake.

PubMed

Rayasam, Geetha Vani; Tulasi, Vamshi Krishna; Sodhi, Reena; Davis, Joseph Alex; Ray, Abhijit

2009-03-01

Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, tau protein and beta catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer's and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target.

SbnG, a citrate synthase in Staphylococcus aureus: A new fold on an old enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kobylarz, Marek J.; Grigg, Jason C.; Sheldon, Jessica R.

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. In this paper, we present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic genemore » clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. Finally, a structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production.« less

SbnG, a citrate synthase in Staphylococcus aureus: A new fold on an old enzyme

DOE PAGES

Kobylarz, Marek J.; Grigg, Jason C.; Sheldon, Jessica R.; ...

2014-10-21

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. In this paper, we present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic genemore » clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. Finally, a structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production.« less

SbnG, a citrate synthase in Staphylococcus aureus: a new fold on an old enzyme.

PubMed

Kobylarz, Marek J; Grigg, Jason C; Sheldon, Jessica R; Heinrichs, David E; Murphy, Michael E P

2014-12-05

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. We present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic gene clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. A structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased and Altered Fragrance of Tobacco Plants after Metabolic Engineering Using Three Monoterpene Synthases from Lemon

PubMed Central

Lücker, Joost; Schwab, Wilfried; van Hautum, Bianca; Blaas, Jan; van der Plas, Linus H. W.; Bouwmeester, Harro J.; Verhoeven, Harrie A.

2004-01-01

Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting β-pinene, limonene, and γ-terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes. PMID:14718674

Bovine protoporphyria: documentation of autosomal recessive inheritance and comparison with the human disease through measurement of heme synthase activity.

PubMed Central

Bloomer, J R; Morton, K O; Reuter, R J; Ruth, G R

1982-01-01

Protoporphyria is an autosomal dominant disease in man in which protoporphyrin accumulated because of a defect in heme synthase (ferrochelatase) activity. A disease has been described in cattle that has the same manifestations as does the human disease. We measured heme synthase activity in sonicates of cultured skin fibroblasts and whole liver homogenates from animals with protoporphyria, their unaffected parents, and normal cattle in order to examine the mode of inheritance and compare it with human protoporphyria. The mean activity (+/- SEM) in fibroblasts from the three groups was 2.0 +/- 0.4, 47 +/- 12, and 149 +/- 10 pmol heme formed/mg protein per hr, respectively, consistent with autosomal recessive inheritance. Similarly, the levels of heme synthase activity in livers of the parents were intermediate to those of normal animals and of animals with protoporphyria. When compared with normal human fibroblasts and liver, the specific activity of heme synthase in normal bovine tissue was significantly higher. These studies indicate that manifestations of protoporphyria do not occur in cattle unless the animal is homozygous for the gene defect, whereas in humans, the heterozygous condition is sufficient. This is probably because the specific activity of heme synthase in cells of heterozygous animals is not reduced to a level that significantly alters heme metabolism. PMID:7072720

A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grum, Daniel, E-mail: daniel.grum@uni-due.de; Boom, Johannes van den, E-mail: johannes.van-den-boom@stud.uni-due.de; Neumann, Daniel, E-mail: dneuman@gwdg.de

2010-05-07

3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Foerster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurementsmore » employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.« less

Crystallization and X-ray diffraction analysis of salicylate synthase, a chorismate-utilizing enyme involved in siderophore biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parsons, James F., E-mail: parsonsj@umbi.umd.edu; Shi, Katherine; Calabrese, Kelly

2006-03-01

Salicylate synthase, which catalyzes the first step in the synthesis of the siderophore yersiniabactin, has been crystallized. Diffraction data have been collected to 2.5 Å. Bacteria have evolved elaborate schemes that help them thrive in environments where free iron is severely limited. Siderophores such as yersiniabactin are small iron-scavenging molecules that are deployed by bacteria during iron starvation. Several studies have linked siderophore production and virulence. Yersiniabactin, produced by several Enterobacteriaceae, is derived from the key metabolic intermediate chorismic acid via its conversion to salicylate by salicylate synthase. Crystals of salicylate synthase from the uropathogen Escherichia coli CFT073 have beenmore » grown by vapour diffusion using polyethylene glycol as the precipitant. The monoclinic (P2{sub 1}) crystals diffract to 2.5 Å. The unit-cell parameters are a = 57.27, b = 164.07, c = 59.04 Å, β = 108.8°. The solvent content of the crystals is 54% and there are two molecules of the 434-amino-acid protein in the asymmetric unit. It is anticipated that the structure will reveal key details about the reaction mechanism and the evolution of salicylate synthase.« less

In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line.

PubMed

Ishikawa, Fumihiro; Sugimoto, Hiroyasu; Kakeya, Hideaki

2016-11-17

Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl-CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl-CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self-malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self-acylation. This lack of self-malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein-protein interaction between the fatty acid and polyketide synthases in the Adm assembly line. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation of aldosterone-synthase inhibition in rats.

PubMed

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

2006-06-01

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

Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mayer, R.; Ross, P.; Weinhouse, H.

1991-06-15

To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either ({sup 32}P)c-di-GMP or ({alpha}-{sup 32}P)UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- andmore » 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-k-Da peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. The authors suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.« less

Isolation and identification of a thermophilic strain producing trehalose synthase from geothermal water in China.

PubMed

Zhu, Yueming; Zhang, Jun; Wei, Dongsheng; Wang, Yufan; Chen, Xiaoyun; Xing, Laijun; Li, Mingchun

2008-08-01

A slightly thermophilic strain, CBS-01, producing trehalose synthase (TreS), was isolated from geothermal water in this study. According to the phenotypic characteristics and phylogenetic analysis of the 16s rRNA gene sequence, it was identified as Meiothermus ruber. The trehalose synthase gene of Meiothermus ruber CBS-01 was cloned by polymerase chain reaction and sequenced. The TreS gene consisted of 2,895 nucleotides, which specified a 964-amino-acid protein. This novel TreS catalyzed reversible interconversion of maltose and trehalose.

The Expression of Type-1 and Type-2 Nitric Oxide Synthase in Selected Tissues of the Gastrointestinal Tract during Mixed Mycotoxicosis

PubMed Central

Gajęcka, Magdalena; Stopa, Ewa; Tarasiuk, Michał; Zielonka, Łukasz; Gajęcki, Maciej

2013-01-01

The aim of the study was to verify the hypothesis that intoxication with low doses of mycotoxins leads to changes in the mRNA expression levels of nitric oxide synthase-1 and nitric oxide synthase-2 genes in tissues of the gastrointestinal tract and the liver. The experiment involved four groups of immature gilts (with body weight of up to 25 kg) which were orally administered zearalenone in a daily dose of 40 μg/kg BW (group Z, n = 18), deoxynivalenol at 12 μg/kg BW (group D, n = 18), zearalenone and deoxynivalenol (group M, n = 18) or placebo (group C, n = 21) over a period of 42 days. The lowest mRNA expression levels of nitric oxide synthase-1 and nitric oxide synthase-2 genes were noted in the sixth week of the study, in particular in group M. Our results suggest that the presence of low mycotoxin doses in feed slows down the mRNA expression of both nitric oxide synthase isomers, which probably lowers the concentrations of nitric oxide, a common precursor of inflammation. PMID:24284830

Structure and Mechanism of MbtI, the Salicylate Synthase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zwahlen,J.; Kolappan, S.; Zhou, R.

2007-01-01

MbtI (rv2386c) from Mycobacterium tuberculosis catalyzes the initial transformation in mycobactin biosynthesis by converting chorismate to salicylate. We report here the structure of MbtI at 2.5 {angstrom} resolution and demonstrate that isochorismate is a kinetically competent intermediate in the synthesis of salicylate from chorismate. At pH values below 7.5 isochorismate is the dominant product while above this pH value the enzyme converts chorismate to salicylate without the accumulation of isochorismate in solution. The salicylate and isochorismate synthase activities of MbtI are Mg{sup 2+}-dependent, and in the absence of Mg{sup 2+} MbtI has a promiscuous chorismate mutase activity similar to thatmore » of the isochorismate pyruvate lyase, PchB, from Pseudomonas aeruginosa. MbtI is part of a larger family of chorismate-binding enzymes descended from a common ancestor (the MST family), that includes the isochorismate synthases and anthranilate synthases. The lack of active site residues unique to pyruvate eliminating members of this family, combined with the observed chorismate mutase activity, suggests that MbtI may exploit a sigmatropic pyruvate elimination mechanism similar to that proposed for PchB. Using a combination of structural, kinetic, and sequence based studies we propose a mechanism for MbtI applicable to all members of the MST enzyme family.« less

Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif.

PubMed

Hurd, Matthew C; Kwon, Moonhyuk; Ro, Dae-Kyun

2017-08-26

Lippia dulcis (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of L. dulcis leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (LdTPS3 and LdTPS9) were isolated from L. dulcis leaf cDNA. To elucidate their catalytic functions, E. coli-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). In vitro enzyme assays showed that LdTPS3 encodes bornyl diphosphate synthase (thus named LdBPPS) while LdTPS9 encodes linalool synthase. Interestingly, the N-terminus of LdBPPS possesses two arginine-rich (RRX 8 W) motifs, and enzyme assays showed that the presence of both RRX 8 W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX 8 W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and L. dulcis. The discovery of LdBPPS opens up the possibility of engineering L. dulcis to remove the undesirable product, camphor. Copyright © 2017 Elsevier Inc. All rights reserved.

Nitric oxide synthase expression in foetal placentas of cows with retained fetal membranes.

PubMed

Shixin, Fu; Li, Zhang; Chunhai, Luo; Chuang, Xu; Cheng, Xia; Zhe, Wang; Xiaobing, Li

2011-10-01

The objectives of this study were to investigate relationship of retained fetal membranes (RFM) to expression of NOS and NOS mRNA and to analyze pathohistological changes and the distribution of nitric oxide synthase (NOS) in foetal placentas of cows with RFM. Twenty cows were assigned to two groups, a control group (no retained fetal membranes, NRFM, n = 10) and a diseased group (RFM, n = 10). The endpoint method was used to detect the nitric oxide (NO) content and nitric oxide synthase (NOS) activity in foetal placental tissue fluid and the fluorescent quantitation PCR was used to measure the expression of NOS mRNA. Immunohistochemistry and hematoxylin-eosin staining were used to observe pathohistological changes. Tissue from RFM cows showed fibronecrosis of the chorionic villi, and a decreased number of trophoblastic cells. The majority of trophoblastic cells displayed vacuolar degeneration. Interstitium vessels were distended and congested. Expression of induced nitric oxide synthase (iNOS) protein and iNOS mRNA was significantly higher (P < 0.05) in the cytoplasm of placental villus trophoblastic cells in the RFM group. But expression of endothelial nitric oxide synthase (eNOS) protein and eNOS mRNA was significantly lower (P<0.05) in the RFM group. The NO content and NOS activity of cows with RFM were significantly higher (P < 0.05). A high expression of iNOS protein and iNOS mRNA in the cow foetal placenta could produce high content of NO, which might inhibit uterine contraction. So over expression of iNOS protein and iNOS mRNA might be an important agent of retained fetal membranes in cows, and it may be a potential diagnosis biomarker. Copyright © 2010 Elsevier Ltd. All rights reserved.

Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence.

PubMed Central

DeWitt, D L; Smith, W L

1988-01-01

Prostaglandin G/H synthase (8,11,14-icosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1) catalyzes the first step in the formation of prostaglandins and thromboxanes, the conversion of arachidonic acid to prostaglandin endoperoxides G and H. This enzyme is the site of action of nonsteroidal anti-inflammatory drugs. We have isolated a 2.7-kilobase complementary DNA (cDNA) encompassing the entire coding region of prostaglandin G/H synthase from sheep vesicular glands. This cDNA, cloned from a lambda gt 10 library prepared from poly(A)+ RNA of vesicular glands, hybridizes with a single 2.75-kilobase mRNA species. The cDNA clone was selected using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the purified enzyme. The full-length cDNA encodes a protein of 600 amino acids, including a signal sequence of 24 amino acids. Identification of the cDNA as coding for prostaglandin G/H synthase is based on comparison of amino acid sequences of seven peptides comprising 103 amino acids with the amino acid sequence deduced from the nucleotide sequence of the cDNA. The molecular weight of the unglycosylated enzyme lacking the signal peptide is 65,621. The synthase is a glycoprotein, and there are three potential sites for N-glycosylation, two of them in the amino-terminal half of the molecule. The serine reported to be acetylated by aspirin is at position 530, near the carboxyl terminus. There is no significant similarity between the sequence of the synthase and that of any other protein in amino acid or nucleotide sequence libraries, and a heme binding site(s) is not apparent from the amino acid sequence. The availability of a full-length cDNA clone coding for prostaglandin G/H synthase should facilitate studies of the regulation of expression of this enzyme and the structural features important for catalysis and for interaction with anti-inflammatory drugs. Images PMID:3125548

Affinity comparison of different THCA synthase to CBGA using modeling computational approaches.

PubMed

Alaoui, Moulay Abdelaziz El; Ibrahimi, Azeddine; Semlali, Oussama; Tarhda, Zineb; Marouane, Melloul; Najwa, Alaoui; Soulaymani, Abdelmajid; Fahime, Elmostafa El

2014-01-01

The Δ(9-)Tetrahydrocannabinol (THCA) is the primary psychoactive compound of Cannabis Sativa. It is produced by Δ(1-) Tetrahydrocannabinolic acid synthase (THCA) which catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) the precursor of the THCA. In this study, we were interested by the three dimensional structure of THCA synthase protein. Generation of models were done by MODELLER v9.11 and homology modeling with Δ1-tetrahydrocannabinolic acid (THCA) synthase X ray structure (PDB code 3VTE) on the basis of sequences retrieved from GenBank. Procheck, Errat, and Verify 3D tools were used to verify the reliability of the six 3D models obtained, the overall quality factor and the Prosa Z-score were also used to check the quality of the six modeled proteins. The RMSDs for C-alpha atoms, main-chain atoms, side-chain atoms and all atoms between the modeled structures and the corresponding template ranged between 0.290 Å-1.252 Å, reflecting the good quality of the obtained models. Our study of the CBGA-THCA synthase docking demonstrated that the active site pocket was successfully recognized using computational approach. The interaction energy of CBGA computed in 'fiber types' proteins ranged between -4.1 95 kcal/mol and -5.95 kcal/mol whereas in the 'drug type' was about -7.02 kcal/mol to -7.16 kcal/mol, which maybe indicate the important role played by the interaction energy of CBGA in the determination of the THCA level in Cannabis Sativa L. varieties. Finally, we have proposed an experimental design in order to explore the binding energy source of ligand-enzyme in Cannabis Sativa and the production level of the THCA in the absence of any information regarding the correlation between the enzyme affinity and THCA level production. This report opens the doors to more studies predicting the binding site pocket with accuracy from the perspective of the protein affinity and THCA level produced in Cannabis Sativa.

The crystal structure of human GDP-L-fucose synthase.

PubMed

Zhou, Huan; Sun, Lihua; Li, Jian; Xu, Chunyan; Yu, Feng; Liu, Yahui; Ji, Chaoneng; He, Jianhua

2013-09-01

Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

PubMed Central

Winter, E; Brummel, M; Schuch, R; Spener, F

1997-01-01

In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

PubMed

Winter, E; Brummel, M; Schuch, R; Spener, F

1997-01-15

In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP.

Disruption of Bcchs4, Bcchs6 or Bcchs7 chitin synthase genes in Botrytis cinerea and the essential role of class VI chitin synthase (Bcchs6).

PubMed

Morcx, Serena; Kunz, Caroline; Choquer, Mathias; Assie, Sébastien; Blondet, Eddy; Simond-Côte, Elisabeth; Gajek, Karina; Chapeland-Leclerc, Florence; Expert, Dominique; Soulie, Marie-Christine

2013-03-01

Chitin synthases play critical roles in hyphal development and fungal pathogenicity. Previous studies on Botrytis cinerea, a model organism for necrotrophic pathogens, have shown that disruption of Bcchs1 and more particularly Bcchs3a genes have a drastic impact on virulence (Soulié et al., 2003, 2006). In this work, we investigate the role of other CHS including BcCHS4, BcCHS6 and BcCHS7 during the life cycle of B. cinerea. Single deletions of corresponding genes were carried out. Phenotypic analysis indicates that: (i) BcCHS4 enzyme is not essential for development and pathogenicity of the fungus; (ii) BcCHS7 is required for pathogenicity in a host dependant manner. For Bcchs6 gene disruption, we obtained only heterokaryotic strains. Indeed, sexual or asexual purification assays were unsuccessful. We concluded that class VI chitin synthase could be essential for B. cinerea and therefore BcCHS6 represents a valuable antifungal target. Copyright © 2012 Elsevier Inc. All rights reserved.

The rice terpene synthase gene OsTPS19 functions as an (S)-limonene synthase in planta, and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae.

PubMed

Chen, Xujun; Chen, Hao; Yuan, Joshua S; Köllner, Tobias G; Chen, Yuying; Guo, Yufen; Zhuang, Xiaofeng; Chen, Xinlu; Zhang, Yong-Jun; Fu, Jianyu; Nebenführ, Andreas; Guo, Zejian; Chen, Feng

2018-03-06

Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up-regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)-limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)-limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)-limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Biochemical characterization of microbial type terpene synthases in two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis.

