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Rodney Croteau - One of the best experts on this subject based on the ideXlab platform.
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inhibition of monoterpene cyclases by inert analogues of Geranyl Diphosphate and linalyl Diphosphate
Archives of Biochemistry and Biophysics, 2007Co-Authors: Frank Karp, Bryce Assink, Robert M Coates, Yuxin Zhao, Bindu Santhamma, Rodney CroteauAbstract:Abstract The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from Geranyl Diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl Diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluoroGeranyl Diphosphate, (±)-2-fluorolinalyl Diphosphate, and (3 R )- and (3 S )-homolinalyl Diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl Diphosphates) were prepared, and compared to the previously described substrate analogue 3-azaGeranyl Diphosphate (3-aza-2,3-dihydroGeranyl Diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (–)-limonene synthase and (+)-bornyl Diphosphate synthase. Although these enantioselective synthases readily distinguished between (3 R )- and (3 S )-homolinalyl Diphosphates, both of which were more effective inhibitors than was 3-azaGeranyl Diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.
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Structure of limonene synthase, a simple model for terpenoid cyclase catalysis
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: David Hyatt, Rodney Croteau, Robert M Coates, Yuxin Zhao, Bindu Santhamma, Buhyun Youn, Chulhee KangAbstract:The crystal structure of (4S)-limonene synthase from Mentha spic ata, a metal ion-dependent monoterpene cyclase that catalyzes the coupled isomerization and cyclization of Geranyl Diphosphate, is reported at 2.7-A; resolution in two forms liganded to the substrate and intermediate analogs, 2-fluoroGeranyl Diphosphate and 2-fluorolinalyl Diphosphate, respectively. The implications of these findings are described for domain interactions in the homodimer and for changes in Diphosphate–metal ion coordination and substrate binding conformation in the course of the multistep reaction.
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organization of monoterpene biosynthesis in mentha immunocytochemical localizations of Geranyl Diphosphate synthase limonene 6 hydroxylase isopiperitenol dehydrogenase and pulegone reductase
Plant Physiology, 2004Co-Authors: Glenn W Turner, Rodney CroteauAbstract:We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) Geranyl Diphosphate synthase, spearmint (Mentha spicata) (−)-(4S)-limonene-6-hydroxylase, peppermint (−)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of Geranyl Diphosphate synthase occurred within secretory cell leucoplasts, (−)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (−)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.
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heteromeric Geranyl Diphosphate synthase from mint construction of a functional fusion protein and inhibition by bisphosphonate substrate analogs
Archives of Biochemistry and Biophysics, 2004Co-Authors: Charles Burke, Karin Klettke, Rodney CroteauAbstract:Abstract Geranyl Diphosphate synthase catalyzes the condensation of dimethylallyl Diphosphate (C 5 ) with isopentenyl Diphosphate (C 5 ) to produce Geranyl Diphosphate (C 10 ), the essential precursor of monoterpenes. The enzyme from peppermint and spearmint ( Mentha × piperita and Mentha spicata , respectively) functions as a heterodimer or heterotetramer consisting of a 40 kDa subunit and 33 kDa subunit. The DNAs encoding each subunit were joined with different sized linkers and in both possible orders, and expressed in Escherichia coli to yield the corresponding fused protein. The properties of the recombinant fused version, in which the small subunit was followed by the large subunit with a 10 amino acid linker, resembled those of the native heteromeric enzyme in kinetics, product chain-length specificity, and architecture, and this form thus provided a suitable single gene transcript for biotechnological purposes. Bisphosphonate substrate analogs of the type that inhibit farnesyl Diphosphate synthase (C 15 ) and GeranylGeranyl Diphosphate synthase (C 20 ) also inhibited the fused Geranyl Diphosphate synthase, apparently by interacting at both the allylic and homoallylic co-substrate binding sites. The results of inhibition studies, along with the previously established role of the small subunit and related mutagenesis experiments, suggest that Geranyl Diphosphate synthase employs a different mechanism for chain-length determination than do other short-chain prenyltransferases.
