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Hj Bouwmeester - One of the best experts on this subject based on the ideXlab platform.
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genetic mapping and characterization of the globe artichoke Germacrene a synthase gene encoding the first dedicated enzyme for biosynthesis of the bitter sesquiterpene lactone cynaropicrin
Plant Science, 2012Co-Authors: Barbara Menin, Hj Bouwmeester, Cinzia Comino, E Portis, Andrea Moglia, Katarina Cankar, Sergio Lanteri, Jules BeekwilderAbstract:Abstract Globe artichoke (Cynara cardunculus var. scolymus L., Asteraceae) is a perennial crop traditionally consumed as a vegetable in the Mediterranean countries and rich in nutraceutically and pharmaceutically active compounds, including phenolic and terpenoid compounds. Its bitter taste is caused by its high content of sesquiterpene lactones (STLs), such as cynaropicrin. The biosynthetic pathway responsible for STL biosynthesis in globe artichoke is unknown, but likely proceeds through Germacrene A, as has been shown for other Asteraceae species. Here, we investigated the accumulation of cynaropicrin in different tissues of globe artichoke, and compared it to accumulation of phenolic compounds. Cynaropicrin concentration was highest in old leaves. A putative Germacrene A synthase (GAS) gene was identified in a set of ∼19,000 globe artichoke unigenes. When heterologously expressed in Escherichia coli, the putative globe artichoke GAS converted farnesyl diphosphate (FPP) into (+)-Germacrene A. Among various tissues assayed, the level of globe artichoke GAS expression was highest in mature (six week old) leaves. A sequence polymorphism within a mapping population parent allowed the corresponding GAS gene to be positioned on a genetic map. This study reports the isolation, expression and mapping of a key gene involved in STL biosynthesis in C. cardunculus. This is a good basis for further investigation of this pathway.
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Genetic mapping and characterization of the globe artichoke (+)-Germacrene A synthase gene, encoding the first dedicated enzyme for biosynthesis of the bitter sesquiterpene lactone cynaropicrin
Plant Science, 2012Co-Authors: Barbara Menin, Hj Bouwmeester, Cinzia Comino, E Portis, Andrea Moglia, Katarina Cankar, Sergio Lanteri, Jules BeekwilderAbstract:Abstract Globe artichoke (Cynara cardunculus var. scolymus L., Asteraceae) is a perennial crop traditionally consumed as a vegetable in the Mediterranean countries and rich in nutraceutically and pharmaceutically active compounds, including phenolic and terpenoid compounds. Its bitter taste is caused by its high content of sesquiterpene lactones (STLs), such as cynaropicrin. The biosynthetic pathway responsible for STL biosynthesis in globe artichoke is unknown, but likely proceeds through Germacrene A, as has been shown for other Asteraceae species. Here, we investigated the accumulation of cynaropicrin in different tissues of globe artichoke, and compared it to accumulation of phenolic compounds. Cynaropicrin concentration was highest in old leaves. A putative Germacrene A synthase (GAS) gene was identified in a set of ∼19,000 globe artichoke unigenes. When heterologously expressed in Escherichia coli, the putative globe artichoke GAS converted farnesyl diphosphate (FPP) into (+)-Germacrene A. Among various tissues assayed, the level of globe artichoke GAS expression was highest in mature (six week old) leaves. A sequence polymorphism within a mapping population parent allowed the corresponding GAS gene to be positioned on a genetic map. This study reports the isolation, expression and mapping of a key gene involved in STL biosynthesis in C. cardunculus. This is a good basis for further investigation of this pathway.
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Enantiospecific (+)- and (-)-Germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif.
Archives of Biochemistry and Biophysics, 2004Co-Authors: Ian M Prosser, Hj Bouwmeester, Wilfried A Konig, Iris G Altug, Andrew L Phillips, Michael H BealeAbstract:The naturally occurring, volatile sesquiterpene hydrocarbon Germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of Germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-Germacrene D synthase and Sc19 as (−)-Germacrene D synthase. Thus, the enantiomers of Germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-Germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.