PubMed

Xiong, Wangdan; Fu, Jianyu; Köllner, Tobias G; Chen, Xinlu; Jia, Qidong; Guo, Haobo; Qian, Ping; Guo, Hong; Wu, Guojiang; Chen, Feng

2018-05-01

Microbial terpene synthase-like (MTPSL) genes are a type of terpene synthase genes only recently identified in plants. In contrast to typical plant terpene synthase genes, which are ubiquitous in land plants, MTPSL genes appear to occur only in nonseed plants. Our knowledge of catalytic functions of MTPSLs is very limited. Here we report biochemical characterization of the enzymes encoded by MTPSL genes from two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis. Seven full-length MTPSL genes were identified in A. punctatus (ApMTPSL1-7) based on the analysis of its genome sequence. Using homology-based cloning, the apparent orthologs for six of the ApMTPSL genes, except ApMTPSL2, were cloned from A. agrestis. They were designated AaMTPSL1, 3-7. The coding sequences for each of the 13 Anthoceros MTPSL genes were cloned into a protein expression vector. Escherichia coli-expressed recombinant MTPSLs from hornworts were assayed for terpene synthase activities. Six ApMTPSLs and five AaMTPSLs, except for ApMTPSL5 and AaMTPSL5, showed catalytic activities with one or more isoprenyl diphosphate substrates. All functional MTPSLs exhibited sesquiterpene synthase activities. In contrast, only ApMTPSL7 and AaMTPSL7 showed monoterpene synthase activity and only ApMTPSL2, ApMTPSL6 and AaMTPSL6 showed diterpene synthase activity. Most MTPSLs from Anthoceros contain uncanonical aspartate-rich motif in the form of either 'DDxxxD' or 'DDxxx'. Homology-based structural modeling analysis of ApMTPSL1 and ApMTPSL7, which contain 'DDxxxD' and 'DDxxx' motif, respectively, showed that 'DDxxxD' and 'DDxxx' motifs are localized in the similar positions as the canonical 'DDxxD' motif in known terpene synthases. To further understand the role of individual aspartate residues in the motifs, ApMTPSL1 and ApMTPSL7 were selected as two representatives for site-directed mutagenesis studies. No activities were detected when any of the conserved aspartic acid was

Characterization of three chalcone synthase-like genes from apple (Malus x domestica Borkh.).

PubMed

Yahyaa, Mosaab; Ali, Samah; Davidovich-Rikanati, Rachel; Ibdah, Muhammad; Shachtier, Alona; Eyal, Yoram; Lewinsohn, Efraim; Ibdah, Mwafaq

2017-08-01

Apple (Malus x domestica Brokh.) is a widely cultivated deciduous tree species of significant economic importance. Apple leaves accumulate high levels of flavonoids and dihydrochalcones, and their formation is dependent on enzymes of the chalcone synthase family. Three CHS genes were cloned from apple leaves and expressed in Escherichia coli. The encoded recombinant enzymes were purified and functionally characterized. In-vitro activity assays indicated that MdCHS1, MdCHS2 and MdCHS3 code for proteins exhibiting polyketide synthase activity that accepted either p-dihydrocoumaroyl-CoA, p-coumaroyl-CoA, or cinnamoyl-CoA as starter CoA substrates in the presence of malonyl-CoA, leading to production of phloretin, naringenin chalcone, and pinocembrin chalcone. MdCHS3 coded a chalcone-dihydrochalcone synthase enzyme with narrower substrate specificity than the previous ones. The apparent Km values of MdCHS3 for p-dihydrocoumaryl-CoA and p-coumaryl-CoA were both 5.0 μM. Expression analyses of MdCHS genes varied according to tissue type. MdCHS1, MdCHS2 and MdCHS3 expression levels were associated with the levels of phloretin accumulate in the respective tissues. Copyright © 2017 Elsevier Ltd. All rights reserved.

GMP Synthase Is Required for Virulence Factor Production and Infection by Cryptococcus neoformans.

PubMed

Chitty, Jessica L; Tatzenko, Tayla L; Williams, Simon J; Koh, Y Q Andre E; Corfield, Elizabeth C; Butler, Mark S; Robertson, Avril A B; Cooper, Matthew A; Kappler, Ulrike; Kobe, Bostjan; Fraser, James A

2017-02-17

Over the last four decades the HIV pandemic and advances in medical treatments that also cause immunosuppression have produced an ever-growing cohort of individuals susceptible to opportunistic pathogens. Of these, AIDS patients are particularly vulnerable to infection by the encapsulated yeast Cryptococcus neoformans Most commonly found in the environment in purine-rich bird guano, C. neoformans experiences a drastic change in nutrient availability during host infection, ultimately disseminating to colonize the purine-poor central nervous system. Investigating the consequences of this challenge, we have characterized C. neoformans GMP synthase, the second enzyme in the guanylate branch of de novo purine biosynthesis. We show that in the absence of GMP synthase, C. neoformans becomes a guanine auxotroph, the production of key virulence factors is compromised, and the ability to infect nematodes and mice is abolished. Activity assays performed using recombinant protein unveiled differences in substrate binding between the C. neoformans and human enzymes, with structural insights into these kinetic differences acquired via homology modeling. Collectively, these data highlight the potential of GMP synthase to be exploited in the development of new therapeutic agents for the treatment of disseminated, life-threatening fungal infections. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Para-Aminobenzoic Acid (PABA) Synthase Enhances Thermotolerance of Mushroom Agaricus bisporus

PubMed Central

Lu, Zhonglei; Kong, Xiangxiang; Lu, Zhaoming; Xiao, Meixiang; Chen, Meiyuan; Zhu, Liang; Shen, Yuemao; Hu, Xiangyang; Song, Siyang

2014-01-01

Most mushrooms are thermo-sensitive to temperatures over 23°C, which greatly restricts their agricultural cultivation. Understanding mushroom’s innate heat-tolerance mechanisms may facilitate genetic improvements of their thermotolerance. Agaricus bisporus strain 02 is a relatively thermotolerant mushroom strain, while strain 8213 is quite thermo-sensitive. Here, we compared their responses at proteomic level to heat treatment at 33°C. We identified 73 proteins that are differentially expressed between 02 and 8213 or induced upon heat stress in strain 02 itself, 48 of which with a known identity. Among them, 4 proteins are constitutively more highly expressed in 02 than 8213; and they can be further upregulated in response to heat stress in 02, but not in 8213. One protein is encoded by the para-aminobenzoic acid (PABA) synthase gene Pabs, which has been shown to scavenge the reactive oxygen species in vitro. Pabs mRNA and its chemical product PABA show similar heat stress induction pattern as PABA synthase protein and are more abundant in 02, indicating transcriptional level upregulation of Pabs upon heat stress. A specific inhibitor of PABA synthesis impaired thermotolerance of 02, while exogenous PABA or transgenic overexpression of 02 derived PABA synthase enhanced thermotolerance of 8213. Furthermore, compared to 8213, 02 accumulated less H2O2 but more defense-related proteins (e.g., HSPs and Chitinase) under heat stress. Together, these results demonstrate a role of PABA in enhancing mushroom thermotolerance by removing H2O2 and elevating defense-related proteins. PMID:24614118

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers

DOE PAGES

Olek, Anna T.; Rayon, Catherine; Makowski, Lee; ...

2014-07-10

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice ( Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain,more » elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. As a result, the arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.« less

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers.

PubMed

Olek, Anna T; Rayon, Catherine; Makowski, Lee; Kim, Hyung Rae; Ciesielski, Peter; Badger, John; Paul, Lake N; Ghosh, Subhangi; Kihara, Daisuke; Crowley, Michael; Himmel, Michael E; Bolin, Jeffrey T; Carpita, Nicholas C

2014-07-01

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize. © 2014 American Society of Plant Biologists. All rights reserved.

Insights Into the Bifunctional Aphidicolan-16-ß-ol Synthase Through Rapid Biomolecular Modeling Approaches.

PubMed

Hirte, Max; Meese, Nicolas; Mertz, Michael; Fuchs, Monika; Brück, Thomas B

2018-01-01

Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modeling techniques offer an alternative route to study the enzyme's reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modeling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate in silico identification of catalytically relevant amino acids. Guided by our modeling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789, and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade en-route from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modeling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially restricted location of

Insights into the bifunctional Aphidicolan-16-ß-ol synthase through rapid biomolecular modelling approaches

NASA Astrophysics Data System (ADS)

Hirte, Max; Meese, Nicolas; Mertz, Michael; Fuchs, Monika; Brück, Thomas B.

2018-04-01

Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modelling techniques offer an alternative route to study the enzyme’s reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modelling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate in silico identification of catalytically relevant amino acids. Guided by our modelling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789 and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade en-route from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modelling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially restricted location

β-Adrenergic-mediated vasodilation in young men and women: cyclooxygenase restrains nitric oxide synthase

PubMed Central

Limberg, Jacqueline K.; Johansson, Rebecca E.; Peltonen, Garrett L.; Harrell, John W.; Kellawan, J. Mikhail; Eldridge, Marlowe W.; Sebranek, Joshua J.

2016-01-01

We tested the hypothesis that women exhibit greater vasodilator responses to β-adrenoceptor stimulation compared with men. We further hypothesized women exhibit a greater contribution of nitric oxide synthase and cyclooxygenase to β-adrenergic-mediated vasodilation compared with men. Forearm blood flow (Doppler ultrasound) was measured in young men (n = 29, 26 ± 1 yr) and women (n = 33, 25 ± 1 yr) during intra-arterial infusion of isoproterenol (β-adrenergic agonist). In subset of subjects, isoproterenol responses were examined before and after local inhibition of nitric oxide synthase [NG-monomethyl-l-arginine (l-NMMA); 6 male/10 female] and/or cyclooxygenase (ketorolac; 5 male/5 female). Vascular conductance (blood flow ÷ mean arterial pressure) was calculated to assess vasodilation. Vascular conductance increased with isoproterenol infusion (P < 0.01), and this effect was not different between men and women (P = 0.41). l-NMMA infusion had no effect on isoproterenol-mediated dilation in men (P > 0.99) or women (P = 0.21). In contrast, ketorolac infusion markedly increased isoproterenol-mediated responses in both men (P < 0.01) and women (P = 0.04) and this rise was lost with subsequent l-NMMA infusion (men, P < 0.01; women, P < 0.05). β-Adrenergic vasodilation is not different between men and women and sex differences in the independent contribution of nitric oxide synthase and cyclooxygenase to β-mediated vasodilation are not present. However, these data are the first to demonstrate β-adrenoceptor activation of cyclooxygenase suppresses nitric oxide synthase signaling in human forearm microcirculation and may have important implications for neurovascular control in both health and disease. PMID:26747505

Structural basis for glucose-6-phosphate activation of glycogen synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baskaran, Sulochanadevi; Roach, Peter J.; DePaoli-Roach, Anna A.

2010-11-22

Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by themore » binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.« less

Glycogen synthase kinase 3: more than a namesake

PubMed Central

Rayasam, Geetha Vani; Tulasi, Vamshi Krishna; Sodhi, Reena; Davis, Joseph Alex; Ray, Abhijit

2009-01-01

Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, τ protein and β catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer's and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target. British Journal of Pharmacology (2009) doi:10.1111/j.1476-5381.2008.00085.x PMID:19366350

Thymidylate synthase and methionine synthase polymorphisms are not associated with susceptibility to childhood acute lymphoblastic leukemia in Kurdish population from Western Iran.

PubMed

Rahimi, Zohreh; Ahmadian, Zainab; Akramipour, Reza; Vaisi-Raygani, Asad; Rahimi, Ziba; Parsian, Abbas

2012-03-01

In order to determine the influence of polymorphism in thymidylate synthase (TS 28-bp repeat) and methionine synthase (MS A2756G) genes on the susceptibility to acute lymphoblastic leukemia (ALL), 73 children with ALL and 128 age and sex matched unrelated healthy individuals from the Kermanshah Province of Iran were screened. The genotyping of TS 28-bp repeat and MS A2756G polymorphisms were performed by polymerase chain reaction (PCR) and PCR-RFLP, respectively. The frequency of TS 2R allele in patients and controls were 41.5 and 38%, respectively (Odds ratios (OR) = 1.13, 95%CI 0.73-1.74, P = 0.56). The allelic frequency of G allele of MS was higher (25%) in patients compared with healthy subjects (23%) (OR = 1.09, 95%CI 0.67-1.75, P = 0.71). Considering MS AA and TS 3R3R genotypes as reference indicated that individuals with MS GG + TS 2R2R genotypes have 1.3-fold increase in the risk of ALL (OR = 1.3, 95%CI 0.6-2.7, P = 0.5). Our results showed that neither TS 28-bp repeat nor MS A2756G polymorphisms are risk factors for susceptibility to ALL in Western Iran.

Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase: Acceleration of fracture healing via inducible nitric oxide synthase.

PubMed

Rajfer, R A; Kilic, A; Neviaser, A S; Schulte, L M; Hlaing, S M; Landeros, J; Ferrini, M G; Ebramzadeh, E; Park, S-H

2017-02-01

We investigated the effects on fracture healing of two up-regulators of inducible nitric oxide synthase (iNOS) in a rat model of an open femoral osteotomy: tadalafil, a phosphodiesterase inhibitor, and the recently reported nutraceutical, COMB-4 (consisting of L-citrulline, Paullinia cupana, ginger and muira puama), given orally for either 14 or 42 days. Unilateral femoral osteotomies were created in 58 male rats and fixed with an intramedullary compression nail. Rats were treated daily either with vehicle, tadalafil or COMB-4. Biomechanical testing of the healed fracture was performed on day 42. The volume, mineral content and bone density of the callus were measured by quantitative CT on days 14 and 42. Expression of iNOS was measured by immunohistochemistry. When compared with the control group, the COMB-4 group exhibited 46% higher maximum strength ( t -test, p = 0.029) and 92% higher stiffness ( t -test, p = 0.023), but no significant changes were observed in the tadalafil group. At days 14 and 42, there was no significant difference between the three groups with respect to callus volume, mineral content and bone density. Expression of iNOS at day 14 was significantly higher in the COMB-4 group which, as expected, had returned to baseline levels at day 42. This study demonstrates an enhancement in fracture healing by an oral natural product known to augment iNOS expression. Cite this article: R. A. Rajfer, A. Kilic, A. S. Neviaser, L. M. Schulte, S. M. Hlaing, J. Landeros, M. G. Ferrini, E. Ebramzadeh, S-H. Park. Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase: Acceleration of fracture healing via inducible nitric oxide synthase. Bone Joint Res 2017:6:-97. DOI: 10.1302/2046-3758.62.BJR-2016-0164.R2. © 2017 Park et al.

Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

PubMed Central

Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E.

2012-01-01

Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) and cis-vaccenic (C18:1) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I. PMID:22194290

A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces

PubMed Central

Arrach, Nabil; Fernández-Martín, Rafael; Cerdá-Olmedo, Enrique; Avalos, Javier

2001-01-01

Previous complementation and mapping of mutations that change the usual yellow color of the Zygomycete Phycomyces blakesleeanus to white or red led to the definition of two structural genes for carotene biosynthesis. We have cloned one of these genes, carRA, by taking advantage of its close linkage to the other, carB, responsible for phytoene dehydrogenase. The sequences of the wild type and six mutants have been established, compared with sequences in other organisms, and correlated with the mutant phenotypes. The carRA and carB coding sequences are separated by 1,381 untranslated nucleotides and are divergently transcribed. Gene carRA contains separate domains for two enzymes, lycopene cyclase and phytoene synthase, and regulates the overall activity of the pathway and its response to physical and chemical stimuli from the environment. The lycopene cyclase domain of carRA derived from a duplication of a gene from a common ancestor of fungi and Brevibacterium linens; the phytoene synthase domain is similar to the phytoene and squalene synthases of many organisms; but the regulatory functions appear to be specific to Phycomyces. PMID:11172012

Sequence of a cDNA and expression of the gene encoding a putative epidermal chitin synthase of Manduca sexta.

PubMed

Zhu, Yu-Cheng; Specht, Charles A; Dittmer, Neal T; Muthukrishnan, Subbaratnam; Kanost, Michael R; Kramer, Karl J

2002-11-01

Glycosyltransferases are enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. One type of glycosyltransferase is chitin synthase, a very important enzyme in biology, which is utilized by insects, fungi, and other invertebrates to produce chitin, a polysaccharide of beta-1,4-linked N-acetylglucosamine. Chitin is an important component of the insect's exoskeletal cuticle and gut lining. To identify and characterize a chitin synthase gene of the tobacco hornworm, Manduca sexta, degenerate primers were designed from two highly conserved regions in fungal and nematode chitin synthase protein sequences and then used to amplify a similar region from Manduca cDNA. A full-length cDNA of 5152 nucleotides was assembled for the putative Manduca chitin synthase gene, MsCHS1, and sequencing of genomic DNA verified the contiguity of the sequence. The MsCHS1 cDNA has an ORF of 4692 nucleotides that encodes a transmembrane protein of 1564 amino acid residues with a mass of approximately 179 kDa (GenBank no. AY062175). It is most similar, over its entire length of protein sequence, to putative chitin synthases from other insects and nematodes, with 68% identity to enzymes from both the blow fly, Lucilia cuprina, and the fruit fly, Drosophila melanogaster. The similarity with fungal chitin synthases is restricted to the putative catalytic domain, and the MsCHS1 protein has, at equivalent positions, several amino acids that are essential for activity as revealed by mutagenesis of the fungal enzymes. A 5.3-kb transcript of MsCHS1 was identified by northern blot hybridization of RNA from larval epidermis, suggesting that the enzyme functions to make chitin deposited in the cuticle. Further examination by RT-PCR showed that MsCHS1 expression is regulated in the epidermis, with the amount of transcript increasing during phases of cuticle deposition.

Starch synthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion

PubMed Central

Crumpton-Taylor, Matilda; Pike, Marilyn; Lu, Kuan-Jen; Hylton, Christopher M; Feil, Regina; Eicke, Simona; Lunn, John E; Zeeman, Samuel C; Smith, Alison M

2013-01-01

Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants. Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume. We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape. SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules. PMID:23952675

Crystallization and preliminary X-ray analysis of the isomerase domain of glucosamine-6-phosphate synthase from Candida albicans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olchowy, Jaroslaw; Jedrzejczak, Robert; Milewski, Slawomir

2005-11-01

The isomerase domain of glucosamine-6-phosphate synthase from C. albicans has been crystallized and X-ray diffraction data have been collected. Preliminary analysis of the data reveals the oligomeric structure of the eukaryotic synthase to be a ‘dimer’ of prokaryotic-like dimers. Glucosamine-6-phosphate synthase (EC 2.6.1.16) catalyses the first and practically irreversible step in the hexosamine metabolism pathway, the end product of which, uridine 5′-diphospho-N-acetyl d-glucosamine, is an essential substrate for assembly of the cell wall. The isomerase domain, consisting of residues 346–712 (42 kDa), of glucosamine-6-phosphate synthase from Candida albicans has been crystallized. X-ray analysis revealed that the crystals belonged to spacemore » group I4, with unit-cell parameters a = b = 149, c = 103 Å. Diffraction data were collected to 3.8 Å. Preliminary results from molecular replacement using the homologous bacterial monomer reveal that the asymmetric unit contains two monomers that resemble a bacterial dimer. The crystal lattice consists of pairs of such symmetry-related dimers forming elongated tetramers.« less

Rational design of aminoacyl-tRNA synthetase specific for p-acetyl-L-phenylalanine.