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Mechanism of monoterpene cyclization: stereochemistry of the transformation of noncyclizable substrate analogs by recombinant (−)-limonene synthase, (+)-bornyl Diphosphate synthase, and (−)-pinene synthase
Journal of Molecular Catalysis B-enzymatic, 2002Co-Authors: Wilfried Schwab, David C Williams, Rodney CroteauAbstract:All monoterpene cyclases investigated to date are capable of overcoming the topological impediment to direct cyclization of the universal, acyclic C10 intermediate of isoprenoid biosynthesis Geranyl Diphosphate. Although strong suggestive evidence has been accumulated for the intermediary linalyl Diphosphate in cyclase catalysis, all previous efforts to directly observe this product at the mandatory isomerization step have failed. (-)-4S-Limonene synthase from spearmint (Mentha spicata), (+)-bornyl Diphosphate synthase from sage (Salvia officinalis), and (-)-pinene synthase from grand fir (Abies grandis) have been expressed in Escherichia coli and the recombinant enzymes have been isolated and purified. These enzymes were examined with the noncyclizable substrate analogs 6,7-dihydroGeranyl Diphosphate and 2,3-methanoGeranyl Diphosphate to gain insight into the normally cryptic isomerization step of the reaction sequence. The analogs were catalytically active, affording acyclic olefins and alcohols as products. Chiral phase gas chromatography and mass spectrometry analysis provided evidence that the normal cyclization of Geranyl Diphosphate by (-)-4S-limonene synthase and by (-)-pinene synthase proceeds via preliminary isomerization to the bound tertiary intermediate 3S-linalyl Diphosphate, whereas the cyclization catalyzed by (+)-bornyl Diphosphate synthase proceeds via the intermediate 3R-linalyl Diphosphate.
Jonathan Gershenzon - One of the best experts on this subject based on the ideXlab platform.
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Overexpression of an Isoprenyl Diphosphate Synthase in Spruce Leads to Unexpected Terpene Diversion Products That Function in Plant Defense
Plant Physiology, 2013Co-Authors: Raimund Nagel, Jonathan Gershenzon, Aileen Berasategui, Christian Paetz, Axel SchmidtAbstract:Spruce (Picea spp.) and other conifers employ terpenoid-based oleoresin as part of their defense against herbivores and pathogens. The short-chain isoprenyl Diphosphate synthases (IDS) are situated at critical branch points in terpene biosynthesis, producing the precursors of the different terpenoid classes. To determine the role of IDS and to create altered terpene phenotypes for assessing the defensive role of terpenoids, we overexpressed a bifunctional spruce IDS, a Geranyl Diphosphate and GeranylGeranyl Diphosphate synthase in white spruce (Picea glauca) saplings. While transcript level (350-fold), enzyme activity level (7-fold), and in planta Geranyl Diphosphate and GeranylGeranyl Diphosphate levels (4- to 8-fold) were significantly increased in the needles of transgenic plants, there was no increase in the major monoterpenes and diterpene acids of the resin and no change in primary isoprenoids, such as sterols, chlorophylls, and carotenoids. Instead, large amounts of GeranylGeranyl fatty acid esters, known from various gymnosperm and angiosperm plant species, accumulated in needles and were shown to act defensively in reducing the performance of larvae of the nun moth (Lymantria monacha), a conifer pest in Eurasia. These results show the impact of overexpression of an IDS and the defensive role of an unexpected accumulation product of terpenoid biosynthesis with the potential for a broader function in plant protection.
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Characterization of three novel isoprenyl Diphosphate synthases from the terpenoid rich mango fruit
Plant Physiology and Biochemistry, 2013Co-Authors: Ram Kulkarni, Axel Schmidt, Jonathan Gershenzon, Sagar Subhash Pandit, Hemangi G. Chidley, Raimund Nagel, Keshav H. Pujari, Ashok K. Giri, Vidya S. GuptaAbstract:Mango (cv. Alphonso) is popular due to its highly attractive, terpenoid-rich flavor. Although Alphonso is clonally propagated, its fruit-flavor composition varies when plants are grown in different geo-climatic zones. Isoprenyl Diphosphate synthases catalyze important branch-point reactions in terpenoid biosynthesis, providing precursors for common terpenoids such as volatile terpenes, sterols and carotenoids. Two Geranyl Diphosphate synthases and a farnesyl Diphosphate synthase were isolated from Alphonso fruits, cloned for recombinant expression and found to produce the respective products. Although, one of the Geranyl Diphosphate synthases showed high sequence similarity to the GeranylGeranyl Diphosphate synthases, it did not exhibit GeranylGeranyl Diphosphate synthesizing activity. When modeled, this Geranyl Diphosphate synthase and farnesyl Diphosphate synthase structures were found to be homologous with the reference structures, having all the catalytic side chains appropriately oriented. The optimum temperature for both the Geranyl Diphosphate synthases was 40 � C and that for farnesyl Diphosphate synthase was 25 � C. This finding correlated well with the dominance of monoterpenes in comparison to sesquiterpenes in the fruits of Alphonso mango in which the mesocarp temperature is higher during ripening than development. The absence of activity of these enzymes with the divalent metal ion other than Mg 2þ indicated their adaptation to the Mg 2þ rich mesocarp. The typical