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enantiospecific and Germacrene d synthases cloned from goldenrod reveal a functionally active variant of the universal isoprenoid biosynthesis aspartate rich motif
Archives of Biochemistry and Biophysics, 2004Co-Authors: Ian M Prosser, Hj Bouwmeester, Wilfried A Konig, Iris G Altug, Andrew L Phillips, Michael H BealeAbstract:The naturally occurring, volatile sesquiterpene hydrocarbon Germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of Germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-Germacrene D synthase and Sc19 as (−)-Germacrene D synthase. Thus, the enantiomers of Germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-Germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.
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isolation and characterization of two Germacrene a synthase cdna clones from chicory
Plant Physiology, 2002Co-Authors: Hj Bouwmeester, Iris G Altug, J.w. De Kraker, J Kodde, Fwa Verstappen, Thorvald WallaartAbstract:Chicory ( Cichorium intybus ) sesquiterpene lactones were recently shown to be derived from a common sesquiterpene intermediate, (+)-Germacrene A. Germacrene A is of interest because of its key role in sesquiterpene lactone biosynthesis and because it is an enzyme-bound intermediate in the biosynthesis of a number of phytoalexins. Using polymerase chain reaction with degenerate primers, we have isolated two sesquiterpene synthases from chicory that exhibited 72% amino acid identity. Heterologous expression of the genes in Escherichia coli has shown that they both catalyze exclusively the formation of (+)-Germacrene A, making this the first report, to our knowledge, on the isolation of (+)-Germacrene A synthase (GAS)-encoding genes. Northern analysis demonstrated that both genes were expressed in all chicory tissues tested albeit at varying levels. Protein isolation and partial purification from chicory heads demonstrated the presence of two GAS proteins. On MonoQ, these proteins co-eluted with the two heterologously produced proteins. The K m value, pH optimum, and MonoQ elution volume of one of the proteins produced in E. coli were similar to the values reported for the GAS protein that was recently purified from chicory roots. Finally, the two deduced amino acid sequences were modeled, and the resulting protein models were compared with the crystal structure of tobacco ( Nicotiana tabacum ) 5- epi -aristolochene synthase, which forms Germacrene A as an enzyme-bound intermediate en route to 5- epi -aristolochene. The possible involvement of a number of amino acids in sesquiterpene synthase product specificity is discussed.
Wilfried A Konig - One of the best experts on this subject based on the ideXlab platform.
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enantiospecific and Germacrene d synthases cloned from goldenrod reveal a functionally active variant of the universal isoprenoid biosynthesis aspartate rich motif
Archives of Biochemistry and Biophysics, 2004Co-Authors: Ian M Prosser, Hj Bouwmeester, Wilfried A Konig, Iris G Altug, Andrew L Phillips, Michael H BealeAbstract:The naturally occurring, volatile sesquiterpene hydrocarbon Germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of Germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-Germacrene D synthase and Sc19 as (−)-Germacrene D synthase. Thus, the enantiomers of Germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-Germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.
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Enantiospecific (+)- and (-)-Germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif.
Archives of Biochemistry and Biophysics, 2004Co-Authors: Ian M Prosser, Hj Bouwmeester, Wilfried A Konig, Iris G Altug, Andrew L Phillips, Michael H BealeAbstract:The naturally occurring, volatile sesquiterpene hydrocarbon Germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of Germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-Germacrene D synthase and Sc19 as (−)-Germacrene D synthase. Thus, the enantiomers of Germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-Germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.