PubMed

Sun, Renhua; Zheng, Heng; Fang, Zhengzhi; Yao, Wenbing

2010-01-01

The Methanococcus jannaschii tRNA(Tyr)/tyrosyl-tRNA synthetase pair has been engineered to incorporate unnatural amino acids into proteins in Escherichia coli site-specifically. In order to add other unnatural amino acids into proteins by this approach, the amino acid binding site of M. jannaschii tyrosyl-tRNA synthetase need to be mutated. The crystal structures of M. jannaschii tyrosyl-tRNA synthetase and its mutations were determined, which provided an opportunity to design aminoacyl-tRNA synthetases specific for other unnatural amino acids. In our study, we attempted to design aminoacyl-tRNA synthetases being able to deliver p-acetyl-L-phenylalanine into proteins. p-Acetyl-L-phenylalanine was superimposed on tyrosyl in M. jannaschii tyrosyl-tRNA synthetase-tyrosine complex. Tyr32 needed to be changed to non-polar amino acid with shorter side chain, Val, Leu, Ile, Gly or Ala, in order to reduce steric clash and provide hydrophobic environment to acetyl on p-acetyl-L-phenylalanine. Asp158 and Ile159 would be changed to specific amino acids for the same reason. So we designed 60 aminoacyl-tRNA synthetases. Binding of these aminoacyl-tRNA synthetases with p-acetyl-L-phenylalanine indicated that only 15 of them turned out to be able to bind p-acetyl-L-phenylalanine with reasonable poses. Binding affinity computation proved that the mutation of Tyr32Leu and Asp158Gly benefited p-acetyl-L-phenylalanine binding. And two of the designed aminoacyl-tRNA synthetases had considerable binding affinities. They seemed to be very promising to be able to incorporate p-acetyl-L-phenylalanine into proteins in E. coli. The results show that the combination of homology modeling and molecular docking is a feasible method to filter inappropriate mutations in molecular design and point out beneficial mutations. Copyright 2009 Elsevier Inc. All rights reserved.

Circadian variation in the effects of nitric oxide synthase inhibitors on body temperature, feeding and activity in rats.

PubMed

Kamerman, Peter; Mitchell, Duncan; Laburn, Helen

2002-02-01

We have investigated whether there is circadian variation in the effects of nitric oxide synthase inhibitors on body temperature, physical activity and feeding. We used nocturnally active Sprague-Dawley rats, housed at approximately 24 degrees C with a 12:12 h light:dark cycle (lights on 07:00 hours) and provided with food and water ad libitum. Nitric oxide synthesis was inhibited by intraperitoneal injection of the unspecific nitric oxide synthase inhibitor N-nitro- L-arginine methyl ester ( L-NAME, 100, 50, 25, 10 mg/kg), or the relatively selective inducible nitric oxide synthase inhibitor aminoguanidine (100, 50 mg/kg), during the day ( approximately 09:00 hours) or night ( approximately 21:00 hours). Body temperature and physical activity were measured using radiotelemetry, while food intake was calculated by weighing each animal's food before as well as 12 and 24 h after each injection. We found that daytime injection of L-NAME and aminoguanidine had no effect on daytime body temperature. However, daytime injection of both drugs did decrease nocturnal food intake ( P<0.05) and activity ( P<0.05). When injected at night, L-NAME reduced night-time body temperature ( P<0.01), activity ( P<0.05) and food intake ( P<0.05) in a dose-dependent manner, but night-time injection of aminoguanidine inhibited only night-time activity ( P<0.05). The effects of nitric oxide synthase inhibition on body temperature, feeding and activity therefore are primarily a consequence of inhibiting constitutively expressed nitric oxide synthase, and are subject to circadian variation.

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors*

PubMed Central

Sielaff, Hendrik; Martin, James; Singh, Dhirendra; Biuković, Goran; Grüber, Gerhard; Frasch, Wayne D.

2016-01-01

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α3β3γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A3B3DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A3B3DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits. PMID:27729450

X-ray Crystal Structure of Aristolochene Synthase from Aspergillus terreus and Evolution of Templates for the Cyclization of Farnesyl Diphosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shishova,E.; Di Costanzo, L.; Cane, D.

2007-01-01

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 {angstrom} resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the {alpha}-helical class I terpene synthase fold with the active site in the 'open', solvent-exposed conformation. Intriguingly, the 2.15 {angstrom} resolution crystal structure of the complex with Mg{sup 2+}{sub 3}-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the 'closed' conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition frommore » the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved {alpha}-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.« less

Exploiting CELLULOSE SYNTHASE (CESA) Class Specificity to Probe Cellulose Microfibril Biosynthesis.

PubMed

Kumar, Manoj; Mishra, Laxmi; Carr, Paul; Pilling, Michael; Gardner, Peter; Mansfield, Shawn D; Turner, Simon

2018-05-01

Cellulose microfibrils are the basic units of cellulose in plants. The structure of these microfibrils is at least partly determined by the structure of the cellulose synthase complex. In higher plants, this complex is composed of 18 to 24 catalytic subunits known as CELLULOSE SYNTHASE A (CESA) proteins. Three different classes of CESA proteins are required for cellulose synthesis and for secondary cell wall cellulose biosynthesis these classes are represented by CESA4, CESA7, and CESA8. To probe the relationship between CESA proteins and microfibril structure, we created mutant cesa proteins that lack catalytic activity but retain sufficient structural integrity to allow assembly of the cellulose synthase complex. Using a series of Arabidopsis ( Arabidopsis thaliana ) mutants and genetic backgrounds, we found consistent differences in the ability of these mutant cesa proteins to complement the cellulose-deficient phenotype of the cesa null mutants. The best complementation was observed with catalytically inactive cesa4, while the equivalent mutation in cesa8 exhibited significantly lower levels of complementation. Using a variety of biophysical techniques, including solid-state nuclear magnetic resonance and Fourier transform infrared microscopy, to study these mutant plants, we found evidence for changes in cellulose microfibril structure, but these changes largely correlated with cellulose content and reflected differences in the relative proportions of primary and secondary cell walls. Our results suggest that individual CESA classes have similar roles in determining cellulose microfibril structure, and it is likely that the different effects of mutating members of different CESA classes are the consequence of their different catalytic activity and their influence on the overall rate of cellulose synthesis. © 2018 American Society of Plant Biologists. All Rights Reserved.

Glycogen synthase kinase-3β inhibition of 6-(methylsulfinyl)hexyl isothiocyanate derived from wasabi (Wasabia japonica Matsum).

PubMed

Yoshida, Jun; Nomura, Satomi; Nishizawa, Naoyuki; Ito, Yoshiaki; Kimura, Ken-ichi

2011-01-01

A new biological activity of 6-(methylsulfinyl)hexyl isothiocyanate derived from Wasabia japonica was discovered as an inhibitor of glycogen synthase kinase-3β. The most potent isothiocyanate, 9-(methylsulfinyl)hexyl isothiocyanate, inhibited glycogen synthase kinase-3β at a K(i) value of 10.5 µM and showed ATP competitive inhibition. The structure-activity relationship revealed an inhibitory potency of methylsulfinyl isothiocyanate dependent on the alkyl chain length and the sulfoxide, sulfone, and/or the isothiocyanate moiety.

Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase.

PubMed

Gemperlein, Katja; Zipf, Gregor; Bernauer, Hubert S; Müller, Rolf; Wenzel, Silke C

2016-01-01

Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sá-Moura, Bebiana; Albuquerque, Luciana; Empadinhas, Nuno

2008-08-01

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

Effects of Tributyltin Chloride on Cybrids with or without an ATP Synthase Pathologic Mutation

PubMed Central

López-Gallardo, Ester; Llobet, Laura; Emperador, Sonia; Montoya, Julio; Ruiz-Pesini, Eduardo

2016-01-01

Background: The oxidative phosphorylation system (OXPHOS) includes nuclear chromosome (nDNA)– and mitochondrial DNA (mtDNA)–encoded polypeptides. Many rare OXPHOS disorders, such as striatal necrosis syndromes, are caused by genetic mutations. Despite important advances in sequencing procedures, causative mutations remain undetected in some patients. It is possible that etiologic factors, such as environmental toxins, are the cause of these cases. Indeed, the inhibition of a particular enzyme by a poison could imitate the biochemical effects of pathological mutations in that enzyme. Moreover, environmental factors can modify the penetrance or expressivity of pathological mutations. Objectives: We studied the interaction between mitochondrially encoded ATP synthase 6 (p.MT-ATP6) subunit and an environmental exposure that may contribute phenotypic differences between healthy individuals and patients suffering from striatal necrosis syndromes or other mitochondriopathies. Methods: We analyzed the effects of the ATP synthase inhibitor tributyltin chloride (TBTC), a widely distributed environmental factor that contaminates human food and water, on transmitochondrial cell lines with or without an ATP synthase mutation that causes striatal necrosis syndrome. Doses were selected based on TBTC concentrations previously reported in human whole blood samples. Results: TBTC modified the phenotypic effects caused by a pathological mtDNA mutation. Interestingly, wild-type cells treated with this xenobiotic showed similar bioenergetics when compared with the untreated mutated cells. Conclusions: In addition to the known genetic causes, our findings suggest that environmental exposure to TBTC might contribute to the etiology of striatal necrosis syndromes. Citation: López-Gallardo E, Llobet L, Emperador S, Montoya J, Ruiz-Pesini E. 2016. Effects of tributyltin chloride on cybrids with or without an ATP synthase pathologic mutation. Environ Health Perspect 124:1399–1405;�

Effects of Tributyltin Chloride on Cybrids with or without an ATP Synthase Pathologic Mutation.

PubMed

López-Gallardo, Ester; Llobet, Laura; Emperador, Sonia; Montoya, Julio; Ruiz-Pesini, Eduardo

2016-09-01

The oxidative phosphorylation system (OXPHOS) includes nuclear chromosome (nDNA)- and mitochondrial DNA (mtDNA)-encoded polypeptides. Many rare OXPHOS disorders, such as striatal necrosis syndromes, are caused by genetic mutations. Despite important advances in sequencing procedures, causative mutations remain undetected in some patients. It is possible that etiologic factors, such as environmental toxins, are the cause of these cases. Indeed, the inhibition of a particular enzyme by a poison could imitate the biochemical effects of pathological mutations in that enzyme. Moreover, environmental factors can modify the penetrance or expressivity of pathological mutations. We studied the interaction between mitochondrially encoded ATP synthase 6 (p.MT-ATP6) subunit and an environmental exposure that may contribute phenotypic differences between healthy individuals and patients suffering from striatal necrosis syndromes or other mitochondriopathies. We analyzed the effects of the ATP synthase inhibitor tributyltin chloride (TBTC), a widely distributed environmental factor that contaminates human food and water, on transmitochondrial cell lines with or without an ATP synthase mutation that causes striatal necrosis syndrome. Doses were selected based on TBTC concentrations previously reported in human whole blood samples. TBTC modified the phenotypic effects caused by a pathological mtDNA mutation. Interestingly, wild-type cells treated with this xenobiotic showed similar bioenergetics when compared with the untreated mutated cells. In addition to the known genetic causes, our findings suggest that environmental exposure to TBTC might contribute to the etiology of striatal necrosis syndromes. López-Gallardo E, Llobet L, Emperador S, Montoya J, Ruiz-Pesini E. 2016. Effects of tributyltin chloride on cybrids with or without an ATP synthase pathologic mutation. Environ Health Perspect 124:1399-1405; http://dx.doi.org/10.1289/EHP182.

Structure of human thymidylate synthase under low-salt conditions.

PubMed

Lovelace, Leslie L; Minor, Wladek; Lebioda, Lukasz

2005-05-01

Human thymidylate synthase, a target in cancer chemotherapy, was crystallized from PEG 3350 with 30 mM ammonium sulfate (AS) in the crystallization medium. The crystals are isomorphous with the high-salt crystals ( approximately 2.0 M AS) and the structure has been solved and refined (R = 22.6%, R(free) = 24.3%) at 1.8 A resolution. The high- and low-AS-concentration structures are quite similar, with loop 181-197 is in the inactive conformation. Also, residues 95-106 and 129-135 (eukaryotic inserts region) show high mobility as assessed by poor electron density and high values of crystallographic temperature factors (residues 1-25 and 108-129 are disordered in both structures). The high mobility of this region may reflect the situation at physiological ionic strength. Of the four sulfate ions observed bound at 2.0 M AS, only two are present at 30 mM AS. The inactive conformation appears to be stabilized by the side chain of Val3 or a leucine residue from the disordered regions. The low-salt conditions of these crystals should be much more suitable for the study of thymidylate synthase inhibitors, especially those that utilize sulfate-binding sites to stabilize the inactive conformation of loop 181-197.

Controlling Citrate Synthase Expression by CRISPR/Cas9 Genome Editing for n-Butanol Production in Escherichia coli.

PubMed

Heo, Min-Ji; Jung, Hwi-Min; Um, Jaeyong; Lee, Sang-Woo; Oh, Min-Kyu

2017-02-17

Genome editing using CRISPR/Cas9 was successfully demonstrated in Esherichia coli to effectively produce n-butanol in a defined medium under microaerobic condition. The butanol synthetic pathway genes including those encoding oxygen-tolerant alcohol dehydrogenase were overexpressed in metabolically engineered E. coli, resulting in 0.82 g/L butanol production. To increase butanol production, carbon flux from acetyl-CoA to citric acid cycle should be redirected to acetoacetyl-CoA. For this purpose, the 5'-untranslated region sequence of gltA encoding citrate synthase was designed using an expression prediction program, UTR designer, and modified using the CRISPR/Cas9 genome editing method to reduce its expression level. E. coli strains with decreased citrate synthase expression produced more butanol and the citrate synthase activity was correlated with butanol production. These results demonstrate that redistributing carbon flux using genome editing is an efficient engineering tool for metabolite overproduction.

Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation

PubMed Central

Dusi, Sabrina; Valletta, Lorella; Haack, Tobias B.; Tsuchiya, Yugo; Venco, Paola; Pasqualato, Sebastiano; Goffrini, Paola; Tigano, Marco; Demchenko, Nikita; Wieland, Thomas; Schwarzmayr, Thomas; Strom, Tim M.; Invernizzi, Federica; Garavaglia, Barbara; Gregory, Allison; Sanford, Lynn; Hamada, Jeffrey; Bettencourt, Conceição; Houlden, Henry; Chiapparini, Luisa; Zorzi, Giovanna; Kurian, Manju A.; Nardocci, Nardo; Prokisch, Holger; Hayflick, Susan; Gout, Ivan; Tiranti, Valeria

2014-01-01

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA. PMID:24360804

Inhibition of inducible nitric oxide synthase expression by novel nonsteroidal anti-inflammatory derivatives with gastrointestinal-sparing properties.

PubMed Central

Cirino, G.; Wheeler-Jones, C. P.; Wallace, J. L.; Del Soldato, P.; Baydoun, A. R.

1996-01-01

1. The effects of novel nitric oxide-releasing nonsteroidal anti-inflammatory compounds (NO-NSAIDs) on induction of nitric oxide (NO) synthase by bacterial lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line, J774. 2. LPS-induced nitrite production was markedly attenuated by the nitroxybutylester derivatives of flurbiprofen (FNBE), aspirin, ketoprofen, naproxen, diclofenac and ketorolac, with each compound reducing accumulated nitrite levels by > 40% at the maximum concentrations (100 micrograms ml-1) used. 3. Further examination revealed that nitrite production was inhibited in a concentration-dependent (1-100 micrograms ml-1) manner by FNBE which at 100 micrograms ml-1 decreased LPS-stimulated levels by 63.3 +/- 8.6% (n = 7). The parent compound flurbiprofen was relatively ineffective over the same concentration-range, inhibiting nitrite accumulation by 24 +/- 0.9% (n = 3) at the maximum concentration used (100 micrograms ml-1). 4. FNBE reduced LPS-induced nitrite production when added to cells up to 4 h after LPS. Thereafter, FNBE caused very little or no reduction in nitrite levels. Furthermore NO-NSAIDs (100 micrograms ml-1) did not inhibit the metabolism of L-[3H]-arginine to citrulline by NO synthase isolated from LPS-activated macrophages. 5. Western blot analysis demonstrated that NO synthase expression was markedly attenuated following co-incubation of J774 cell with LPS (1 microgram ml-1; 24 h) and FNBE (100 micrograms ml-1; 24 h). Thus taken together, these findings indicate that NO-NSAIDs inhibit induction of NO synthase without directly affecting enzyme activity. 6. In conclusion our results indicate that NO-NSAIDs can inhibit the inducible L-arginine-NO pathway, and are capable of suppressing NO synthesis by inhibiting expression of NO synthase. The clinical implications of these findings remain to be established. Images Figure 4 PMID:8730734

Characterization and evolutionary analysis of ent-kaurene synthase like genes from the wild rice species Oryza rufipogon.

PubMed

Toyomasu, Tomonobu; Miyamoto, Koji; Shenton, Matthew R; Sakai, Arisa; Sugawara, Chizu; Horie, Kiyotaka; Kawaide, Hiroshi; Hasegawa, Morifumi; Chuba, Masaru; Mitsuhashi, Wataru; Yamane, Hisakazu; Kurata, Nori; Okada, Kazunori

2016-11-18

Cultivated rice (Oryza sativa) possesses various labdane-related diterpene synthase genes, homologs of ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) that are responsible for the biosynthesis of phytohormone gibberellins. The CPS homologs and KS like (KSL) homologs successively converted geranylgeranyl diphosphate to cyclic diterpene hydrocarbons via ent-copalyl diphosphate or syn-copalyl diphosphate in O. sativa. Consequently, a variety of labdane-related diterpenoids, including phytoalexin phytocassanes, momilactones and oryzalexins, have been identified from cultivated rice. Our previous report indicated that the biosynthesis of phytocassanes and momilactones is conserved in Oryza rufipogon, the progenitor of Asian cultivated rice. Moreover, their biosynthetic gene clusters, containing OsCPS2 and OsKSL7 for phytocassane biosynthesis and OsCPS4 and OsKSL4 for momilactone biosynthesis, are also present in the O. rufipogon genome. We herein characterized O. rufipogon homologs of OsKSL5, OsKSL6, OsKSL8 responsible for oryzalexin S biosynthesis, and OsKSL10 responsible for oryzalexins A-F biosynthesis, to obtain more evolutionary insight into diterpenoid biosynthesis in O. sativa. Our phytoalexin analyses showed that no accumulation of oryzalexins was detected in extracts from O. rufipogon leaf blades. In vitro functional analyses indicated that unlike OsKSL10, O. rufipogon KSL10 functions as an ent-miltiradiene synthase, which explains the lack of accumulation of oryzalexins A-F in O. rufipogon. The different functions of KSL5 and KSL8 in O. sativa japonica to those in indica are conserved in each type of O. rufipogon, while KSL6 functions (ent-isokaurene synthases) are well conserved. Our study suggests that O. sativa japonica has evolved distinct specialized diterpenoid metabolism, including the biosynthesis of oryzalexins. Copyright © 2016 Elsevier Inc. All rights reserved.