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Nonradioactive assay for detecting isoprenyl Diphosphate synthase activity in crude plant extracts using liquid chromatography coupled with tandem mass spectrometry
Analytical Biochemistry, 2012Co-Authors: Raimund Nagel, Jonathan Gershenzon, Axel SchmidtAbstract:Terpenoids form the largest class of plant metabolites involved in primary and secondary metabolism. Isoprenyl Diphosphate synthases (IDSs) catalyze the condensation of the C5 terpenoid building blocks, isopentenyl Diphosphate and dimethylallyl Diphosphate, to form Geranyl Diphosphate (C10), farnesyl Diphosphate (C15), and GeranylGeranyl Diphosphate (C20). These branch point reactions control the flow of metabolites that act as precursors to each of the major terpene classes—monoterpenes, sequiterpenes, and diterpenes, respectively. Thus accurate and easily performed assays of IDS enzyme activity are critical to increase our knowledge about the regulation of terpene biosynthesis. Here we describe a new and sensitive nonradioactive method for carrying out IDS assays using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) to detect the short-chain prenyl Diphosphate products directly without dephosphorylation. Furthermore, we were able to separate cisoid and transoid isomers of both C10 enzyme products (Geranyl Diphosphate and neryl Diphosphate) and three C15 products [(E,E)-, (Z,E)-, and (Z,Z)-farnesyl Diphosphate]. By applying the method to crude protein extracts from various organs of Arabidopsis thaliana, Nicotiana attenuata, Populus trichocarpa, and Picea abies, we could deter
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a bifunctional Geranyl and GeranylGeranyl Diphosphate synthase is involved in terpene oleoresin formation in picea abies
Plant Physiology, 2010Co-Authors: Axel Schmidt, Betty Wachtler, Ulrike Temp, Trygve Krekling, Armand Seguin, Jonathan GershenzonAbstract:The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin composed chiefly of monoterpenes (C10) and diterpenes (C20). An important group of enzymes in oleoresin biosynthesis are the short-chain isoprenyl Diphosphate synthases that produce Geranyl Diphosphate (C10), farnesyl Diphosphate (C15), and GeranylGeranyl Diphosphate (C20) as precursors of different terpenoid classes. We isolated a gene from P. abies via a homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl Diphosphate synthase making an unusual mixture of two products, Geranyl Diphosphate (C10) and GeranylGeranyl Diphosphate (C20). This bifunctionality was confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this isoprenyl Diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to single-product conifer Geranyl Diphosphate and GeranylGeranyl Diphosphate synthases. Its catalytic properties and reaction mechanism resemble those of conifer GeranylGeranyl Diphosphate synthases, except that significant quantities of the intermediate Geranyl Diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product Geranyl Diphosphate and GeranylGeranyl Diphosphate synthases, specific amino acid residues were identified that alter the relative composition of Geranyl to GeranylGeranyl Diphosphate.
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cloning and characterization of two different types of Geranyl Diphosphate synthases from norway spruce picea abies
Phytochemistry, 2008Co-Authors: Axel Schmidt, Jonathan GershenzonAbstract:Geranyl Diphosphate (GPP), the universal precursor of monoterpenes, is formed from isopentenyl Diphosphate and dimethylallyl Diphosphate by the action of Geranyl Diphosphate synthase, one of the key branchpoint enzymes of terpene biosynthesis. Three types of GPP synthase can be distinguished in plants based on sequence similarity and subunit architecture, but until now individual species have been reported to contain only one of these types. Here we show that the conifer, Norway spruce (Picea abies), contains two different types of GPP synthase belonging to two separate groups of homodimeric proteins. One enzyme, designated PaIDS2 (P. abies isoprenyl Diphosphate synthase 2), has high sequence similarity to other gymnosperm GPP synthases. It produces solely GPP in in vitro assays after expression in Escherichia coli and likely participates in monoterpene biosynthesis accompanying induced oleoresin formation, based on dramatic increases in transcript level after methyl jasmonate application. The other enzyme, designated PaIDS3, has highest similarity to the previously reported Arabidopsis thaliana GPP synthase and several other angiosperm sequences, and is not associated with induced oleoresin formation in Norway spruce. In vitro assay of this protein and one encoded by a similar gene sequence from Quercus robur gave substantial amounts of the larger prenyl Diphosphates, FPP and GGPP, in addition to GPP. Hence these proteins may not be involved in monoterpene formation and could conceivably form products in addition to GPP in planta.