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Germacrene d receptor neurones in three species of heliothine moths structure activity relationships
Journal of Comparative Physiology A-neuroethology Sensory Neural and Behavioral Physiology, 2003Co-Authors: M Stranden, Wilfried A Konig, Annakarin Borgkarlson, Ilme Liblikas, Tor Jorgen Almaas, Hanna MustapartaAbstract:Specificity of olfactory receptor neurones plays an important role in food and host preferences of a species, and may have become conserved or changed in the evolution of polyphagy and oligophagy. We have identified a major type of plant odour receptor neurones responding to the sesquiterpene Germacrene D in three species of heliothine moths, the polyphagous Heliothis virescens and Helicoverpa armigera and the oligophagous Helicoverpa assulta. The neurones respond with high sensitivity and selectivity to (–)-Germacrene D, as demonstrated by screening via gas chromatography with numerous mixtures of plant volatiles. Germacrene D was present in both host and non-host plants, but only in half of the tested species. The specificity of the neurones was similar in the three species, as shown by the "secondary" responses to a few other sesquiterpenes. The effect of (–)-Germacrene D was about ten times stronger than that of the (+)-enantiomer, which again was about ten times stronger than that of (–)-α-ylangene. Weaker effects were obtained for (+)-β-ylangene, (+)-α-copaene, β-copaene and two unidentified sesquiterpenes. The structure-activity relationship shows that the important properties of (–)-Germacrene D in activating the neurones are the ten-membered ring system and the three double bonds acting as electron-rich centres, in addition to the direction of the isopropyl-group responsible for the different effects of the Germacrene D enantiomers.
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(–)-Germacrene D receptor neurones in three species of heliothine moths: structure-activity relationships
Journal of Comparative Physiology A-neuroethology Sensory Neural and Behavioral Physiology, 2003Co-Authors: M Stranden, Wilfried A Konig, Ilme Liblikas, Tor Jorgen Almaas, Anna-karin Borg-karlson, Hanna MustapartaAbstract:Specificity of olfactory receptor neurones plays an important role in food and host preferences of a species, and may have become conserved or changed in the evolution of polyphagy and oligophagy. We have identified a major type of plant odour receptor neurones responding to the sesquiterpene Germacrene D in three species of heliothine moths, the polyphagous Heliothis virescens and Helicoverpa armigera and the oligophagous Helicoverpa assulta. The neurones respond with high sensitivity and selectivity to (–)-Germacrene D, as demonstrated by screening via gas chromatography with numerous mixtures of plant volatiles. Germacrene D was present in both host and non-host plants, but only in half of the tested species. The specificity of the neurones was similar in the three species, as shown by the "secondary" responses to a few other sesquiterpenes. The effect of (–)-Germacrene D was about ten times stronger than that of the (+)-enantiomer, which again was about ten times stronger than that of (–)-α-ylangene. Weaker effects were obtained for (+)-β-ylangene, (+)-α-copaene, β-copaene and two unidentified sesquiterpenes. The structure-activity relationship shows that the important properties of (–)-Germacrene D in activating the neurones are the ten-membered ring system and the three double bonds acting as electron-rich centres, in addition to the direction of the isopropyl-group responsible for the different effects of the Germacrene D enantiomers.
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the role of Germacrene d as a precursor in sesquiterpene biosynthesis investigations of acid catalyzed photochemically and thermally induced rearrangements
Phytochemistry, 2000Co-Authors: Nils Bulow, Wilfried A KonigAbstract:Abstract Germacrene D is considered as a precursor of many sesquiterpene hydrocarbons. We have investigated the acid catalyzed as well as the photochemically and thermally induced rearrangement processes of Germacrene D isolated from several Solidago species, which contain both enantiomers of Germacrene D. Enantiomeric mixtures of sesquiterpenes of the cadinane, eudesmane (selinane), oppositane, axane, isodaucane, and bourbonane group as well as isoGermacrene D were identified as main products and made available as reference compounds for structure investigations and stereochemical assignments of plant constituents. δ-Amorphene, one of the rearrangement products, was identified as a natural product for the first time. The absolute configuration of γ-amorphene was revised by correlation with the absolute configuration of Germacrene D. The mechanisms of the rearrangement reactions are discussed.
Michael H Beale - One of the best experts on this subject based on the ideXlab platform.