Glyphosate sensitivity of 5-enol-pyruvylshikimate-3-phosphate synthase from Bacillus subtilis depends upon state of activation induced by monovalent cations.

PubMed

Fischer, R S; Rubin, J L; Gaines, C G; Jensen, R A

1987-07-01

The 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase from Bacillus subtilis was activated by monovalent cations, catalytic activity being negligible in the absence of monovalent cations. The order of cation effectiveness (NH4+ greater than K+ greater than Rb+ greater than Na+ = Cs+ = Li+) indicated that the extent of activation was directly related to the unhydrated cation radius. Ammonium salts, at physiological concentrations, were dramatically more effective than other cations. Activation by ammonium was instantaneous, was not influenced by the counter ion, and gave a hyperbolic saturation curve. Hill plots did not show detectable cooperativity in the binding of ammonium. Double-reciprocal plots indicated that ammonium increases the maximal velocity and decreases the apparent Michaelis constants of EPSP synthase with respect to both phosphoenol pyruvate (PEP) and shikimate 3-phosphate (S3P). A direct relationship between sensitivity to inhibition by glyphosate and the activation state of EPSP synthase was demonstrated. Hill plots indicated a single value for glyphosate binding throughout the range of ammonium activation. Double-reciprocal plots of substrate saturation data obtained with ammonium-activated enzyme in the presence of glyphosate showed glyphosate to behave as a competitive inhibitor with respect to PEP and as a mixed-type inhibitor relative to S3P. The increased glyphosate sensitivity of ammonium-activated EPSP synthase is attributed to a lowering of the inhibitor constant of glyphosate with respect to PEP. Erroneous underestimates of sensitivities of some bacterial EPSP synthases to inhibition by glyphosate may result from failure to recognize cation requirements of EPSP synthases.

The effect of high pressure on the intracellular trehalose synthase activity of Thermus aquaticus.

PubMed

Dong, Yongsheng; Ma, Lei; Duan, Yuanliang

2016-01-01

To understand the effect of high pressure on the intracellular trehalose synthase activity, Thermus aquaticus (T. aquaticus) in the logarithmic growth phase was treated with high-pressure air, and its intracellular trehalose synthase (TSase) activity was determined. Our results indicated that pressure is a factor strongly affecting the cell growth. High pressure significantly attenuated the growth rate of T. aquaticus and shortened the duration of stationary phase. However, after 2 h of culture under 1.0 MPa pressure, the activity of intracellular TSase in T. aquaticus reached its maximum value, indicating that pressure can significantly increase the activity of intracellular TSase in T. aquaticus. Thus the present study provides an important guide for the enzymatic production of trehalose.

Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii

PubMed Central

Huynh, Frederick; Tan, Tien-Chye; Swaminathan, Kunchithapadam; Patel, Bharat K. C.

2005-01-01

This is the first report of the crystallization of a sucrose phosphate synthase (SPS; EC 2.4.1.14). It also constitutes the first study of a sucrose phosphate synthase from a non-photosynthetic thermohalophilic anaerobic bacterium, Halothermothrix orenii. The purified recombinant spsA protein has been crystallized in the monoclinic space group C2, with unit-cell parameters a = 154.2, b = 47.9, c = 72.3 Å, β = 103.16°, using the hanging-drop vapour-diffusion method. The crystal diffracts X-rays to a resolution limit of 3.01 Å. Heavy-metal and halide-soaking trials are currently in progress to solve the structure. PMID:16508108

A First Analysis of Metallome Biosignatures of Hyperthermophilic Archaea

PubMed Central

Cameron, Vyllinniskii; House, Christopher H.; Brantley, Susan L.

2012-01-01

To date, no experimental data has been reported for the metallome of hyperthermophilic microorganisms although their metal requirements for growth are known to be unique. Here, experiments were conducted to determine (i) cellular trace metal concentrations of the hyperthermophilic Archaea Methanococcus jannaschii and Pyrococcus furiosus, and (ii) a first estimate of the metallome for these hyperthermophilic species via ICP-MS. The metal contents of these cells were compared to parallel experiments using the mesophilic bacterium Escherichia coli grown under aerobic and anaerobic conditions. Fe and Zn were typically the most abundant metals in cells. Metal concentrations for E. coli grown aerobically decreased in the order Fe > Zn > Cu > Mo > Ni > W > Co. In contrast, M. jannaschii and P. furiosus show almost the reverse pattern with elevated Ni, Co, and W concentrations. Of the three organisms, a biosignature is potentially demonstrated for the methanogen M. jannaschii that may, in part, be related to the metallome requirements of methanogenesis. The bioavailability of trace metals more than likely has varied through time. If hyperthermophiles are very ancient, then the trace metal patterns observed here may begin to provide some insights regarding Earth's earliest cells and in turn, early Earth chemistry. PMID:23243390

Identification and characterization of a class III chitin synthase gene of Moniliophthora perniciosa, the fungus that causes witches' broom disease of cacao.

PubMed

Souza, Catiane S; Oliveira, Bruno M; Costa, Gustavo G L; Schriefer, Albert; Selbach-Schnadelbach, Alessandra; Uetanabaro, Ana Paula T; Pirovani, Carlos P; Pereira, Gonçalo A G; Taranto, Alex G; Cascardo, Júlio Cézar de M; Góes-Neto, Aristóteles

2009-08-01

Chitin synthase (CHS) is a glucosyltransferase that converts UDP-N-acetylglucosamine into chitin, one of the main components of fungal cell wall. Class III chitin synthases act directly in the formation of the cell wall. They catalyze the conversion of the immediate precursor of chitin and are responsible for the majority of chitin synthesis in fungi. As such, they are highly specific molecular targets for drugs that can inhibit the growth and development of fungal pathogens. In this work, we have identified and characterized a chitin synthase gene of Moniliophthora perniciosa (Mopchs) by primer walking. The complete gene sequence is 3,443 bp, interrupted by 13 small introns, and comprises a cDNA with an ORF with 2,739 bp, whose terminal region was experimentally determined, encoding a protein with 913 aa that harbors all the motifs and domains typically found in class III chitin synthases. This is the first report on the characterization of a chitin synthase gene, its mature transcription product, and its putative protein in basidioma and secondary mycelium stages of M. perniciosa, a basidiomycotan fungus that causes witches' broom disease of cacao.

Aromatic polyketide synthases from 127 Fusarium: pas de deux for chemical diversity

USDA-ARS?s Scientific Manuscript database

Fusarium species collectively cause disease on almost all crop plants and produce numerous natural products (NPs), including mycotoxins, of great concern. Many Fusarium NPs are derived from polyketide synthases (PKSs), large enzymes that catalyze the condensation of simple carboxylic acids. To gain ...

Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions.

PubMed

Du, Jinping; Rehm, Bernd H A

2017-11-02

Recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction. By translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b). Here a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.

Arrangement of photosystem II and ATP synthase in chloroplast membranes of spinach and pea.

PubMed

Daum, Bertram; Nicastro, Daniela; Austin, Jotham; McIntosh, J Richard; Kühlbrandt, Werner

2010-04-01

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

PubMed Central

De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

2014-01-01

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase.

PubMed

Wang, Yang; Desai, Janish; Zhang, Yonghui; Malwal, Satish R; Shin, Christopher J; Feng, Xinxin; Sun, Hong; Liu, Guizhi; Guo, Rey-Ting; Oldfield, Eric

2016-10-19

We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED 50 ∼0.15 μg mL -1 ) acted synergistically with seven antibiotics known to target bacterial cell-wall biosynthesis (a fractional inhibitory concentration index (FICI) of ∼0.35, on average) but had indifferent effects in combinations with six non-cell-wall biosynthesis inhibitors (average FICI∼1.45). The most active compounds were found to inhibit two enzymes involved in isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP), but not farnesyl diphosphate synthase, and there were good correlations between bacterial cell growth inhibition, UPPS inhibition, and UPPP inhibition. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of the uridine 5'-diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, F.C.; Brown, R.M. Jr.; Drake, R.R. Jr.

1990-03-25

Photoaffinity labeling of purified cellulose synthase with (beta-32P)5-azidouridine 5'-diphosphoglucose (UDP-Glc) has been used to identify the UDP-Glc binding subunit of the cellulose synthase from Acetobacter xylinum strain ATCC 53582. The results showed exclusive labeling of an 83-kDa polypeptide. Photoinsertion of (beta-32P)5-azido-UDP-Glc is stimulated by the cellulose synthase activator, bis-(3'----5') cyclic diguanylic acid. Addition of increasing amounts of UDP-Glc prevents photolabeling of the 83-kDa polypeptide. The reversible and photocatalyzed binding of this photoprobe also showed saturation kinetics. These studies demonstrate that the 83-kDa polypeptide is the catalytic subunit of the cellulose synthase in A. xylinum strain ATCC 53582.

Effect of selective inhibition of renal inducible nitric oxide synthase on renal blood flow and function in experimental hyperdynamic sepsis.

PubMed

Ishikawa, Ken; Calzavacca, Paolo; Bellomo, Rinaldo; Bailey, Michael; May, Clive N

2012-08-01

Nitric oxide plays an important role in the control of renal blood flow and renal function. In sepsis, increased levels of inducible nitric oxide synthase produce excessive nitric oxide, which may contribute to the development of acute kidney injury. We, therefore, examined the effects of intrarenal infusion of selective inducible nitric oxide synthase inhibitors in a large animal model of hyperdynamic sepsis in which acute kidney injury occurs in the presence of increased renal blood flow. Prospective crossover randomized controlled interventional studies. University-affiliated research institute. Twelve unilaterally nephrectomized Merino ewes. Infusion of a selective (1400W) and a partially selective inducible nitric oxide synthase inhibitor (aminoguanidine) into the renal artery for 2 hrs after the induction of sepsis, and comparison with a nonselective inhibitor (Nω-nitro-L-arginine methyl ester). In sheep with nonhypotensive hyperdynamic sepsis, creatinine clearance halved (32 to 16 mL/min, ratio [95% confidence interval] 0.51 [0.28-0.92]) despite increased renal blood flow (241 to 343 mL/min, difference [95% confidence interval] 102 [78-126]). Infusion of 1400W did not change renal blood flow, urine output, or creatinine clearance, whereas infusion of Nω-nitro-L-arginine methyl ester and a high dose of aminoguanidine normalized renal blood flow, but did not alter creatinine clearance. In hyperdynamic sepsis, intrarenal infusion of a highly selective inducible nitric oxide synthase inhibitor did not reduce the elevated renal blood flow or improve renal function. In contrast, renal blood flow was reduced by infusion of a nonselective NOS inhibitor or a high dose of a partially selective inducible nitric oxide synthase inhibitor. The renal vasodilatation in septic acute kidney injury may be due to nitric oxide derived from the endothelial and neural isoforms of nitric oxide synthase, but their blockade did not restore renal function.

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis.

PubMed

Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

2013-10-29

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200-300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis

PubMed Central

Omadjela, Okako; Narahari, Adishesh; Strumillo, Joanna; Mélida, Hugo; Mazur, Olga; Bulone, Vincent; Zimmer, Jochen

2013-01-01

Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200–300. Catalytic activity critically depends on the presence of the allosteric activator cyclic-di-GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP. PMID:24127606

Functional characterization and expression of GASCL1 and GASCL2, two anther-specific chalcone synthase like enzymes from Gerbera hybrida.

PubMed

Kontturi, Juha; Osama, Raisa; Deng, Xianbao; Bashandy, Hany; Albert, Victor A; Teeri, Teemu H

2017-02-01

The chalcone synthase superfamily consists of type III polyketidesynthases (PKSs), enzymes responsible for producing plant secondary metabolites with various biological and pharmacological activities. Anther-specific chalcone synthase-like enzymes (ASCLs) represent an ancient group of type III PKSs involved in the biosynthesis of sporopollenin, the main component of the exine layer of moss spores and mature pollen grains of seed plants. In the latter, ASCL proteins are localized in the tapetal cells of the anther where they participate in sporopollenin biosynthesis and exine formation within the locule. It is thought that the enzymes responsible for sporopollenin biosynthesis are highly conserved, and thus far, each angiosperm species with a genome sequenced has possessed two ASCL genes, which in Arabidopsis thaliana are PKSA and PKSB. The Gerbera hybrida (gerbera) PKS protein family consists of three chalcone synthases (GCHS1, GCHS3 and GCHS4) and three 2-pyrone synthases (G2PS1, G2PS2 and G2PS3). In previous studies we have demonstrated the functions of chalcone synthases in flavonoid biosynthesis, and the involvement of 2-pyrone synthases in the biosynthesis of antimicrobial compounds found in gerbera. In this study we expanded the gerbera PKS-family by functionally characterizing two gerbera ASCL proteins. In vitro enzymatic studies using purified recombinant proteins showed that both GASCL1 and GASCL2 were able to use medium and long-chain acyl-CoA starters and perform two to three condensation reactions of malonyl-CoA to produce tri- and tetraketide 2-pyrones, usually referred to as alpha-pyrones in sporopollenin literature. Both GASCL1 and GASCL2 genes were expressed only in floral organs, with most expression observed in anthers. In the anthers, transcripts of both genes showed strict tapetum-specific localization. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification and analysis of putative polyhydroxyalkanoate synthase (PhaC) in Pseudomonas fluorescens.

PubMed

Lim, Ju Hyoung; Rhie, Ho-Gun; Kim, Jeong Nam

2018-05-11

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. PHA granules mainly consisted of poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate- co -3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas . In vivo expression of the putative PHA synthase gene ( phaC Pf ) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli ( phaC Pf + , pUMS) grown in medium containing glucose accumulated PHA granules mainly consisting of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from E. coli fadR mutant ( phaC Pf + ) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified PhaC Pf , suggesting that the putative PHA synthase of P. fluorescens mainly utilizes short-chain-length PHA precursors as a substrate.

An In Planta-Expressed Polyketide Synthase Produces (R)-Mellein in the Wheat Pathogen Parastagonospora nodorum

PubMed Central

Krill, Christian; Barrow, Russell A.; Chen, Shasha; Trengove, Robert; Oliver, Richard P.; Solomon, Peter S.

2014-01-01

Parastagonospora nodorum is a pathogen of wheat that affects yields globally. Previous transcriptional analysis identified a partially reducing polyketide synthase (PR-PKS) gene, SNOG_00477 (SN477), in P. nodorum that is highly upregulated during infection of wheat leaves. Disruption of the corresponding SN477 gene resulted in the loss of production of two compounds, which we identified as (R)-mellein and (R)-O-methylmellein. Using a Saccharomyces cerevisiae yeast heterologous expression system, we successfully demonstrated that SN477 is the only enzyme required for the production of (R)-mellein. This is the first identification of a fungal PKS that is responsible for the synthesis of (R)-mellein. The P. nodorum ΔSN477 mutant did not show any significant difference from the wild-type strain in its virulence against wheat. However, (R)-mellein at 200 μg/ml inhibited the germination of wheat (Triticum aestivum) and barrel medic (Medicago truncatula) seeds. Comparative sequence analysis identified the presence of mellein synthase (MLNS) homologues in several Dothideomycetes and two sodariomycete genera. Phylogenetic analysis suggests that the MLNSs in fungi and bacteria evolved convergently from fungal and bacterial 6-methylsalicylic acid synthases. PMID:25326302

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit.

PubMed

Dong, J G; Kim, W T; Yip, W K; Thompson, G A; Li, L; Bennett, A B; Yang, S F

1991-08-01

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)(+) RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3'-end was intact, it lacked a portion of sequence at the 5'-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5'-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.

Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.

PubMed

Dusi, Sabrina; Valletta, Lorella; Haack, Tobias B; Tsuchiya, Yugo; Venco, Paola; Pasqualato, Sebastiano; Goffrini, Paola; Tigano, Marco; Demchenko, Nikita; Wieland, Thomas; Schwarzmayr, Thomas; Strom, Tim M; Invernizzi, Federica; Garavaglia, Barbara; Gregory, Allison; Sanford, Lynn; Hamada, Jeffrey; Bettencourt, Conceição; Houlden, Henry; Chiapparini, Luisa; Zorzi, Giovanna; Kurian, Manju A; Nardocci, Nardo; Prokisch, Holger; Hayflick, Susan; Gout, Ivan; Tiranti, Valeria

2014-01-02

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Identification of Bacillus subtilis men mutants which lack O-succinylbenzoyl-coenzyme A synthetase and dihydroxynaphthoate synthase.

PubMed Central

Meganathan, R; Bentley, R; Taber, H

1981-01-01

Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis, whose growth requirement is satisfied by 1,4-dihydroxy-2-naphthoic acid but not by o-succinylbenzoic acid (OSB), have been analyzed for enzymatic defects. Complementation analysis of cell-free extracts of the mutants revealed that there are two groups, as already indicated by genetic analysis. The missing enzyme in each group was identified by complementation of the cell-free extracts with o-succinylbenzoyl-coenzyme A (CoA) synthetase and dihydroxynaphthoate synthase extracted from Mycobacterium phlei. Mutants found to lack dihydroxynaphthoate synthase, and which therefore complement with dihydroxynaphthoate synthase of M. phlei, were designated as menB; those lacking o-succinylbenzoyl-CoA synthetase, and therefore complementing with o-succinylbenzoyl-CoA synthetase, were designated as menE. The menB mutants RB413 (men-325) and RB415 (men-329), when incubated with [2,3-14C2]OSB, produced only the spirodilactone form of OSB in a reaction that was CoA and adenosine 5'-triphosphate dependent. PMID:6780515

Producing a trimethylpentanoic acid using hybrid polyketide synthases

DOEpatents

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

2014-10-07

The present invention provides for a polyketide synthase (PKS) capable of synthesizing trimethylpentanoic acid. The present invention also provides for a host cell comprising the PKS and when cultured produces the trimethylpentanoic acid. The present invention also provides for a method of producing the trimethylpentanoic acid, comprising: providing a host cell of the present invention, and culturing said host cell in a suitable culture medium such that the trimethylpentanoic acid is produced, optionally isolating the trimethylpentanoic acid, and optionally, reducing the isolated trimethylpentanoic acid into a trimethylpentanol or an iso-octane.