Charles Burke - One of the best experts on this subject based on the ideXlab platform.
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heteromeric Geranyl Diphosphate synthase from mint construction of a functional fusion protein and inhibition by bisphosphonate substrate analogs
Archives of Biochemistry and Biophysics, 2004Co-Authors: Charles Burke, Karin Klettke, Rodney CroteauAbstract:Abstract Geranyl Diphosphate synthase catalyzes the condensation of dimethylallyl Diphosphate (C 5 ) with isopentenyl Diphosphate (C 5 ) to produce Geranyl Diphosphate (C 10 ), the essential precursor of monoterpenes. The enzyme from peppermint and spearmint ( Mentha × piperita and Mentha spicata , respectively) functions as a heterodimer or heterotetramer consisting of a 40 kDa subunit and 33 kDa subunit. The DNAs encoding each subunit were joined with different sized linkers and in both possible orders, and expressed in Escherichia coli to yield the corresponding fused protein. The properties of the recombinant fused version, in which the small subunit was followed by the large subunit with a 10 amino acid linker, resembled those of the native heteromeric enzyme in kinetics, product chain-length specificity, and architecture, and this form thus provided a suitable single gene transcript for biotechnological purposes. Bisphosphonate substrate analogs of the type that inhibit farnesyl Diphosphate synthase (C 15 ) and GeranylGeranyl Diphosphate synthase (C 20 ) also inhibited the fused Geranyl Diphosphate synthase, apparently by interacting at both the allylic and homoallylic co-substrate binding sites. The results of inhibition studies, along with the previously established role of the small subunit and related mutagenesis experiments, suggest that Geranyl Diphosphate synthase employs a different mechanism for chain-length determination than do other short-chain prenyltransferases.
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Geranyl Diphosphate synthase from abies grandis cdna isolation functional expression and characterization
Archives of Biochemistry and Biophysics, 2002Co-Authors: Charles Burke, Rodney CroteauAbstract:Abstract Geranyl Diphosphate synthase catalyzes the condensation of dimethylallyl Diphosphate and isopentenyl Diphosphate to generate Geranyl Diphosphate, the essential precursor of monoterpene biosynthesis. Using GeranylGeranyl Diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus GeranylGeranyl Diphosphate synthase (>66%), were isolated from a grand fir ( Abies grandis ) cDNA library. When expressed in Escherichia coli , three of the recombinant enzymes produced Geranyl Diphosphate and one produced GeranylGeranyl Diphosphate as the dominant product when supplied with isopentenyl Diphosphate and dimethylallyl Diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific Geranyl Diphosphate synthase, in that it accepted only dimethylallyl Diphosphate as the allylic cosubstrate and it produced exclusively Geranyl Diphosphate as product, with a k cat of 1.8 s −1 . Gel filtration experiments performed on the recombinant Geranyl Diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric Geranyl Diphosphate synthase of mint.
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interaction with the small subunit of Geranyl Diphosphate synthase modifies the chain length specificity of GeranylGeranyl Diphosphate synthase to produce Geranyl Diphosphate
Journal of Biological Chemistry, 2002Co-Authors: Charles Burke, Rodney CroteauAbstract:Abstract Geranyl Diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl Diphosphate synthase and GeranylGeranyl Diphosphate synthase, that catalyzes the specific formation, from C5 units, of the respective C10, C15, and C20 precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl Diphosphate synthase and GeranylGeranyl Diphosphate synthase, which are homodimers, Geranyl Diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56–75%) with GeranylGeranyl Diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl Diphosphate synthases; yet it is absolutely required for Geranyl Diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha Geranyl Diphosphate synthase small subunit with the phylogenetically distant GeranylGeranyl Diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated Geranyl Diphosphate as product in each case. These results indicate that the Geranyl Diphosphate synthase small subunit is capable of modifying the chain length specificity of GeranylGeranyl Diphosphate synthase (but not, apparently, farnesyl Diphosphate synthase) to favor the production of C10 chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both Geranyl Diphosphate synthase and GeranylGeranyl Diphosphate synthase.