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enantiospecific and Germacrene d synthases cloned from goldenrod reveal a functionally active variant of the universal isoprenoid biosynthesis aspartate rich motif
Archives of Biochemistry and Biophysics, 2004Co-Authors: Ian M Prosser, Hj Bouwmeester, Wilfried A Konig, Iris G Altug, Andrew L Phillips, Michael H BealeAbstract:The naturally occurring, volatile sesquiterpene hydrocarbon Germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of Germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-Germacrene D synthase and Sc19 as (−)-Germacrene D synthase. Thus, the enantiomers of Germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-Germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.
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Enantiospecific (+)- and (-)-Germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif.
Archives of Biochemistry and Biophysics, 2004Co-Authors: Ian M Prosser, Hj Bouwmeester, Wilfried A Konig, Iris G Altug, Andrew L Phillips, Michael H BealeAbstract:The naturally occurring, volatile sesquiterpene hydrocarbon Germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of Germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-Germacrene D synthase and Sc19 as (−)-Germacrene D synthase. Thus, the enantiomers of Germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-Germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.
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10r Germacrene a synthase from goldenrod solidago canadensis cdna isolation bacterial expression and functional analysis
Phytochemistry, 2002Co-Authors: Ian M Prosser, Andrew L Phillips, Simon Gittings, Mervyn J Lewis, Antony M Hooper, J A Pickett, Michael H BealeAbstract:Abstract Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC–MS revealed the presence of both enantiomers of Germacrene D and lesser amounts of Germacrene A, α-humulene, and β-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC–MS analysis of products from of 3H- and 2H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-Germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.
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(+)-(10R )-Germacrene A synthase from goldenrod, Solidago canadensis ; cDNA isolation, bacterial expression and functional analysis
Phytochemistry, 2002Co-Authors: Ian M Prosser, Andrew L Phillips, Simon Gittings, Mervyn J Lewis, Antony M Hooper, J A Pickett, Michael H BealeAbstract:Abstract Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC–MS revealed the presence of both enantiomers of Germacrene D and lesser amounts of Germacrene A, α-humulene, and β-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC–MS analysis of products from of 3H- and 2H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-Germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.
Ian M Prosser - One of the best experts on this subject based on the ideXlab platform.
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enantiospecific and Germacrene d synthases cloned from goldenrod reveal a functionally active variant of the universal isoprenoid biosynthesis aspartate rich motif
Archives of Biochemistry and Biophysics, 2004Co-Authors: Ian M Prosser, Hj Bouwmeester, Wilfried A Konig, Iris G Altug, Andrew L Phillips, Michael H BealeAbstract:The naturally occurring, volatile sesquiterpene hydrocarbon Germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of Germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-Germacrene D synthase and Sc19 as (−)-Germacrene D synthase. Thus, the enantiomers of Germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-Germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.
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Enantiospecific (+)- and (-)-Germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif.
Archives of Biochemistry and Biophysics, 2004Co-Authors: Ian M Prosser, Hj Bouwmeester, Wilfried A Konig, Iris G Altug, Andrew L Phillips, Michael H BealeAbstract:The naturally occurring, volatile sesquiterpene hydrocarbon Germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of Germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-Germacrene D synthase and Sc19 as (−)-Germacrene D synthase. Thus, the enantiomers of Germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-Germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.
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10r Germacrene a synthase from goldenrod solidago canadensis cdna isolation bacterial expression and functional analysis
Phytochemistry, 2002Co-Authors: Ian M Prosser, Andrew L Phillips, Simon Gittings, Mervyn J Lewis, Antony M Hooper, J A Pickett, Michael H BealeAbstract:Abstract Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC–MS revealed the presence of both enantiomers of Germacrene D and lesser amounts of Germacrene A, α-humulene, and β-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC–MS analysis of products from of 3H- and 2H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-Germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.