Enzymatic synthesis of S-phenyl-L-cysteine from keratin hydrolysis industries wastewater with tryptophan synthase.

PubMed

Xu, Lisheng; Wang, Zhiyuan; Mao, Pingting; Liu, Junzhong; Zhang, Hongjuan; Liu, Qian; Jiao, Qing-Cai

2013-04-01

An economical method for production of S-phenyl-L-cysteine from keratin acid hydrolysis wastewater (KHW) containing L-serine was developed by recombinant tryptophan synthase. This study provides us with an alternative KHW utilization strategy to synthesize S-phenyl-L-cysteine. Tryptophan synthase could efficiently convert L-serine contained in KHW to S-phenyl-L-cysteine at pH 9.0, 40°C and Trion X-100 of 0.02%. In a scale up study, L-serine conversion rate reach 97.1% with a final S-phenyl-L-cysteine concentration of 38.6 g l(-1). Copyright © 2013 Elsevier Ltd. All rights reserved.

Two Distinct Waxy Alleles Impact the Granule-Bound Starch Synthase in Sorghum

USDA-ARS?s Scientific Manuscript database

The granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in starch granules. Loss of GBSS activity results in starch granules containing mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles we...

The role of glycogen synthase kinase 3 beta in brain injury induced by myocardial ischemia/reperfusion injury in a rat model of diabetes mellitus.

PubMed

Zhao, Bo; Gao, Wen-Wei; Liu, Ya-Jing; Jiang, Meng; Liu, Lian; Yuan, Quan; Hou, Jia-Bao; Xia, Zhong-Yuan

2017-10-01

Myocardial ischemia/reperfusion injury can lead to severe brain injury. Glycogen synthase kinase 3 beta is known to be involved in myo-cardial ischemia/reperfusion injury and diabetes mellitus. However, the precise role of glycogen synthase kinase 3 beta in myocardial ischemia/reperfusion injury-induced brain injury is unclear. In this study, we observed the effects of glycogen synthase kinase 3 beta on brain injury induced by myocardial ischemia/reperfusion injury in diabetic rats. Rat models of diabetes mellitus were generated via intraperitoneal injection of streptozotocin. Models of myocardial ischemia/reperfusion injury were generated by occluding the anterior descending branch of the left coronary artery. Post-conditioning comprised three cycles of ischemia/reperfusion. Immunohistochemical staining and western blot assays demonstrated that after 48 hours of reperfusion, the structure of the brain was seriously damaged in the experimental rats compared with normal controls. Expression of Bax, interleukin-6, interleukin-8, terminal deoxynucleotidyl transferase dUTP nick end labeling, and cleaved caspase-3 in the brain was significantly increased, while expression of Bcl-2, interleukin-10, and phospho-glycogen synthase kinase 3 beta was decreased. Diabetes mellitus can aggravate inflammatory reactions and apoptosis. Ischemic post-conditioning with glycogen synthase kinase 3 beta inhibitor lithium chloride can effectively reverse these changes. Our results showed that myocardial ischemic post-conditioning attenuated myocardial ischemia/reperfusion injury-induced brain injury by activating glyco-gen synthase kinase 3 beta. According to these results, glycogen synthase kinase 3 beta appears to be an important factor in brain injury induced by myocardial ischemia/reperfusion injury.

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors.

PubMed

Sielaff, Hendrik; Martin, James; Singh, Dhirendra; Biuković, Goran; Grüber, Gerhard; Frasch, Wayne D

2016-12-02

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A 3 B 3 DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α 3 β 3 γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A 3 B 3 DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A 3 B 3 DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Use of linalool synthase in genetic engineering of scent production

DOEpatents

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.

Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis.

PubMed

Morehouse, Benjamin R; Kumar, Ramasamy P; Matos, Jason O; Olsen, Sarah Naomi; Entova, Sonya; Oprian, Daniel D

2017-03-28

Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C 10 ) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.

Effect of Hammerhead Ribozyme against Human Thymidylate Synthase on the Cytotoxicity of Thymidylate Synthase Inhibitors

PubMed Central

Takemura, Yuzuru; Miyachi, Hayato; Skelton, Lorraine; Jackman, Ann L.

1995-01-01

One of the resistance mechanisms to folate‐based thymidylate synthase (TS) inhibitors is the increase in TS activity in tumor cells. Human B lymphoblastoid cell line (W1L2) was made resistant to a lipophilic non‐polyglutamatable TS inhibitor (ZM249148), and the subline (W1L2:R179) showed a 20‐fold increase in TS enzyme activity with concomitant overexpression of TS mRNA. To overcome the resistance, we designed a ribozyme that can cleave the CUC sequences in a triple tandemly repeated sequence of TS mRNA. Expression of this ribozyme in W1L2:R179 cells transfected with Epstein Barr virus‐based expression vector resulted in sensitization to TS inhibitors concomitantly with a decrease of TS expression. The ribozyme expressed in transfectants was shown to be functional in cleaving artificial TS RNA in vitro. PMID:8567390

Structure-based virtual screening of hypothetical inhibitors of the enzyme longiborneol synthase-a potential target to reduce Fusarium head blight disease.

PubMed

Bresso, E; Leroux, V; Urban, M; Hammond-Kosack, K E; Maigret, B; Martins, N F

2016-07-01

Fusarium head blight (FHB) is one of the most destructive diseases of wheat and other cereals worldwide. During infection, the Fusarium fungi produce mycotoxins that represent a high risk to human and animal health. Developing small-molecule inhibitors to specifically reduce mycotoxin levels would be highly beneficial since current treatments unspecifically target the Fusarium pathogen. Culmorin possesses a well-known important synergistically virulence role among mycotoxins, and longiborneol synthase appears to be a key enzyme for its synthesis, thus making longiborneol synthase a particularly interesting target. This study aims to discover potent and less toxic agrochemicals against FHB. These compounds would hamper culmorin synthesis by inhibiting longiborneol synthase. In order to select starting molecules for further investigation, we have conducted a structure-based virtual screening investigation. A longiborneol synthase structural model is first built using homology modeling, followed by molecular dynamics simulations that provided the required input for a protein-ligand ensemble docking procedure. From this strategy, the three most interesting compounds (hits) were selected among the 25 top-ranked docked compounds from a library of 15,000 drug-like compounds. These putative inhibitors of longiborneol synthase provide a sound starting point for further studies involving molecular modeling coupled to biochemical experiments. This process could eventually lead to the development of novel approaches to reduce mycotoxin contamination in harvested grain.

Crystallization and preliminary crystallographic analysis of an acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morita, Hiroyuki; Kondo, Shin; Kato, Ryohei

2007-07-01

An acridone-producing novel type III polyketide synthase from H. serrata has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 2.0 Å. Polyketide synthase 1 (PKS1) from Huperzia serrata is a plant-specific type III polyketide synthase that shows an unusually versatile catalytic potential, producing various aromatic tetraketides, including chalcones, benzophenones, phlorogulucinols and acridones. Recombinant H. serrata PKS1 expressed in Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group I222 or I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 73.3, b = 85.0, c = 137.7 Å, α =more » β = γ = 90.0°. Diffraction data were collected to 2.0 Å resolution using synchrotron radiation at BL24XU of SPring-8.« less

The structure of subunit E of the Pyrococcus horikoshii OT3 A-ATP synthase gives insight into the elasticity of the peripheral stalk.

PubMed

Balakrishna, Asha Manikkoth; Hunke, Cornelia; Grüber, Gerhard

2012-07-13

A(1)A(O) ATP synthases are the major energy converters of archaea. They are composed of an A(1) region that synthesizes ATP and an integral part A(O) that conducts ions. Subunit E is a component of the peripheral stalk that links the A(1) with the A(O) part of the A-ATP synthase. We have determined the crystal structure of the entire subunit E (PhE) of the Pyrococcus horikoshii OT3 A-ATP synthase at 3.6 Å resolution. The structure reveals an extended S-shaped N-terminal α-helix with 112.29 Å in length, followed by a globular head group. The S-shaped feature, common in elastic connectors and spacers, would facilitate the storage of transient elastic energy during rotary motion in the enzyme. The structure has been superimposed into the asymmetric peripheral stalks of the three-dimensional reconstruction of the Pyrococcus furiosus enzyme, revealing that the S-shaped subunit PhE fits well into the bent peripheral stalk, whereas the previously solved E subunit structure (3.1 Å resolution) of Thermus thermophilus A-ATP synthase is well accommodated in the density of the straight stator domain. The different features of the two stalk subunits are discussed in light of a novel coupling mechanism in A-ATP synthases proposed to differ from the Wankel engine of F-ATP synthases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of a novel hedycaryol synthase gene isolated from Camellia brevistyla flowers and floral scent of Camellia cultivars.

PubMed

Hattan, Jun-ichiro; Shindo, Kazutoshi; Ito, Tomoko; Shibuya, Yurica; Watanabe, Arisa; Tagaki, Chie; Ohno, Fumina; Sasaki, Tetsuya; Ishii, Jun; Kondo, Akihiko; Misawa, Norihiko

2016-04-01

A novel terpene synthase (Tps) gene isolated from Camellia brevistyla was identified as hedycaryol synthase, which was shown to be expressed specifically in flowers. Camellia plants are very popular because they bloom in winter when other plants seldom flower. Many ornamental cultivars of Camellia have been bred mainly in Japan, although the fragrance of their flowers has not been studied extensively. We analyzed floral scents of several Camellia cultivars by gas chromatography-mass spectrometry (GC-MS) and found that Camellia brevistyla produced various sesquiterpenes in addition to monoterpenes, whereas Camellia japonica and its cross-lines produced only monoterpenes, including linalool as the main product. From a flower of C. brevistyla, we isolated one cDNA encoding a terpene synthase (TPS) comprised of 554 amino acids, which was phylogenetically positioned to a sole gene clade. The cDNA, designated CbTps1, was expressed in mevalonate-pathway-engineered Escherichia coli, which carried the Streptomyces mevalonate-pathway gene cluster in addition to the acetoacetate-CoA ligase gene. A terpene product was purified from recombinant E. coli cultured with lithium acetoacetate, and analyzed by (1)H-nulcear magnetic resonance spectroscopy ((1)H-NMR) and GC-MS. It was shown that a sesquiterpene hedycaryol was produced, because (1)H-NMR signals of the purified product were very broad, and elemol, a thermal rearrangement product from hedycaryol, was identified by GC-MS analysis. Spectroscopic data of elemol were also determined. These results indicated that the CbTps1 gene encodes hedycaryol synthase. Expression analysis of CbTps1 showed that it was expressed specifically in flowers, and hedycaryol is likely to be one of the terpenes that attract insects for pollination of C. brevistyla. A linalool synthase gene, which was isolated from a flower of Camellia saluenensis, is also described.

Prostaglandin E(2) synthase inhibition as a therapeutic target.

PubMed

Iyer, Jitesh P; Srivastava, Punit K; Dev, Rishabh; Dastidar, Sunanda G; Ray, Abhijit

2009-07-01

Most NSAIDs function by inhibiting biosynthesis of PGE(2) by inhibition of COX-1 and/or COX-2. Since COX-1 has a protective function in the gastro-intestinal tract (GIT), non-selective inhibition of both cycloxy genases leads to moderate to severe gastro-intestinal intolerance. Attempts to identify selective inhibitors of COX-2, led to the identification of celecoxib and rofecoxib. However, long-term use of these drugs has serious adverse effects of sudden myocardial infarction and thrombosis. Drug-mediated imbalance in the levels of prostaglandin I(2) (PGI(2)) and thromboxane A(2) (TXA(2)) with a bias towards TXA(2) may be the primary reason for these events. This resulted in the drugs being withdrawn from the market, leaving a need for an effective and safe anti-inflammatory drug. Recently, the focus of research has shifted to enzymes downstream of COX in the prosta glandin biosynthetic pathway such as prostaglandin E(2) synthases. Microsomal prostaglandin E(2) synthase-1 (mPGES-1) specifically isomerizes PGH(2) to PGE(2), under inflammatory conditions. In this review, we examine the biology of mPGES-1 and its role in disease. Progress in designing molecules that can selectively inhibit mPGES-1 is reviewed. mPGES-1 has the potential to be a target for anti-inflammatory therapy, devoid of adverse GIT and cardiac effects and warrants further investigation.

Cyclooxygenases, microsomal prostaglandin E synthase-1, and cardiovascular function

PubMed Central

Cheng, Yan; Wang, Miao; Yu, Ying; Lawson, John; Funk, Colin D.; FitzGerald, Garret A.

2006-01-01

We investigated the mechanisms by which inhibitors of prostaglandin G/H synthase-2 (PGHS-2; known colloquially as COX-2) increase the incidence of myocardial infarction and stroke. These inhibitors are believed to exert both their beneficial and their adverse effects by suppression of PGHS-2–derived prostacyclin (PGI2) and PGE2. Therefore, the challenge remains to identify a mechanism whereby PGI2 and PGE2 expression can be suppressed while avoiding adverse cardiovascular events. Here, selective inhibition, knockout, or mutation of PGHS-2, or deletion of the receptor for PGHS-2–derived PGI2, was shown to accelerate thrombogenesis and elevate blood pressure in mice. These responses were attenuated by COX-1 knock down, which mimics the beneficial effects of low-dose aspirin. PGE2 biosynthesis is catalyzed by the coordinate actions of COX enzymes and microsomal PGE synthase-1 (mPGES-1). We show that deletion of mPGES-1 depressed PGE2 expression, augmented PGI2 expression, and had no effect on thromboxane biosynthesis in vivo. Most importantly, mPGES-1 deletion affected neither thrombogenesis nor blood pressure. These results suggest that inhibitors of mPGES-1 may retain their antiinflammatory efficacy by depressing PGE2, while avoiding the adverse cardiovascular consequences associated with PGHS-2–mediated PGI2 suppression. PMID:16614756

CERAMIDE SYNTHASE 1 IS REGULATED BY PROTEASOMAL MEDIATED TURNOVER

PubMed Central

Sridevi, Priya; Alexander, Hannah; Laviad, Elad L.; Pewzner-Jung, Yael; Hannink, Mark; Futerman, Anthony H.; Alexander, Stephen

2009-01-01

Ceramide is an important bioactive lipid, intimately involved in many cellular functions, including the regulation of cell death, and in cancer and chemotherapy. Ceramide is synthesized de novo from sphinganine and acyl CoA via a family of 6 ceramide synthase enzymes, each having a unique preference for different fatty acyl CoA substrates and a unique tissue distribution. However, little is known regarding the regulation of these important enzymes. In this study we focus on ceramide synthase 1 (CerS1) which is the most structurally and functionally distinct of the enzymes, and describe a regulatory mechanism that specifically controls the level of CerS1 via ubiquitination and proteasome dependent protein turnover. We show that both endogenous and ectopically expressed CerS1 have rapid basal turnover and that diverse stresses including chemotherapeutic drugs, UV light and DTT can induce CerS1 turnover. The turnover requires CerS1 activity and is regulated by the opposing actions of p38 MAP kinase and protein kinase C (PKC). p38 MAP kinase is a positive regulator of turnover, while PKC is a negative regulator of turnover. CerS1 is phosphorylated in vivo and activation of PKC increases the phosphorylation of the protein. This study reveals a novel and highly specific mechanism by which CerS1 protein levels are regulated and which directly impacts ceramide homeostasis. PMID:19393694

Superresolution microscopy reveals spatial separation of UCP4 and F0F1-ATP synthase in neuronal mitochondria

PubMed Central

Klotzsch, Enrico; Smorodchenko, Alina; Löfler, Lukas; Moldzio, Rudolf; Parkinson, Elena; Schütz, Gerhard J.; Pohl, Elena E.

2015-01-01

Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regulation of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical reconstruction microscopy) to visualize several mitochondrial proteins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F0F1-ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F0F1-ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various expression levels in different mitochondria, supporting the hypothesis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F0F1-ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from acting as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production. PMID:25535394

The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

PubMed Central

Zíková, Alena; Schnaufer, Achim; Dalley, Rachel A.; Panigrahi, Aswini K.; Stuart, Kenneth D.

2009-01-01

The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought. PMID:19436713

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanazawa, Atsuko; Ostendorf, Elisabeth; Kohzuma, Kaori

In wild type plants, decreasing CO 2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b 6f complex. Here in this paper, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO 2. The increased thylakoid proton conductivitymore » (g H +) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO 2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.« less

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

DOE PAGES

Kanazawa, Atsuko; Ostendorf, Elisabeth; Kohzuma, Kaori; ...

2017-05-03

In wild type plants, decreasing CO 2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b 6f complex. Here in this paper, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO 2. The increased thylakoid proton conductivitymore » (g H +) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO 2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.« less

Deletion of phytochelatin synthase modulates the metal accumulation pattern of cadmium exposed C. elegans

DOE PAGES

Essig, Yona J.; Webb, Samuel M.; Stürzenbaum, Stephen R.

2016-02-19

Here, environmental metal pollution is a growing health risk to flora and fauna. It is therefore important to fully elucidate metal detoxification pathways. Phytochelatin synthase (PCS), an enzyme involved in the biosynthesis of phytochelatins (PCs), plays an important role in cadmium detoxification. The PCS and PCs are however not restricted to plants, but are also present in some lower metazoans. The model nematode Caenorhabditis elegans, for example, contains a fully functional phytochelatin synthase and phytochelatin pathway. By means of a transgenic nematode strain expressing a pcs-1 promoter-tagged GFP ( pcs-1::GFP) and a pcs-1 specific qPCR assay, further evidence is presentedmore » that the expression of the C. elegans phytochelatin synthase gene (pcs-1) is transcriptionally non-responsive to a chronic (48 h) insult of high levels of zinc (500 μM) or acute (3 h) exposures to high levels of cadmium (300 μM). However, the accumulation of cadmium, but not zinc, is dependent on the pcs-1 status of the nematode. Synchrotron based X-ray fluorescence imaging uncovered that the cadmium body burden increased significantly in the pcs-1(tm1748) knockout allele. Taken together, this suggests that whilst the transcription of pcs-1 may not be mediated by an exposure zinc or cadmium, it is nevertheless an integral part of the cadmium detoxification pathway in C. elegans.« less

Mitochondrial β-Cyanoalanine Synthase Is Essential for Root Hair Formation in Arabidopsis thaliana[W

PubMed Central

García, Irene; Castellano, José María; Vioque, Blanca; Solano, Roberto; Gotor, Cecilia; Romero, Luis C.