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Geranyl Diphosphate synthase cloning expression and characterization of this prenyltransferase as a heterodimer
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Charles Burke, Mark R Wildung, Rodney CroteauAbstract:Geranyl Diphosphate synthase, which catalyzes the condensation of dimethylallyl Diphosphate and isopentenyl Diphosphate to Geranyl Diphosphate, the key precursor of monoterpene biosynthesis, was purified from isolated oil glands of spearmint. Peptide fragments generated from the pure proteins of 28 and 37 kDa revealed amino acid sequences that matched two cDNA clones obtained by random screening of a peppermint-oil gland cDNA library. The deduced sequences of both proteins showed some similarity to existing prenyltransferases, and both contained a plastid-targeting sequence. Expression of each cDNA individually yielded no detectable prenyltransferase activity; however, coexpression of the two together produced functional Geranyl Diphosphate synthase. Antibodies raised against each protein were used to demonstrate that both subunits were required to produce catalytically active native and recombinant enzymes, thus confirming that Geranyl Diphosphate synthase is a heterodimer.
C. Ambid - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of Geranyl Diphosphate synthase from vitis vinifera l cv muscat de frontignan cell cultures
Plant Physiology, 1993Co-Authors: Marc Clastre, Gilles Feron, Brigitte Bantignies, Eric Soler, C. AmbidAbstract:A Geranyl Diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of Geranyl Diphosphate from dimethylallyl Diphosphate and isopentenyl Diphosphate, was isolated from Vitis vinifera L. cv Muscat de Frontignan cell cultures. Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels. The enzyme formed only Geranyl Diphosphate as a product. In all cases, neither neryl Diphosphate, the cis isomer, nor farnesyl Diphosphate was detected. The enzyme showed a native molecular mass of 68 [plus or minus] 5 kD as determined by gel permeation. On sodium dodecyl sulfate polyacrylamide gels, Geranyl Diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 [plus or minus] 2 kD. Michaelis constants for isopentenyl Diphosphate and dimethylallyl Diphosphate were 8.5 and 56.8 [mu]M, respectively. The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100. Inorganic pyrophosphate, aminophenylethyl Diphosphate, and Geranyl Diphosphate had inhibitory effects on the enzyme.
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Evidence for a Geranyl-Diphosphate synthase located within the plastids of Vitis vinifera L. cultivated in vitro
Planta, 1992Co-Authors: E. Soler, R. Dargent, M. Gleizes, Marc Clastre, Gilles Feron, C. AmbidAbstract:Intact plastids from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan, free of detectable contamination by other particles as judged by the distribution of organelle-specific marker enzymes and by electron microscopy, exhibit Geranyl-Diphosphate synthase activity (EC 2.5.1.1). This synthase activity remains stable after tryptic digestion of unlysed organelles and is enhanced by plastid disruption. We conclude that the enzyme is located within the organelle. The possibility of an iso pentenyl Diphosphate/dimethylallyl Diphosphate translocating system which would play a major role in the regulation of monoterpene metabolism is discussed.
Mark D Distefano - One of the best experts on this subject based on the ideXlab platform.
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site specific labeling of proteins and peptides with trans cyclooctene containing handles capable of tetrazine ligation
Chemical Biology & Drug Design, 2014Co-Authors: James W Wollack, Benjamin J Monson, Jonathan K Dozier, Joseph J Dalluge, Kristina Poss, Scott A Hilderbrand, Mark D DistefanoAbstract:There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide-alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper’s toxicity.1 Here we report the synthesis of trans-cyclooctene Geranyl Diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site-specially modified with trans-cyclooctene Geranyl Diphosphate were subsequently targeted for further modification via tetrazine ligation. Since tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.
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Site‐Specific Labeling of Proteins and Peptides with Trans‐cyclooctene Containing Handles Capable of Tetrazine Ligation
Chemical Biology & Drug Design, 2014Co-Authors: James W Wollack, Benjamin J Monson, Jonathan K Dozier, Joseph J Dalluge, Kristina Poss, Scott A Hilderbrand, Mark D DistefanoAbstract:There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide-alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper’s toxicity.1 Here we report the synthesis of trans-cyclooctene Geranyl Diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site-specially modified with trans-cyclooctene Geranyl Diphosphate were subsequently targeted for further modification via tetrazine ligation. Since tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.