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(+)-(10R )-Germacrene A synthase from goldenrod, Solidago canadensis ; cDNA isolation, bacterial expression and functional analysis
Phytochemistry, 2002Co-Authors: Ian M Prosser, Andrew L Phillips, Simon Gittings, Mervyn J Lewis, Antony M Hooper, J A Pickett, Michael H BealeAbstract:Abstract Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC–MS revealed the presence of both enantiomers of Germacrene D and lesser amounts of Germacrene A, α-humulene, and β-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC–MS analysis of products from of 3H- and 2H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-Germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.
Maurice C. R. Franssen - One of the best experts on this subject based on the ideXlab platform.
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Germacrenes from fresh costus roots
Phytochemistry, 2001Co-Authors: Janwillem De Kraker, Maurice C. R. Franssen, Aede De Groot, Toshiro Shibata, Hj BouwmeesterAbstract:Abstract Four Germacrenes, previously shown to be intermediates in sesquiterpene lactone biosynthesis, were isolated from fresh costus roots ( Saussurea lappa ). The structures of (+)-Germacrene A, germacra-1(10),4,11(13)-trien-12-ol, germacra-1(10),4,11(13)-trien-12-al, and germacra-1(10),4,11(13)-trien-12-oic acid were deduced by a combination of spectral data and chemical transformations. Heating of these compounds yields (−)-β-elemene, (−)-elema-1,3,11(13)-trien-12-ol, (−)-elema-1,3,11(13)-trien-12-al, and elema-1,3,11(13)-trien-12-oic acid respectively, in addition to small amounts of their diastereomers. Acid induced cyclisation of the Germacrenes yields selinene, costol, costal, and costic acid respectively. It is highly probable that the elemenes reported in literature for costus root oil are artefacts.
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biosynthesis of Germacrene a carboxylic acid in chicory roots demonstration of a cytochrome p450 Germacrene a hydroxylase and nadp dependent sesquiterpenoid dehydrogenase s involved in sesquiterpene lactone biosynthesis
Plant Physiology, 2001Co-Authors: Janwillem De Kraker, Maurice C. R. Franssen, Aede De Groot, Marcella C F Dalm, Hj BouwmeesterAbstract:Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-Germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the Germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the Germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-Germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized Germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-Germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates β-elemene with a modest degree of enantioselectivity.
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Biosynthesis of Germacrene A Carboxylic Acid in Chicory Roots. Demonstration of a Cytochrome P450 (+)-Germacrene A Hydroxylase and NADP+-Dependent Sesquiterpenoid Dehydrogenase(s) Involved in Sesquiterpene Lactone Biosynthesis
Plant Physiology, 2001Co-Authors: Janwillem De Kraker, Maurice C. R. Franssen, Aede De Groot, Marcella C F Dalm, Hj BouwmeesterAbstract:Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-Germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the Germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the Germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-Germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized Germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-Germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates β-elemene with a modest degree of enantioselectivity.
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Germacrene a synthesis in chicory cichorium intybus l the first step in sesquiterpene lactone biosynthesis
Acta Botanica Gallica, 1999Co-Authors: J.w. De Kraker, Hj Bouwmeester, Maurice C. R. Franssen, C P G M De GrootAbstract:Abstract The slightly bitter taste of Chicorium intybus L. originates from sesquiterpene lactones with come through precursors formed via fanesyl diphosphate by a sesquiterpene cyclase. The first step of this biosynthesis is the (+)-Germacrene A.
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(+)-Germacrene A synthesis in chicory (Cichorium intybus L.); the first step in sesquiterpene lactone biosynthesis
Acta Botanica Gallica, 1999Co-Authors: J.w. De Kraker, Hj Bouwmeester, Maurice C. R. Franssen, C P G M De GrootAbstract:Abstract The slightly bitter taste of Chicorium intybus L. originates from sesquiterpene lactones with come through precursors formed via fanesyl diphosphate by a sesquiterpene cyclase. The first step of this biosynthesis is the (+)-Germacrene A.