2010-01-01

Cyanide is stoichiometrically produced as a coproduct of the ethylene biosynthesis pathway and is detoxified by β-cyanoalanine synthase enzymes. The molecular and phenotypical analysis of T-DNA insertion mutants of the mitochondrial β-cyanoalanine synthase CYS-C1 suggests that discrete accumulation of cyanide is not toxic for the plant and does not alter mitochondrial respiration rates but does act as a strong inhibitor of root hair development. The cys-c1 null allele is defective in root hair formation and accumulates cyanide in root tissues. The root hair defect is phenocopied in wild-type plants by the exogenous addition of cyanide to the growth medium and is reversed by the addition of hydroxocobalamin or by genetic complementation with the CYS-C1 gene. Hydroxocobalamin not only recovers the root phenotype of the mutant but also the formation of reactive oxygen species at the initial step of root hair tip growth. Transcriptional profiling of the cys-c1 mutant reveals that cyanide accumulation acts as a repressive signal for several genes encoding enzymes involved in cell wall rebuilding and the formation of the root hair tip as well as genes involved in ethylene signaling and metabolism. Our results demonstrate that mitochondrial β-cyanoalanine synthase activity is essential to maintain a low level of cyanide for proper root hair development. PMID:20935247

Deletion of phytochelatin synthase modulates the metal accumulation pattern of cadmium exposed C. elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Essig, Yona J.; Webb, Samuel M.; Stürzenbaum, Stephen R.

Here, environmental metal pollution is a growing health risk to flora and fauna. It is therefore important to fully elucidate metal detoxification pathways. Phytochelatin synthase (PCS), an enzyme involved in the biosynthesis of phytochelatins (PCs), plays an important role in cadmium detoxification. The PCS and PCs are however not restricted to plants, but are also present in some lower metazoans. The model nematode Caenorhabditis elegans, for example, contains a fully functional phytochelatin synthase and phytochelatin pathway. By means of a transgenic nematode strain expressing a pcs-1 promoter-tagged GFP ( pcs-1::GFP) and a pcs-1 specific qPCR assay, further evidence is presentedmore » that the expression of the C. elegans phytochelatin synthase gene (pcs-1) is transcriptionally non-responsive to a chronic (48 h) insult of high levels of zinc (500 μM) or acute (3 h) exposures to high levels of cadmium (300 μM). However, the accumulation of cadmium, but not zinc, is dependent on the pcs-1 status of the nematode. Synchrotron based X-ray fluorescence imaging uncovered that the cadmium body burden increased significantly in the pcs-1(tm1748) knockout allele. Taken together, this suggests that whilst the transcription of pcs-1 may not be mediated by an exposure zinc or cadmium, it is nevertheless an integral part of the cadmium detoxification pathway in C. elegans.« less

Terpene synthases from Cannabis sativa.

PubMed

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

2017-01-01

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

Use of linalool synthase in genetic engineering of scent production

DOEpatents

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.

Phosphoproteomics links glycogen synthase kinase-3 to RNA splicing.

PubMed

Khoa, Le Tran Phuc; Dou, Yali

2017-11-03

Protein kinases play essential biological roles by phosphorylating a diverse range of signaling molecules, but deciphering their direct physiological targets remains a challenge. A new study by Shinde et al. uses phosphoproteomics to identify glycogen synthase kinase-3 (GSK-3) substrates in mouse embryonic stem cells (mESCs), providing a broad profile of GSK-3 activity and defining a new role for this central kinase in regulating RNA splicing. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular cloning of the human UMP synthase gene and characterization of point mutations in two hereditary orotic aciduria families

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suchi, Mariko; Mizuno, Haruo; Tsuboi, Takashi

Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzing the last two steps of de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5{prime}-monophosphate decarboxylase (ODC). Loss of either enzymatic activity results in hereditary orotic aciduria, a rare autosomal recessive disorder characterized by retarded growth, anemia, and excessive urinary excretion of orotic acid. We have isolated the UMP synthase chromosomal gene from a {lambda}EMBL-3 human genomic library and report a single-copy gene spanning {approximately}15 kb. The UMP synthase genomic structure encodes six exons ranging in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AGmore » rule. Cognate promoter elements implicated in glucocorticoid- and cAMP-mediated regulation as well as in liver-, myeloid-, and lymphocyte-specific expression are located within the 5{prime} flanking sequence. Molecular investigation of UMP synthase deficiency in a Japanese orotic aciduria patient revealed mutations R96G (A- to-G transition; nt 286) and G429R (G-to-C transversion; nt 1285) in one allele and V109G (T-to-G transversion; nt 326) in the other allele. Expression of human UMP synthase cDNAs containing these mutations in pyrimidine auxotrophic Escherichia coli and in recombinant baculovirus-infected Sf21 cells demonstrates impaired activity presumably associated with the urinary orotic acid substrate accumulations observed in vivo. We further establish the identity of two polymorphisms, G213A ({nu} = .26) and 440 Gpoly ({nu} = .27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function. 76 refs., 5 figs., 7 tabs.« less

Directed evolution and expression tuning of geraniol synthase for efficient geraniol production in Escherichia coli.

PubMed

Tashiro, Miki; Fujii, Akira; Kawai-Noma, Shigeko; Saito, Kyoichi; Umeno, Daisuke

2017-11-17

To achieve an efficient production of geraniol and its derivatives in Escherichia coli, we aimed to improve the activity of geraniol synthase (GES) through a single round of mutagenesis and screening for higher substrate consumption. We isolated GES variants that outperform their parent in geraniol production. The analysis of GES variants indicated that the expression level of GES was the bottleneck for geraniol synthesis. Over-expression of the mutant GES M53 with a 5'-untranslated sequence designed for high translational efficiency, along with the additional expression of mevalonate pathway enzymes, isopentenyl pyrophosphate isomerase, and geranyl pyrophosphate synthase, yielded 300 mg/L/12 h geraniol and its derivatives (>1000 mg/L/42 h in total) in a shaking flask.

Nuclear glycogen and glycogen synthase kinase 3.

PubMed

Ragano-Caracciolo, M; Berlin, W K; Miller, M W; Hanover, J A

1998-08-19

Glycogen is the principal storage form of glucose in animal cells. It accumulates in electron-dense cytoplasmic granules and is synthesized by glycogen synthase (GS), the rate-limiting enzyme of glycogen deposition. Glycogen synthase kinase-3 (GSK-3) is a protein kinase that phosphorylates GS. Two nearly identical forms of GSK-3 exist: GSK-3 alpha and GSK-3 beta. Both are constitutively active in resting cells and their activity can be modulated by hormones and growth factors. GSK-3 is implicated in the regulation of many physiological responses in mammalian cells by phosphorylating substrates including neuronal cell adhesion molecule, neurofilaments, synapsin I, and tau. Recent observations point to functions for glycogen and glycogen metabolism in the nucleus. GSK-3 phosphorylates several transcription factors, and we have recently shown that it modifies the major nuclear pore protein p62. It also regulates PK1, a protein kinase required for maintaining the interphase state and for DNA replication in cycling Xenopus egg extracts. Recently, glycogen was shown to be required for nuclear reformation in vitro using ovulated Xenopus laevis egg lysates. Because neither glycogen nor GSK-3 has been localized to the nuclear envelope or intranuclear sites, glycogen and GSK-3 activites were measured in rat liver nuclei and nuclear reformation extracts. Significant quantities of glycogen-like material co-purified with the rat-liver nuclear envelope. GSK-3 is also highly enriched in the glycogen pellet of egg extracts of Xenopus that is required for nuclear assembly in vitro. Based on the finding that enzymes of glycogen metabolism copurify with glycogen, we propose that glycogen may serve a structural role as a scaffold for nuclear assembly and sequestration of critical kinases and phosphatases in the nucleus. Copyright 1998 Academic Press.

Analysis of polyhydroxyalkanoate (PHA) synthase gene and PHA-producing bacteria in activated sludge that produces PHA containing 3-hydroxydodecanoate.

PubMed

Yang, Chao; Zhang, Wei; Liu, Ruihua; Zhang, Chi; Gong, Ting; Li, Qiang; Wang, Shufang; Song, Cunjiang

2013-09-01

Activated sludge is an alternative to pure cultures for polyhydroxyalkanoate (PHA) production due to the presence of many PHA-producing bacteria in activated sludge community. In this study, activated sludge was submitted to aerobic dynamic feeding in a sequencing batch reactor. During domestication, the changes of bacterial community structure were observed by terminal restriction fragment length polymorphism analysis. Furthermore, some potential PHA-producing bacteria, such as Thauera, Acinetobacter and Pseudomonas, were identified by denaturing gradient gel electrophoresis analysis. The constructed PHA synthase gene library was analyzed by DNA sequencing. Of the 80 phaC genes obtained, 76 belonged to the Class I PHA synthase, and four to the Class II PHA synthase. Gas chromatography-mass spectrometry analysis showed that PHA produced by activated sludge was composed of three types of monomers: 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxydodecanoate (3HDD). This is the first report of production of medium-chain-length PHAs (PHAMCL ) containing 3HDD by activated sludge. Further studies suggested that a Pseudomonas strain may play an important role in the production of PHAMCL containing 3HDD. Moreover, a Class II PHA synthase was found to have a correlation with the production of 3HDD-containing PHAMCL . © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Predicted cycloartenol synthase protein from Kandelia obovata and Rhizophora stylosa using online software of Phyre2 and Swiss-model

NASA Astrophysics Data System (ADS)

Basyuni, M.; Sulistiyono, N.; Wati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

2018-03-01

Cloning of Kandelia obovata KcCAS gene (previously known as Kandelia candel) and Rhizophora stylosa RsCAS have already have been reported and encoded cycloartenol synthases. In this study, the predicted KcCAS and RsCAS protein were analyzed using online software of Phyre2 and Swiss-model. The protein modelling for KcCAS and RsCAS cycloartenol synthases was determined using Pyre2 had similar results with slightly different in sequence identity. By contrast, the Swiss-model for KcCAS slightly had higher sequence identity (47.31%) and Qmean (0.70) compared to RsCAS. No difference of ligands binding site which is considered as modulators for both cycloartenol synthases. The range of predicted protein derived from 91-757 amino acid residues with coverage sequence similarities 0.86, respectively from template model of lanosterol synthase from the human. Homology modelling revealed that 706 residues (93% of the amino acid sequence) had been modelled with 100.0% confidence by the single highest scoring template for both KcCAS and RsCAS using Phyre2. This coverage was more elevated than swiss-model predicted (86%). The present study suggested that both genes are responsible for the genesis of cycloartenol in these mangrove plants.

Glycogen Synthase in Sertoli Cells: More Than Glycogenesis?

PubMed

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.

The rotation of cellulose synthase trajectories is microtubule dependent and influences the texture of epidermal cell walls in Arabidopsis hypocotyls.

PubMed

Chan, Jordi; Crowell, Elizabeth; Eder, Magdalena; Calder, Grant; Bunnewell, Susan; Findlay, Kim; Vernhettes, Samantha; Höfte, Herman; Lloyd, Clive

2010-10-15

Plant shoots have thick, polylamellate outer epidermal walls based on crossed layers of cellulose microfibrils, but the involvement of microtubules in such wall lamellation is unclear. Recently, using a long-term movie system in which Arabidopsis seedlings were grown in a biochamber, the tracks along which cortical microtubules move were shown to undergo slow rotary movements over the outer surface of hypocotyl epidermal cells. Because microtubules are known to guide cellulose synthases over the short term, we hypothesised that this previously unsuspected microtubule rotation could, over the longer term, help explain the cross-ply structure of the outer epidermal wall. Here, we test that hypothesis using Arabidopsis plants expressing the cellulose synthase GFP-CESA3 and show that cellulose synthase trajectories do rotate over several hours. Neither microtubule-stabilising taxol nor microtubule-depolymerising oryzalin affected the linear rate of GFP-CESA3 movement, but both stopped the rotation of cellulose synthase tracks. Transmission electron microscopy revealed that drug-induced suppression of rotation alters the lamellation pattern, resulting in a thick monotonous wall layer. We conclude that microtubule rotation, rather than any hypothetical mechanism for wall self-assembly, has an essential role in developing cross-ply wall texture.

Isolation and characterization of an oxidosqualene cyclase gene encoding a β-amyrin synthase involved in Polygala tenuifolia Willd. saponin biosynthesis.

PubMed

Jin, Mei Lan; Lee, Dae Young; Um, Yurry; Lee, Jeong Hoon; Park, Chun Geun; Jetter, Reinhard; Kim, Ok Tae

2014-03-01

Expression of PtBS (Polygala tenuifolia β-amyrin synthase) led to the production of β-amyrin as sole product. Polygala tenuifolia Willdenow is a rich source of triterpene saponins, onjisaponins and polygalasaponins, used as herbal medicine to treat phlegms and for detumescence in traditional Asian healing. The Polygala saponins share the oleanane backbone structure and are, therefore, likely synthesized via β-amyrin as a common precursor. We hypothesized that, in analogy to diverse other plant species, this central intermediate should be formed by a β-amyrin synthase catalyzing the complex cyclization of oxidosqualene. This member of the oxidosqualene cyclase (OSC) family of enzymes is thus defining an important branch point between primary and secondary metabolisms, and playing a crucial role in the control of oleanane-type triterpene saponin biosynthesis. From P. tenuifolia roots, we isolated an OSC cDNA containing a reading frame of 2,289 bp nucleotides. The predicted protein of 763 amino acids (molecular weight 87.353 kDa) showed particularly high amino acid sequence identities to known β-amyrin synthases (85-87 %) and was, therefore, named PtBS. Expression of PtBS in the triterpenoid synthase-deficient yeast mutant GIL77 led to the production of β-amyrin as sole product. qRT-PCR analysis of various P. tenuifolia organs showed that PtBS transcript levels were highest in the roots, consistent with onjisaponin accumulation patterns. Therefore, we conclude that PtBS is the β-amyrin synthase enzyme catalyzing the first committed step in the biosynthesis of onjisaponins and polygalasaponins in P. tenuifolia.

Light and Fungal Elicitor Induce 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase mRNA in Suspension Cultured Cells of Parsley (Petroselinum crispum L.) 1

PubMed Central

Henstrand, John M.; McCue, Kent F.; Brink, Kent; Handa, Avtar K.; Herrmann, Klaus M.; Conn, Eric E.

1992-01-01

Light and fungal elicitor induce mRNA encoding 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase in suspension cultured cells of parsley (Petroselinum crispum L.). The kinetics and dose response of mRNA accumulation were similar for DAHP synthase and phenylalanine ammonia-lyase (PAL). Six micrograms of elicitor from Phytophthora megasperma f. glycinia gave a detectable induction within 1 hour. Induction of DAHP synthase and PAL mRNAs by light was transient, reaching maximal levels at 4 hours and returning to pretreatment levels after 24 hours. Our data suggest that either light or fungal elicitor transcriptionally activate DAHP synthase. A coordinate regulation for key enzymes in the synthesis of primary and secondary metabolites is indicated. ImagesFigure 1 PMID:16668708

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

PubMed Central

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

2009-01-01

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

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

PubMed

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-01-01

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

Quantitative Proteomic Analysis of Human Lung Tumor Xenografts Treated with the Ectopic ATP Synthase Inhibitor Citreoviridin

PubMed Central

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-01-01

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy. PMID:23990911

Tomato linalool synthase is induced in trichomes by jasmonic acid

PubMed Central

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

Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase

PubMed Central

Sauge-Merle, Sandrine; Cuiné, Stéphan; Carrier, Patrick; Lecomte-Pradines, Catherine; Luu, Doan-Trung; Peltier, Gilles

2003-01-01

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes. PMID:12514032

Evolution of Homospermidine Synthase in the Convolvulaceae: A Story of Gene Duplication, Gene Loss, and Periods of Various Selection Pressures[C][W][OA

PubMed Central

Kaltenegger, Elisabeth; Eich, Eckart; Ober, Dietrich

2013-01-01

Homospermidine synthase (HSS), the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, is known to have its origin in the duplication of a gene encoding deoxyhypusine synthase. To study the processes that followed this gene duplication event and gave rise to HSS, we identified sequences encoding HSS and deoxyhypusine synthase from various species of the Convolvulaceae. We show that HSS evolved only once in this lineage. This duplication event was followed by several losses of a functional gene copy attributable to gene loss or pseudogenization. Statistical analyses of sequence data suggest that, in those lineages in which the gene copy was successfully recruited as HSS, the gene duplication event was followed by phases of various selection pressures, including purifying selection, relaxed functional constraints, and possibly positive Darwinian selection. Site-specific mutagenesis experiments have confirmed that the substitution of sites predicted to be under positive Darwinian selection is sufficient to convert a deoxyhypusine synthase into a HSS. In addition, analyses of transcript levels have shown that HSS and deoxyhypusine synthase have also diverged with respect to their regulation. The impact of protein–protein interaction on the evolution of HSS is discussed with respect to current models of enzyme evolution. PMID:23572540

Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

PubMed

Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

2012-07-27

Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

Two Polyhydroxyalkanoate Synthases from Distinct Classes from the Aromatic Degrader Cupriavidus pinatubonensis JMP134 Exhibit the Same Substrate Preference.

PubMed

Jiang, Xuan; Luo, Xi; Zhou, Ning-Yi

2015-01-01

Cupriavidus pinatubonensis JMP134 utilizes a variety of aromatic substrates as sole carbon sources, including meta-nitrophenol (MNP). Two polyhydroxyalkanoate (PHA) synthase genes, phaC1 and phaC2, were annotated and categorized as class I and class II PHA synthase genes, respectively. In this study, both His-tagged purified PhaC1 and PhaC2 were shown to exhibit typical class I PHA synthase substrate specificity to make short-chain-length (SCL) PHA from 3-hydroxybutyryl-CoA and failed to make medium-chain-length (MCL) PHA from 3-hydroxyoctanoyl-CoA. The phaC1 or phaC2 deletion strain could also produce SCL PHA when grown in fructose or octanoate, but the double mutant of phaC1 and phaC2 lost this ability. The PhaC2 also exhibited substrate preference towards SCL substrates when expressed in Pseudomonas aeruginosa PAO1 phaC mutant strain. On the other hand, the transcriptional level of phaC1 was 70-fold higher than that of phaC2 in MNP-grown cells, but 240-fold lower in octanoate-grown cells. Further study demonstrated that only phaC1 was involved in PHA synthesis in MNP-grown cells. These findings suggested that phaC1 and phaC2 genes were differentially regulated under different growth conditions in this strain. Within the phaC2-containing gene cluster, a single copy of PHA synthase gene was present clustering with genes encoding enzymes in the biosynthesis of PHA precursors. This is markedly different from the genetic organization of all other previously reported class II PHA synthase gene clusters and this cluster likely comes from a distinct evolutionary path.

Two Polyhydroxyalkanoate Synthases from Distinct Classes from the Aromatic Degrader Cupriavidus pinatubonensis JMP134 Exhibit the Same Substrate Preference

PubMed Central

Jiang, Xuan; Luo, Xi; Zhou, Ning-Yi

2015-01-01

Cupriavidus pinatubonensis JMP134 utilizes a variety of aromatic substrates as sole carbon sources, including meta-nitrophenol (MNP). Two polyhydroxyalkanoate (PHA) synthase genes, phaC1 and phaC2, were annotated and categorized as class I and class II PHA synthase genes, respectively. In this study, both His-tagged purified PhaC1 and PhaC2 were shown to exhibit typical class I PHA synthase substrate specificity to make short-chain-length (SCL) PHA from 3-hydroxybutyryl-CoA and failed to make medium-chain-length (MCL) PHA from 3-hydroxyoctanoyl-CoA. The phaC1 or phaC2 deletion strain could also produce SCL PHA when grown in fructose or octanoate, but the double mutant of phaC1 and phaC2 lost this ability. The PhaC2 also exhibited substrate preference towards SCL substrates when expressed in Pseudomonas aeruginosa PAO1 phaC mutant strain. On the other hand, the transcriptional level of phaC1 was 70-fold higher than that of phaC2 in MNP-grown cells, but 240-fold lower in octanoate-grown cells. Further study demonstrated that only phaC1 was involved in PHA synthesis in MNP-grown cells. These findings suggested that phaC1 and phaC2 genes were differentially regulated under different growth conditions in this strain. Within the phaC2-containing gene cluster, a single copy of PHA synthase gene was present clustering with genes encoding enzymes in the biosynthesis of PHA precursors. This is markedly different from the genetic organization of all other previously reported class II PHA synthase gene clusters and this cluster likely comes from a distinct evolutionary path. PMID:26544851

Identification of a new polyhydroxyalkanoate (PHA) producer Aquitalea sp. USM4 (JCM 19919) and characterization of its PHA synthase.

PubMed

Ng, Lee-Mei; Sudesh, Kumar

2016-11-01

Aquitalea sp. USM4 (JCM 19919) was isolated from a freshwater sample at Lata Iskandar Waterfall in Perak, Malaysia. It is a rod-shaped, gram-negative bacterium with high sequence identity (99%) to Aquitalea magnusonii based on 16S rRNA gene analysis. Aquitalea sp. USM4 also possessed a PHA synthase gene (phaC), which had amino acid sequence identity of 77-78% to the PHA synthase of Chromobacterium violaceum ATCC12472 and Pseudogulbenkiania sp. NH8B. PHA biosynthesis results showed that wild-type Aquitalea sp. USM4 was able to accumulate up to 1.5 g/L of poly(3-hydroxybutyrate), [P(3HB)]. The heterologous expression of the PHA synthase gene of Aquitalea sp. USM4 (phaC Aq ) in Cupriavidus necator PHB - 4 had resulted in PHA accumulation up to 3.2 g/L of P(3HB). It was further confirmed by 1 H nuclear magnetic resonance (NMR) analysis that Aquitalea sp. USM4 and C. necator PHB - 4 transformant were able to produce PHA containing 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB) and 3-hydroxy-4-methylvalerate (3H4MV) monomers from suitable precursor substrates. Interestingly, relatively high PHA synthase activity of 863 U/g and 1402 U/g were determined in wild-type Aquitalea sp. USM4 and C. necator PHB - 4 transformant respectively. This is the first report on the member of genus Aquitalea as a new PHA producer as well as in vitro and in vivo characterization of a novel PHA synthase from Aquitalea sp. USM4. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Cloning of a sesquiterpene synthase from Lavandula x intermedia glandular trichomes.

PubMed

Sarker, Lukman S; Demissie, Zerihun A; Mahmoud, Soheil S

2013-11-01

The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also contain small amounts of sesquiterpenes, depending on species and environmental conditions. For example, the sesquiterpene 9-epi-caryophyllene can make up to 8 % of the EO in a few species, including those commercially propagated for EO production. Here, we report the cloning and functional characterization of 9-epi-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not encode a transit peptide, was expressed in Escherichia coli and the recombinant protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa recombinant protein specifically converted farnesyl diphosphate to 9-epi-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the monoterpene precursor geranyl diphosphate (GPP), but--unlike most monoterpene synthases--was not able to derive detectable amounts of any products from the cis isomer of GPP, neryl diphosphate. The LiCPS transcripts accumulated in developing L. x intermedia flowers and were highly enriched in glandular trichomes, but were not detected in leaves suggesting that the transcriptional expression of this gene is spatially and developmentally regulated.

Structural and Functional Studies of a Pyran Synthase Domain from a trans-Acyltransferase Assembly Line.

PubMed

Wagner, Drew T; Zhang, Zhicheng; Meoded, Roy A; Cepeda, Alexis J; Piel, Jörn; Keatinge-Clay, Adrian T

2018-04-20

trans-Acyltransferase assembly lines possess enzymatic domains often not observed in their better characterized cis-acyltransferase counterparts. Within this repertoire of largely unexplored biosynthetic machinery is a class of enzymes called the pyran synthases that catalyze the formation of five- and six-membered cyclic ethers from diverse polyketide chains. The 1.55 Å resolution crystal structure of a pyran synthase domain excised from the ninth module of the sorangicin assembly line highlights the similarity of this enzyme to the ubiquitous dehydratase domain and provides insight into the mechanism of ring formation. Functional assays of point mutants reveal the central importance of the active site histidine that is shared with the dehydratases as well as the supporting role of a neighboring semiconserved asparagine.

PhaM is the physiological activator of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha.

PubMed

Pfeiffer, Daniel; Jendrossek, Dieter

2014-01-01

Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.

Modulation of hyaluronan synthase activity in cellular membrane fractions.

PubMed

Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

2009-10-30

Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

Nitric Oxide Synthase and Cyclooxygenase Pathways: A Complex Interplay in Cellular Signaling.

PubMed

Sorokin, Andrey

2016-01-01

The cellular reaction to external challenges is a tightly regulated process consisting of integrated processes mediated by a variety of signaling molecules, generated as a result of modulation of corresponding biosynthetic systems. Both, nitric oxide synthase (NOS) and cyclooxygenase (COX) systems, consist of constitutive forms (NOS1, NOS3 and COX-1), which are mostly involved in housekeeping tasks, and inducible forms (NOS2 and COX-2), which shape the cellular response to stress and variety of bioactive agents. The complex interplay between NOS and COX pathways can be observed at least at three levels. Firstly, products of NOS and Cox systems can mediate the regulation and the expression of inducible forms (NOS2 and COX-2) in response of similar and dissimilar stimulus. Secondly, the reciprocal modulation of cyclooxygenase activity by nitric oxide and NOS activity by prostaglandins at the posttranslational level has been shown to occur. Mechanisms by which nitric oxide can modulate prostaglandin synthesis include direct S-nitrosylation of COX and inactivation of prostaglandin I synthase by peroxynitrite, product of superoxide reaction with nitric oxide. Prostaglandins, conversely, can promote an increased association of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase) with NOS1, thereby reducing its activity. The third level of interplay is provided by intracellular crosstalk of signaling pathways stimulated by products of NOS and COX which contributes significantly to the complexity of cellular signaling. Since modulation of COX and NOS pathways was shown to be principally involved in a variety of pathological conditions, the dissection of their complex relationship is needed for better understanding of possible therapeutic strategies. This review focuses on implications of interplay between NOS and COX for cellular function and signal integration.

Characterization of Geraniol Synthase from the Peltate Glands of Sweet Basil1

PubMed Central

Iijima, Yoko; Gang, David R.; Fridman, Eyal; Lewinsohn, Efraim; Pichersky, Eran

2004-01-01

The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn2+ as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. Km values of 21 and 51 μm were obtained for geranyl diphosphate and Mn2+, respectively. In the presence of 18O-labeled water, GES catalyzed the formation of 18O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral. PMID:14657409

The α-Terpineol to 1,8-Cineole Cyclization Reaction of Tobacco Terpene Synthases1

PubMed Central

Piechulla, Birgit; Bartelt, Richard; Brosemann, Anne; Bouwmeester, Harro; Hippauf, Frank

2016-01-01

Flowers of Nicotiana species emit a characteristic blend including the cineole cassette monoterpenes. This set of terpenes is synthesized by multiproduct enzymes, with either 1,8-cineole or α-terpineol contributing most to the volatile spectrum, thus referring to cineole or terpineol synthase, respectively. To understand the molecular and structural requirements of the enzymes that favor the biochemical formation of α-terpineol and 1,8-cineole, site-directed mutagenesis, in silico modeling, and semiempiric calculations were performed. Our results indicate the formation of α-terpineol by a nucleophilic attack of water. During this attack, the α-terpinyl cation is stabilized by π-stacking with a tryptophan side chain (tryptophan-253). The hypothesized catalytic mechanism of α-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (histidine-502 and glutamate-249), acting as a base, and a threonine (threonine-278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (S)-(−)-α-terpineol, which is the favored enantiomer product of the recombinant enzymes. Furthermore, by site-directed mutagenesis, we were able to identify amino acids at positions 147, 148, and 266 that determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase. Since amino acid 266 is more than 10 Å away from the active site, an indirect effect of this amino acid exchange on the catalysis is discussed. PMID:27729471

Expression of nitric oxide synthase-2 in the lungs decreases airway resistance and responsiveness.

PubMed

Hjoberg, Josephine; Shore, Stephanie; Kobzik, Lester; Okinaga, Shoji; Hallock, Arlene; Vallone, Joseph; Subramaniam, Venkat; De Sanctis, George T; Elias, Jack A; Drazen, Jeffrey M; Silverman, Eric S

2004-07-01

Individuals with asthma have increased levels of nitric oxide in their exhaled air. To explore its role, we have developed a regulatable transgenic mouse capable of overexpressing inducible nitric oxide synthase in a lung-specific fashion. The CC10-rtTA-NOS-2 mouse contains two transgenes, a reverse tetracycline transactivator under the control of the Clara cell protein promoter and the mouse nitric oxide synthase-2 (NOS-2) coding region under control of a tetracycline operator. Addition of doxycycline to the drinking water of CC10-rtTA-NOS-2 mice causes an increase in nitric oxide synthase-2 that is largely confined to the airway epithelium. The fraction of expired nitric oxide increases over the first 24 h from approximately 10 parts per billion to a plateau of approximately 20 parts per billion. There were no obvious differences between CC10-rtTA-NOS-2 mice, with or without doxycycline, and wild-type mice in lung histology, bronchoalveolar protein, total cell count, or count differentials. However, airway resistance was lower in CC10-rtTA-NOS-2 mice with doxycycline than in CC10-rtTA-NOS-2 mice without doxycycline or wild-type mice with doxycycline. Moreover, doxycycline-treated CC10-rtTA-NOS-2 mice were hyporesponsive to methacholine compared with other groups. These data suggest that increased nitric oxide in the airways has no proinflammatory effects per se and may have beneficial effects on pulmonary function.

Growth, sucrose synthase, and invertase activities of developing Phaseolus vulgaris L. fruits

Treesearch

Shi-Jean S. Sung; W.J. Sheih; D.R. Geiger; C.C. Black

1994-01-01

Activities of sucrose-cleaving enzymes, acid and neutral invertase and sucrose synthase, were measured in pods and seeds of developing snap bean (Phaseolus vulgaris L.) fruits, and compared with 14C-import, elongation and dry weight accumulation. The data supports the association of specific sucrose-cleaving enzymes with the specific processes that occur in the...

CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN11[OPEN

PubMed Central

Wilkop, Thomas E.; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

2015-01-01

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. PMID:25535279

REVIEWS OF TOPICAL PROBLEMS Molecular energy transducers of the living cell. Proton ATP synthase: a rotating molecular motor

NASA Astrophysics Data System (ADS)

Romanovsky, Yurii M.; Tikhonov, Alexander N.

2010-12-01

The free energy released upon the enzymatic hydrolysis of adenosine triphosphate (ATP) is the main source of energy for the functioning of the living cell and all multicellular organisms. The overwhelming majority of ATP molecules are formed by proton ATP synthases, which are the smallest macromolecular electric motors in Nature. This paper reviews the modern concepts of the molecular structure and functioning of the proton ATP synthase, and real-time biophysical experiments on the rotation of the 'rotor' of this macromolecular motor. Some mathematical models describing the operation of this nanosized macromolecular machine are described.

Pneumocystis jirovecii dihydropteroate synthase (DHPS) genotypes in non-HIV-immunocompromised patients: a tertiary care reference health centre study.

PubMed

Tyagi, A K; Mirdha, B R; Luthra, K; Guleria, R; Mohan, A; Singh, U B; Samantaray, J C; Dar, L; Iyer, V K; Sreenivas, V

2011-02-01

Studies on Pneumocystis jirovecii dihydropteroate synthase (DHPS) genotypes among non-HIV immunocompromised patients from developing countries are rare. In the present prospective investigation, 24 (11.8%) cases were found to be positive for Pneumocystis jirovecii out of 203 non-HIV patients with a clinical suspicion of Pneumocystis pneumonia (PCP). Dihydropteroate synthase (DHPS) genotype 1 (Thr55+Pro57) was noted in 95.8% P. jirovecii isolates in the present study in contrast to only 4.1% of patients with DHPS genotype 4 (Thr55Ala + Pro57Ser).

Rapid discovery and functional characterization of terpene synthases from four endophytic xylariaceae

DOE PAGES

Wu, Weihua; Tran, William; Taatjes, Craig A.; ...

2016-02-17

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

Rapid discovery and functional characterization of terpene synthases from four endophytic xylariaceae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Weihua; Tran, William; Taatjes, Craig A.

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

Genome-wide identification, functional and evolutionary analysis of terpene synthases in pineapple.

PubMed

Chen, Xiaoe; Yang, Wei; Zhang, Liqin; Wu, Xianmiao; Cheng, Tian; Li, Guanglin

2017-10-01

Terpene synthases (TPSs) are vital for the biosynthesis of active terpenoids, which have important physiological, ecological and medicinal value. Although terpenoids have been reported in pineapple (Ananas comosus), genome-wide investigations of the TPS genes responsible for pineapple terpenoid synthesis are still lacking. By integrating pineapple genome and proteome data, twenty-one putative terpene synthase genes were found in pineapple and divided into five subfamilies. Tandem duplication is the cause of TPS gene family duplication. Furthermore, functional differentiation between each TPS subfamily may have occurred for several reasons. Sixty-two key amino acid sites were identified as being type-II functionally divergence between TPS-a and TPS-c subfamily. Finally, coevolution analysis indicated that multiple amino acid residues are involved in coevolutionary processes. In addition, the enzyme activity of two TPSs were tested. This genome-wide identification, functional and evolutionary analysis of pineapple TPS genes provide a new insight into understanding the roles of TPS family and lay the basis for further characterizing the function and evolution of TPS gene family. Copyright © 2017 Elsevier Ltd. All rights reserved.

Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase 1

PubMed Central

Hack, Ethan; Kemp, John D.

1980-01-01

A single enzyme catalyzes the synthesis of all four N2-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N2-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (Km) differ, the ratio V/Km is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:16661312

Transcriptome profiling of the Australian arid-land plant Eremophila serrulata (A.DC.) Druce (Scrophulariaceae) for the identification of monoterpene synthases.

PubMed

Kracht, Octavia Natascha; Ammann, Ann-Christin; Stockmann, Julia; Wibberg, Daniel; Kalinowski, Jörn; Piotrowski, Markus; Kerr, Russell; Brück, Thomas; Kourist, Robert

2017-04-01

Plant terpenoids are a large and highly diverse class of metabolites with an important role in the immune defense. They find wide industrial application as active pharmaceutical ingredients, aroma and fragrance compounds. Several Eremophila sp. derived terpenoids have been documented. To elucidate the terpenoid metabolism, the transcriptome of juvenile and mature Eremophila serrulata (A.DC.) Druce (Scrophulariaceae) leaves was sequenced and a transcript library was generated. We report on the first transcriptomic dataset of an Eremophila plant. IlluminaMiSeq sequencing (2 × 300 bp) revealed 7,093,266 paired reads, which could be assembled to 34,505 isogroups. To enable detection of terpene biosynthetic genes, leaves were separately treated with methyl jasmonate, a well-documented inducer of plant secondary metabolites. In total, 21 putative terpene synthase genes were detected in the transcriptome data. Two terpene synthase isoenzymatic genes, termed ES01 and ES02, were successfully expressed in E. coli. The resulting proteins catalyzed the conversion of geranyl pyrophosphate, the universal substrate of monoterpene synthases to myrcene and Z-(b)-ocimene, respectively. The transcriptomic data and the discovery of the first terpene synthases from Eremophila serrulata are the initial step for the understanding of the terpene metabolism in this medicinally important plant genus. Copyright © 2017 Elsevier Ltd. All rights reserved.

Asymmetric Synthesis of (R)-1-Alkyl Substituted Tetrahydro-ß-carbolines Catalyzed by Strictosidine Synthases.

PubMed

Pressnitz, Desiree; Fischereder, Eva-Maria; Pletz, Jakob; Kofler, Christina; Hammerer, Lucas; Hiebler, Katharina; Lechner, Horst; Richter, Nina; Eger, Elisabeth; Kroutil, Wolfgang

2018-05-31

Stereoselective methods for the synthesis of tetrahydro-ß-carbolines are of significant interest due to the broad spectrum of biological activity of the target molecules. In the plant kingdom strictosidine synthases catalyze the C-C coupling via a Pictet-Spengler reaction of tryptamine and secologanin to exclusively form the (S)-configured tetrahydro-ß-carboline (S)-strictosidine. Investigating the biocatalytic Pictet-Spengler reaction of tryptamine with small-molecular-weight aliphatic aldehydes revealed that the strictosidine synthases gave unexpectedly access to the (R)-configured product. Developing an efficient expression method of the catalyst allowed the preparative transformation of various aldehydes giving the products with up to >98% ee. With this tool in hand a chemoenzymatic two-step synthesis of (R)-harmicine was achieved giving (R)-harmicine in 67% overall yield in optically pure form. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

S-Acylation of the cellulose synthase complex is essential for its plasma membrane localization.

PubMed

Kumar, Manoj; Wightman, Raymond; Atanassov, Ivan; Gupta, Anjali; Hurst, Charlotte H; Hemsley, Piers A; Turner, Simon

2016-07-08

Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. How interactions between membranes and the CSC are regulated is currently unknown. Here, we demonstrate that all catalytic subunits of the CSC, known as cellulose synthase A (CESA) proteins, are S-acylated. Analysis of Arabidopsis CESA7 reveals four cysteines in variable region 2 (VR2) and two cysteines at the carboxy terminus (CT) as S-acylation sites. Mutating both the VR2 and CT cysteines permits CSC assembly and trafficking to the Golgi but prevents localization to the plasma membrane. Estimates suggest that a single CSC contains more than 100 S-acyl groups, which greatly increase the hydrophobic nature of the CSC and likely influence its immediate membrane environment. Copyright © 2016, American Association for the Advancement of Science.

Multiple Glycogen-binding Sites in Eukaryotic Glycogen Synthase Are Required for High Catalytic Efficiency toward Glycogen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baskaran, Sulochanadevi; Chikwana, Vimbai M.; Contreras, Christopher J.

2012-12-10

Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V{sub max}/S{sub 0.5} for glycogen by 40- and 70-fold,more » respectively. Combined mutation of site-1 and site-2 decreased the V{sub max}/S{sub 0.5} for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells ({Delta}gsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a 'toehold mechanism,' keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.« less

Redirection of the Reaction Specificity of a Thermophilic Acetolactate Synthase toward Acetaldehyde Formation

PubMed Central

Cheng, Maria; Yoshiyasu, Hayato; Okano, Kenji; Ohtake, Hisao; Honda, Kohsuke

2016-01-01

Acetolactate synthase and pyruvate decarboxylase are thiamine pyrophosphate-dependent enzymes that convert pyruvate into acetolactate and acetaldehyde, respectively. Although the former are encoded in the genomes of many thermophiles and hyperthermophiles, the latter has been found only in mesophilic organisms. In this study, the reaction specificity of acetolactate synthase from Thermus thermophilus was redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Error-prone PCR and mutant library screening led to the identification of a quadruple mutant with 3.1-fold higher acetaldehyde-forming activity than the wild-type. Site-directed mutagenesis experiments revealed that the increased activity of the mutant was due to H474R amino acid substitution, which likely generated two new hydrogen bonds near the thiamine pyrophosphate-binding site. These hydrogen bonds might result in the better accessibility of H+ to the substrate-cofactor-enzyme intermediate and a shift in the reaction specificity of the enzyme. PMID:26731734

Glycogen synthase kinase-3 as drug target: from wallflower to center of attention.

PubMed

Van Wauwe, Jean; Haefner, Burkhard

2003-11-01

Some 20 years ago, glycogen synthase kinase-3 (GSK-3) was categorized as one of several protein kinases that could phosphorylate glycogen synthase and regulate the glucose metabolism pathway. Today, GSK-3 is being identified as a ubiquitous serine/threonine protein kinase that participates in a multitude of cellular processes, ranging from cell membrane-to-nucleus signaling, gene transcription, translation, cytoskeletal organization to cell cycle progression and survival. Two functional aspects make GSK-3 a peculiar kinase: its activity is constitutive and downregulated after cell activation by phosphorylation or interaction with inhibitory proteins, and the enzyme prefers substrates that are specifically prepared, that is prephosphorylated, by other kinases. Its pleiotropic but unique activities have made GSK-3 a much sought-after target for the treatment of prevalent human diseases such as type 2 diabetes and Alzheimer's disease. Recent drug discovery efforts have identified small-molecule, orally active inhibitors of GSK-3. This accomplishment may represent the first step toward the development of novel therapeutic agents.

Expression Patterns, Activities and Carbohydrate-Metabolizing Regulation of Sucrose Phosphate Synthase, Sucrose Synthase and Neutral Invertase in Pineapple Fruit during Development and Ripening

PubMed Central

Zhang, Xiu-Mei; Wang, Wei; Du, Li-Qing; Xie, Jiang-Hui; Yao, Yan-Li; Sun, Guang-Ming

2012-01-01

Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion. PMID:22949808

CESA TRAFFICKING INHIBITOR inhibits cellulose deposition and interferes with the trafficking of cellulose synthase complexes and their associated proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1.

PubMed

Worden, Natasha; Wilkop, Thomas E; Esteve, Victor Esteva; Jeannotte, Richard; Lathe, Rahul; Vernhettes, Samantha; Weimer, Bart; Hicks, Glenn; Alonso, Jose; Labavitch, John; Persson, Staffan; Ehrhardt, David; Drakakaki, Georgia

2015-02-01

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls. © 2015 American Society of Plant Biologists. All Rights Reserved.

Redox regulation and reaction mechanism of human cystathionine-beta-synthase: a PLP-dependent hemesensor protein.

PubMed

Banerjee, Ruma; Zou, Cheng-Gang

2005-01-01

Cystathionine beta-synthase in mammals lies at a pivotal crossroad in methionine metabolism directing flux toward cysteine synthesis and catabolism. The enzyme exhibits a modular organization and complex regulation. It catalyzes the beta-replacement of the hydroxyl group of serine with the thiolate of homocysteine and is unique in being the only known pyridoxal phosphate-dependent enzyme that also contains heme b as a cofactor. The heme functions as a sensor and modulates enzyme activity in response to redox change and to CO binding. Mutations in this enzyme are the single most common cause of hereditary hyperhomocysteinemia. Elucidation of the crystal structure of a truncated and highly active form of the human enzyme containing the heme- and pyridoxal phosphate binding domains has afforded a structural perspective on mechanistic and mutation analysis studies. The C-terminal regulatory domain containing two CBS motifs exerts intrasteric regulation and binds the allosteric activator, S-adenosylmethionine. Studies with mammalian cells in culture as well as with animal models have unraveled multiple layers of regulation of cystathionine beta-synthase in response to redox perturbations and reveal the important role of this enzyme in glutathione-dependent redox homestasis. This review discusses the recent advances in our understanding of the structure, mechanism, and regulation of cystathionine beta-synthase from the perspective of its physiological function, focusing on the clinically relevant human enzyme.

Activation and inhibition of CTP synthase from Trypanosoma brucei, the causative agent of African sleeping sickness.

PubMed

Steeves, Craig H; Bearne, Stephen L

2011-09-15

CTP Synthase from Trypanosoma brucei (TbCTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the development of antiprotozoal agents. GTP activates glutamine-dependent CTP formation catalyzed by TbCTPS at concentrations below 0.2 mM, but inhibits this activity at concentrations above 0.2 mM. TbCTPS catalyzes ammonia-dependent CTP formation, which is inhibited by purine derivatives such as GTP, guanosine, caffeine, and uric acid with IC(50) values of 460, 380, 480, and 100 μM, respectively. These observations suggest that the purine ring may serve as a useful scaffold for the development of inhibitors of trypanosomal CTP synthase. Copyright © 2011 Elsevier Ltd. All rights reserved.

Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase: In Vivo and In Vitro Studies of Enzymatic Activity and Substrate Specificity

PubMed Central

Chuah, Jo-Ann; Tomizawa, Satoshi; Yamada, Miwa; Tsuge, Takeharu; Doi, Yoshiharu

2013-01-01

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases. PMID:23584780

Treatment of rats with glucagon or mannoheptulose increases mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity and decreases succinyl-CoA content in liver.

PubMed Central

Quant, P A; Tubbs, P K; Brand, M D

1989-01-01

1. The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (EC 4.1.3.5) in extracts of rapidly frozen rat livers was doubled in animals treated in various ways to increase ketogenic flux. 2. Some 90% of the activity measured was mitochondrial, and changes in mitochondrial activity dominated changes in total enzyme activity. 3. The elevated HMG-CoA synthase activities persisted throughout the isolation of liver mitochondria. 4. Intramitochondrial succinyl-CoA content was lower in whole liver homogenates and in mitochondria isolated from animals treated with glucagon or mannoheptulose. 5. HMG-CoA synthase activity in mitochondria from both ox and rat liver was negatively correlated with intramitochondrial succinyl-CoA levels when these were manipulated artificially. Under these conditions, the differences between mitochondria from control and hormone-treated rats were abolished. 6. These findings show that glucagon can decrease intramitochondrial succinyl-CoA concentration, and that this in turn can regulate mitochondrial HMG-CoA synthase. They support the hypothesis that the formation of ketone bodies from acetyl-CoA may be regulated by the extent of succinylation of mitochondrial HMG-CoA synthase. PMID:2573345

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues.

PubMed

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia; Calzia, Daniela; Beitia, Maider; Morelli, Alessandro; Rosano, Camillo

2017-01-22

F 1 F o -ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H + flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H + within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F 1 and F O domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo. Copyright © 2016 Elsevier Inc. All rights reserved.

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia

F{sub 1}F{sub o}-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H{sup +} flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H{sup +} within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F{sub 1} andmore » F{sub O} domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo.« less

MULTI-ANALYTE CHEMISTRY METHODS FOR PESTICIDES WHICH ARE ACETOLACTATE SYNTHASE (ALS) INHIBITORS IN SOIL

EPA Science Inventory

A joint EPA/state/industry working group has developed several multi-analyte methods to analyze soils for low ppb (parts per billion) levels of herbicides (such as sulfonylureas, imidazolinones, and sulfonamides) that are acetolactate synthase (ALS) inhibitors and may cause phyto...

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis)

USGS Publications Warehouse

Zak, Megan A.; Regish, Amy M.; McCormick, Stephen; Manzon, Richard G.

2017-01-01

Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19 °C) or below (8 °C) the thermal optimum (13 °C) and exposure to exogenous thyroid hormone (60 µg T4/g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation.

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis).

PubMed

Zak, Megan A; Regish, Amy M; McCormick, Stephen D; Manzon, Richard G

2017-06-01

Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19°C) or below (8°C) the thermal optimum (13°C) and exposure to exogenous thyroid hormone (60µg T 4 /g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation. Copyright © 2017 Elsevier Inc. All rights reserved.

A multigene family related to chitin synthase genes of yeast in the opportunistic pathogen Aspergillus fumigatus.

PubMed

Mellado, E; Aufauvre-Brown, A; Specht, C A; Robbins, P W; Holden, D W

1995-02-06

Two approaches were used to isolate fragments of chitin synthase genes from the opportunistic human pathogen Aspergillus fumigatus. Firstly, regions of amino acid conservation in chitin synthases of Saccharomyces cerevisiae were used to design degenerate primers for amplification of portions of related genes, and secondly, a segment of the S. cerevisiae CSD2 gene was used to screen an A. fumigatus lambda genomic DNA library. the polymerase chain reaction (PCR)-based approach led to the identification of five different genes, designated chsA, chsB, chsC, chsD and chsE. chsA, chsB, and chsC fall into Classes I, II and III of the 'zymogen type' chitin synthases, respectively. The chsD fragment has approximately 35% amino acid sequence identity to both the zymogen type genes and the non-zymogen type CSD2 gene. chsF appears to be a homologue of CSD2, being 80% identical to CSD2 over 100 amino acids. An unexpected finding was the isolation by heterologous hybridization of another gene (chsE), which also has strong sequence similarity (54% identity at the amino acid level over the same region as chsF) to CSD2. Reverse transcriptase-PCR was used to show that each gene is expressed during hyphal growth in submerged cultures.

Crystallization and preliminary X-ray diffraction studies of polyketide synthase-1 (PKS-1) from Cannabis sativa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taguchi, Chiho; Quantum Beam Science Directorate, Japan Atomic Energy Agency; Taura, Futoshi

Polyketide synthase-1 from C. sativa has been crystallized. The crystal diffracted to 1.55 Å resolution with sufficient quality for further structure determination. Polyketide synthase-1 (PKS-1) is a novel type III polyketide synthase that catalyzes the biosynthesis of hexanoyl triacetic acid lactone in Cannabis sativa (Mexican strain). PKS-1 was overproduced in Escherichia coli, purified and finally crystallized in two different space groups. The crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M calcium acetate and 20%(w/v) polyethylene glycol 3350 diffracted to 1.65 Å resolution and belonged to space group P1, with unit-cell parameters a = 54.3, b =more » 59.3, c = 62.6 Å, α = 69, β = 81, γ = 80°. Another crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M sodium chloride and 20%(w/v) polyethylene glycol 3350 diffracted to 1.55 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 54.3, b = 110, c = 130 Å. These data will enable us to determine the crystal structure of PKS-1.« less

Over-expression of a grape stilbene synthase gene in tomato induces parthenocarpy and causes abnormal pollen development.

PubMed

Ingrosso, Ilaria; Bonsegna, Stefania; De Domenico, Stefania; Laddomada, Barbara; Blando, Federica; Santino, Angelo; Giovinazzo, Giovanna

2011-10-01

A novel strategy to induce parthenocarpy in tomato fruits by the induction of resveratrol biosynthesis in flower tissues was exploited. Two transgenic tomato lines were considered: a higher resveratrol-producing (35SS) line, constitutively expressing a grape stilbene synthase cDNA, and a lower resveratrol-producing (LoxS) line, expressing stilbene synthase under a fruit-specific promoter. The expression of the stilbene synthase gene affected flavonoid metabolism in a different manner in the transgenic lines, and in one of these, the 35SS line, resulted in complete male sterility. Resveratrol was synthesised either in 35SS or LoxS tomato flowers, at an even higher extent (about 8-10 times) in the former line. We further investigated whether stilbene synthase expression may have resulted in impaired naringenin accumulation during flower development. In the 35SS flowers, naringenin was significantly impaired by about 50%, probably due to metabolic competition. Conversely, the amount of glycosylated flavonols increased in transgenic flowers, thereby excluding the diminished production of flavonols as a reason for parthenocarpy in tomato. We further investigated whether resveratrol synthesis may have resulted changes to pollen structure. Microscopic observations revealed the presence of few and abnormal flake-like pollen grains in 35SS flowers with no germination capability. Finally, the analysis of coumaric and ferulic acids, the precursors of lignin and sporopollenin biosynthesis, revealed significant depletion of these compounds, therefore suggesting an impairment in structural compounds as a reason for pollen ablation. These overall outcomes, to the best of our knowledge, reveal for the first time the major role displayed by resveratrol synthesis on parthenocarpy in tomato fruits. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Terpene synthases from Cannabis sativa

PubMed Central

Booth, Judith K.; Page, Jonathan E.

2017-01-01

Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety ‘Finola’ revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of ‘Finola’ resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties. PMID:28355238

Molecular Dynamic Simulation and Inhibitor Prediction of Cysteine Synthase Structured Model as a Potential Drug Target for Trichomoniasis

PubMed Central

Singh, Satendra; Singh, Atul Kumar; Gautam, Budhayash

2013-01-01

In our presented research, we made an attempt to predict the 3D model for cysteine synthase (A2GMG5_TRIVA) using homology-modeling approaches. To investigate deeper into the predicted structure, we further performed a molecular dynamics simulation for 10 ns and calculated several supporting analysis for structural properties such as RMSF, radius of gyration, and the total energy calculation to support the predicted structured model of cysteine synthase. The present findings led us to conclude that the proposed model is stereochemically stable. The overall PROCHECK G factor for the homology-modeled structure was −0.04. On the basis of the virtual screening for cysteine synthase against the NCI subset II molecule, we present the molecule 1-N, 4-N-bis [3-(1H-benzimidazol-2-yl) phenyl] benzene-1,4-dicarboxamide (ZINC01690699) having the minimum energy score (−13.0 Kcal/Mol) and a log P value of 6 as a potential inhibitory molecule used to inhibit the growth of T. vaginalis infection. PMID:24073401

Exploiting CELLULOSE SYNTHASE (CESA) Class Specificity to Probe Cellulose Microfibril Biosynthesis1[OPEN

PubMed Central

Mishra, Laxmi; Carr, Paul; Gardner, Peter

2018-01-01

Cellulose microfibrils are the basic units of cellulose in plants. The structure of these microfibrils is at least partly determined by the structure of the cellulose synthase complex. In higher plants, this complex is composed of 18 to 24 catalytic subunits known as CELLULOSE SYNTHASE A (CESA) proteins. Three different classes of CESA proteins are required for cellulose synthesis and for secondary cell wall cellulose biosynthesis these classes are represented by CESA4, CESA7, and CESA8. To probe the relationship between CESA proteins and microfibril structure, we created mutant cesa proteins that lack catalytic activity but retain sufficient structural integrity to allow assembly of the cellulose synthase complex. Using a series of Arabidopsis (Arabidopsis thaliana) mutants and genetic backgrounds, we found consistent differences in the ability of these mutant cesa proteins to complement the cellulose-deficient phenotype of the cesa null mutants. The best complementation was observed with catalytically inactive cesa4, while the equivalent mutation in cesa8 exhibited significantly lower levels of complementation. Using a variety of biophysical techniques, including solid-state nuclear magnetic resonance and Fourier transform infrared microscopy, to study these mutant plants, we found evidence for changes in cellulose microfibril structure, but these changes largely correlated with cellulose content and reflected differences in the relative proportions of primary and secondary cell walls. Our results suggest that individual CESA classes have similar roles in determining cellulose microfibril structure, and it is likely that the different effects of mutating members of different CESA classes are the consequence of their different catalytic activity and their influence on the overall rate of cellulose synthesis. PMID:29523715

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

PubMed Central

Salmon, Melissa; Thimmappa, Ramesha B.; Minto, Robert E.; Melton, Rachel E.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne