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May R Berenbaum - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of cytochrome p450 mediated detoxification as an evolutionary strategy for specialization on Furanocoumarin containing hostplants cyp6ae89 in parsnip webworms
Insect Molecular Biology, 2020Co-Authors: Bernarda Calla, Mary A Schuler, Charles A E Dean, May R BerenbaumAbstract:The parsnip webworm, Depressaria pastinacella, is restricted to two hostplant genera containing six structurally diverse Furanocoumarins. Of these, imperatorin is detoxified by a specialized cytochrome P450, CYP6AB3. A previous whole-larva transcriptome analysis confirmed the presence of nine transcripts that belong to the CYP6AE subfamily. Here, by examining midgut-specific gene expression patterns we determined that CYP6AE89 transcripts were highly expressed and Furanocoumarin-inducible. Computer docking and energy-minimization of a CYP6AE89 model with all six Furanocoumarins showed that 5-methoxylated bergapten and 8-methoxylated xanthotoxin had the smallest distances from the heme to the proton-donor residue in the catalytic I-helix, and that the 5,8-dimethoxylated isopimpinellin and bergapten had the smallest energy-minimized distance from the heme oxygen to the furan ring double bond. To evaluate this prediction, we expressed the CYP6AE89 protein in an Escherichia coli system, and used it to detect high catalytic activity against the two mono-methoxylated linear Furanocoumarins - bergapten and xanthotoxin - and weak activity against isopimpinellin. Thus, CYP6AE89, like CYP6AB3, is probably specialized for detoxifying only a subset of hostplant Furanocoumarins. A maximum-likelihood tree built with six representative lepidopterans with manually annotated cytochrome P450s shows that CYP6AE89 may have evolved much faster than the other CYP6AE proteins, possibly indicative of host selection pressure.
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cyp6b1 and cyp6b3 of the black swallowtail papilio polyxenes adaptive evolution through subfunctionalization
Molecular Biology and Evolution, 2006Co-Authors: Sanjeewa G Rupasinghe, May R Berenbaum, Mary A SchulerAbstract:Gene duplication provides essential material for functional divergence of proteins and hence allows organisms to adapt to changing environments. Following duplication events, redundant paralogs may undergo different evolutionary paths via processes known as nonfunctionalization, neofunctionalization, or subfunctionalization. Studies of adaptive evolution at the molecular level have progressed rapidly by computationally analyzing nucleotide substitution patterns but such studies are limited by the absence of information relating to alterations of function of the encoded enzymes. In this respect, evolution of the Papilio polyxenes cytochrome P450 monooxygenases (P450s) responsible for the adaptation of this insect to Furanocoumarin-containing host plants provides an excellent model for elucidating the evolutionary fate of duplicated genes. Evidence from sequence and functional analysis in combination with molecular modeling indicates that the paralogous CYP6BI and CYP6B3 genes in P. polyxenes have probably evolved via subfunctionalization after the duplication event by which they arose. Both enzymes have been under independent purifying selection as evidenced by the low dN/dS ratio in both the coding region and substrate recognition sites. Both enzymes have maintained their ability to metabolize linear and angular Furanocoumarins albeit at different efficiencies. Comparisons of molecular models developed for the CYP6B3 and CYP6B1 proteins highlight differences in their binding modes that account for their different activities toward linear and angular Furanocoumarins. That P. polyxenes maintains these 2 Furanocoumarin-metabolizing loci with somewhat different activities and expression patterns provides this species with the potential to acquire P450s with novel functions while maintaining those most critical to its exclusive feeding on its current range of host plants.
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Inhibition of CYP6B1-Mediated Detoxification of Xanthotoxin by Plant Allelochemicals in the Black Swallowtail (Papilio polyxenes)
Journal of Chemical Ecology, 2006Co-Authors: Zhimou Wen, May R Berenbaum, Mary A SchulerAbstract:The structural and biosynthetic diversity of allelochemicals in plants is thought to arise from selection for additive toxicity as a consequence of toxin mixture or for enhanced toxicity as a result of synergism. In order to understand how insects cope with this type of plant defense, we tested the effects of some allelochemicals in host plants of the black swallowtail Papilio polyxenes on the xanthotoxin-metabolic activity of CYP6B1, the principal enzyme responsible for the detoxification of Furanocoumarins in this caterpillar. Additionally, the effects of some synthetic compounds not normally encountered by P. polyxenes on CYP6B1 were tested. These studies demonstrate that the integrity of Furanocoumarin structure is important for competitive binding to the active site of CYP6B1, even though the carbonyl group on the pyranone ring apparently does not affect its inhibitory capacity, as in the case of furanochromones. Angular Furanocoumarins are generally less phototoxic to many organisms than linear Furanocoumarins due to their reduced capacity for cross-linking DNA strands, yet they are more toxic than linear Furanocoumarins to black swallowtail larvae. This enhanced toxicity in vivo may be due to the ability of angular Furanocoumarins to bind to the active site of CYP6B1 without being rapidly metabolized. This binding reduces the availability of CYP6B1 to metabolize other linear Furanocoumarins. The structure-activity relationships for methylenedioxyphenyl compounds, flavonoids, imidazole, and imidazole derivatives are also discussed in light of their capacity to inhibit the xanthotoxin-metabolic activity of CYP6B1.
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Furanocoumarins and their detoxification in a tri-trophic interaction
CHEMOECOLOGY, 2006Co-Authors: Jennifer L. Mcgovern, Arthur R. Zangerl, May R BerenbaumAbstract:The parsnip webworm, Depressaria pastinacella , specializes on wild parsnip, Pastinaca sativa , and several species of Heracleum , hostplants rich in toxic Furanocoumarins. Rates of Furanocoumarin metabolism in this species are among the highest known for any insect. Within its native range in Europe, webworms are heavily parasitized by the polyembryonic parasitoid wasp Copidosoma sosares . In this study, we determined whether these parasitoids are exposed to Furanocoumarins in host hemolymph, whether they can metabolize Furanocoumarins, and whether parasitism influences the ability of webworms to detoxify Furanocoumarins. Hemolymph of webworms fed artificial diet containing 0.3 % fresh weight xanthotoxin, a Furanocoumarin prevalent in wild parsnip hosts, contained trace amounts of this toxin; as well, hemolymph of webworms consuming P. sativa flowers and fruits contained trace amounts of six of seven Furanocoumarins present in the hostplant. Thus, parasitoids likely encounter Furanocoumarins in host hemolymph. Assays of xanthotoxin metabolism in C. sosares failed to show any ability to metabolize this compound. Parasitized webworms, collected from populations of Heracleum sphondylium in the Netherlands in 2004, were on average 55 % larger by weight than unparasitized individuals. This weight is inclusive of host and parasitoid masses. Absolute rates of detoxification (nmoles min^−1) of five different Furanocoumarins were indistinguishable between parasitized and unparasitized ultimate instars, suggesting that the intrinsic rates of metabolism are fixed. Thus, although parasitized larvae are larger, detoxification rates are not commensurate with size; rates in parasitized larvae expressed per gram of larval mass were 25 % lower than in unparasitized larvae.
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Cytochrome P450-Mediated Metabolism of Xanthotoxin by Papilio multicaudatus
Journal of Chemical Ecology, 2006Co-Authors: Mark A. Berhow, Arthur R. Zangerl, Jennifer Mcgovern, May R BerenbaumAbstract:Within the genus Papilio , the P. glaucus group contains the most polyphagous Papilio species within the Papilionidae. The majority of Papilio species are associated with hostplants in the families Rutaceae and Apiaceae, and characterizing most are secondary metabolites called Furanocoumarins. Recent phylogenetic studies suggest that Furanocoumarin metabolism is an ancestral trait, with the glaucus group derived from ancestors associated with Furanocoumarin-containing Rutaceae. In this study, we examined this relationship by conducting a gravimetric analysis of growth that used various concentrations of the Furanocoumarin xanthotoxin. Papilio multicaudatus , the putative ancestor of the glaucus group, includes at least one Furanocoumarin-containing rutaceous species among its hostplants; this species can consume leaf tissue containing up to 0.3% xanthotoxin with no detectable effect on relative growth rate, relative consumption rate, or efficiency of conversion of ingested food. As is the case for other Papilio species, xanthotoxin metabolism is mediated by cytochrome P450 monooxygenases (P450s). Ingestion of xanthotoxin by ultimate instar P. multicaudatus increases activity up to 30-fold in a dose-dependent fashion. Midguts of induced larvae can also effectively metabolize six other Furanocoumarins, including both linear (bergapten, isopimpinellin, imperatorin) and angular (angelicin, sphondin) forms. A metabolite of xanthotoxin in the frass from xanthotoxin-treated larvae, identified as 6-(7-hydroxy-8-methoxycoumaryl)-acetic acid by MS–MS and NMR analyses, is identical to one from the frass of P. polyxenes . The occurrence of this metabolite in two swallowtails and the presence of a second metabolite of xanthotoxin, 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol in the frass of both P. polyxenes and Depressaria pastinacella are consistent with the suggestion that lepidopterans share as the first step of xanthotoxin metabolism the P450-mediated epoxidation of the furan ring 2′–3′ double bond.
Mary A Schuler - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of cytochrome p450 mediated detoxification as an evolutionary strategy for specialization on Furanocoumarin containing hostplants cyp6ae89 in parsnip webworms
Insect Molecular Biology, 2020Co-Authors: Bernarda Calla, Mary A Schuler, Charles A E Dean, May R BerenbaumAbstract:The parsnip webworm, Depressaria pastinacella, is restricted to two hostplant genera containing six structurally diverse Furanocoumarins. Of these, imperatorin is detoxified by a specialized cytochrome P450, CYP6AB3. A previous whole-larva transcriptome analysis confirmed the presence of nine transcripts that belong to the CYP6AE subfamily. Here, by examining midgut-specific gene expression patterns we determined that CYP6AE89 transcripts were highly expressed and Furanocoumarin-inducible. Computer docking and energy-minimization of a CYP6AE89 model with all six Furanocoumarins showed that 5-methoxylated bergapten and 8-methoxylated xanthotoxin had the smallest distances from the heme to the proton-donor residue in the catalytic I-helix, and that the 5,8-dimethoxylated isopimpinellin and bergapten had the smallest energy-minimized distance from the heme oxygen to the furan ring double bond. To evaluate this prediction, we expressed the CYP6AE89 protein in an Escherichia coli system, and used it to detect high catalytic activity against the two mono-methoxylated linear Furanocoumarins - bergapten and xanthotoxin - and weak activity against isopimpinellin. Thus, CYP6AE89, like CYP6AB3, is probably specialized for detoxifying only a subset of hostplant Furanocoumarins. A maximum-likelihood tree built with six representative lepidopterans with manually annotated cytochrome P450s shows that CYP6AE89 may have evolved much faster than the other CYP6AE proteins, possibly indicative of host selection pressure.
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cyp6b1 and cyp6b3 of the black swallowtail papilio polyxenes adaptive evolution through subfunctionalization
Molecular Biology and Evolution, 2006Co-Authors: Sanjeewa G Rupasinghe, May R Berenbaum, Mary A SchulerAbstract:Gene duplication provides essential material for functional divergence of proteins and hence allows organisms to adapt to changing environments. Following duplication events, redundant paralogs may undergo different evolutionary paths via processes known as nonfunctionalization, neofunctionalization, or subfunctionalization. Studies of adaptive evolution at the molecular level have progressed rapidly by computationally analyzing nucleotide substitution patterns but such studies are limited by the absence of information relating to alterations of function of the encoded enzymes. In this respect, evolution of the Papilio polyxenes cytochrome P450 monooxygenases (P450s) responsible for the adaptation of this insect to Furanocoumarin-containing host plants provides an excellent model for elucidating the evolutionary fate of duplicated genes. Evidence from sequence and functional analysis in combination with molecular modeling indicates that the paralogous CYP6BI and CYP6B3 genes in P. polyxenes have probably evolved via subfunctionalization after the duplication event by which they arose. Both enzymes have been under independent purifying selection as evidenced by the low dN/dS ratio in both the coding region and substrate recognition sites. Both enzymes have maintained their ability to metabolize linear and angular Furanocoumarins albeit at different efficiencies. Comparisons of molecular models developed for the CYP6B3 and CYP6B1 proteins highlight differences in their binding modes that account for their different activities toward linear and angular Furanocoumarins. That P. polyxenes maintains these 2 Furanocoumarin-metabolizing loci with somewhat different activities and expression patterns provides this species with the potential to acquire P450s with novel functions while maintaining those most critical to its exclusive feeding on its current range of host plants.
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Inhibition of CYP6B1-Mediated Detoxification of Xanthotoxin by Plant Allelochemicals in the Black Swallowtail (Papilio polyxenes)
Journal of Chemical Ecology, 2006Co-Authors: Zhimou Wen, May R Berenbaum, Mary A SchulerAbstract:The structural and biosynthetic diversity of allelochemicals in plants is thought to arise from selection for additive toxicity as a consequence of toxin mixture or for enhanced toxicity as a result of synergism. In order to understand how insects cope with this type of plant defense, we tested the effects of some allelochemicals in host plants of the black swallowtail Papilio polyxenes on the xanthotoxin-metabolic activity of CYP6B1, the principal enzyme responsible for the detoxification of Furanocoumarins in this caterpillar. Additionally, the effects of some synthetic compounds not normally encountered by P. polyxenes on CYP6B1 were tested. These studies demonstrate that the integrity of Furanocoumarin structure is important for competitive binding to the active site of CYP6B1, even though the carbonyl group on the pyranone ring apparently does not affect its inhibitory capacity, as in the case of furanochromones. Angular Furanocoumarins are generally less phototoxic to many organisms than linear Furanocoumarins due to their reduced capacity for cross-linking DNA strands, yet they are more toxic than linear Furanocoumarins to black swallowtail larvae. This enhanced toxicity in vivo may be due to the ability of angular Furanocoumarins to bind to the active site of CYP6B1 without being rapidly metabolized. This binding reduces the availability of CYP6B1 to metabolize other linear Furanocoumarins. The structure-activity relationships for methylenedioxyphenyl compounds, flavonoids, imidazole, and imidazole derivatives are also discussed in light of their capacity to inhibit the xanthotoxin-metabolic activity of CYP6B1.
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characterization and evolution of Furanocoumarin inducible cytochrome p450s in the parsnip webworm depressaria pastinacella
Insect Molecular Biology, 2004Co-Authors: W. Li, Mary A Schuler, Arthur R. Zangerl, May R BerenbaumAbstract:: Depressaria pastinacella, the parsnip webworm, a specialist on two genera in the Apiaceae, routinely consumes plant tissues high in Furanocoumarin content and is capable of rapid cytochrome P450-mediated detoxification of these compounds. In this study, four cDNAs were cloned from the larval midgut of this insect: two full-length CYP6AB3 and CYP6AE1 cDNAs are closely related to members of the Furanocoumarin-metabolizing CYP6B subfamily and two partial CYP9A6 and CYP9A7 cDNAs are related to members of the CYP9A subfamily that have also been linked to the detoxification of xenobiotics. At least one of these P450s (CYP6AB3) is inducible by dietary Furanocoumarins, indicating its potential involvement in Furanocoumarin metabolism. A homology model of CYP6AB3 was constructed and compared to models of CYP6B1 from the specialist species, Papilio polyxenes, and CYP6B4 from the generalist species, P. glaucus. Structural superpositioning of these models has revealed very high spatial similarity of elements, including the B helix, B'-C loop, I helix and C-terminal domain, within the catalytic sites of these proteins. Most importantly, key amino acid residues that can potentially come into contact with Furanocoumarin substrates display conservation in their spatial positioning and side chain polarities. Three of these residues, Val103, Leu113 and Phe118 (numbered according to CYP6AB3), are conserved in all three of these proteins, further implicating CYP6AB3 in Furanocoumarin metabolism by parsnip webworms. Characterization of these P450 cDNAs will allow for functional analyses aimed at elucidating the molecular mechanisms underlying the coevolutionary interactions between this herbivore and its principal host plant.
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diversification of Furanocoumarin metabolizing cytochrome p450 monooxygenases in two papilionids specificity and substrate encounter rate
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Mary A Schuler, May R BerenbaumAbstract:Diversification of cytochrome P450 monooxygenases (P450s) is thought to result from antagonistic interactions between plants and their herbivorous enemies. However, little direct evidence demonstrates the relationship between selection by plant toxins and adaptive changes in herbivore P450s. Here we show that the Furanocoumarin-metabolic activity of CYP6B proteins in two species of swallowtail caterpillars is associated with the probability of encountering host plant Furanocoumarins. Catalytic activity was compared in two closely related CYP6B4 and CYP6B17 groups in the polyphagous congeners Papilio glaucus and Papilio canadensis. Generally, P450s from P. glaucus, which feeds occasionally on Furanocoumarin-containing host plants, display higher activities against Furanocoumarins than those from P. canadensis, which normally does not encounter Furanocoumarins. These P450s in turn catalyze a larger range of Furanocoumarins at lower efficiency than CYP6B1, a P450 from Papilio polyxenes, which feeds exclusively on Furanocoumarin-containing host plants. Reconstruction of the ancestral CYP6B sequences using maximum likelihood predictions and comparisons of the sequence and geometry of their active sites to those of contemporary CYP6B proteins indicate that host plant diversity is directly related to P450 activity and inversely related to substrate specificity. These predictions suggest that, along the lineage leading to Papilio P450s, the ancestral, highly versatile CYP6B protein presumed to exist in a polyphagous species evolved through time into a more efficient and specialized CYP6B1-like protein in Papilio species with continual exposure to Furanocoumarins. Further diversification of Papilio CYP6Bs has likely involved interspersed events of positive selection in oligophagous species and relaxation of functional constraints in polyphagous species.
Arthur R. Zangerl - One of the best experts on this subject based on the ideXlab platform.
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Furanocoumarins and their detoxification in a tri-trophic interaction
CHEMOECOLOGY, 2006Co-Authors: Jennifer L. Mcgovern, Arthur R. Zangerl, May R BerenbaumAbstract:The parsnip webworm, Depressaria pastinacella , specializes on wild parsnip, Pastinaca sativa , and several species of Heracleum , hostplants rich in toxic Furanocoumarins. Rates of Furanocoumarin metabolism in this species are among the highest known for any insect. Within its native range in Europe, webworms are heavily parasitized by the polyembryonic parasitoid wasp Copidosoma sosares . In this study, we determined whether these parasitoids are exposed to Furanocoumarins in host hemolymph, whether they can metabolize Furanocoumarins, and whether parasitism influences the ability of webworms to detoxify Furanocoumarins. Hemolymph of webworms fed artificial diet containing 0.3 % fresh weight xanthotoxin, a Furanocoumarin prevalent in wild parsnip hosts, contained trace amounts of this toxin; as well, hemolymph of webworms consuming P. sativa flowers and fruits contained trace amounts of six of seven Furanocoumarins present in the hostplant. Thus, parasitoids likely encounter Furanocoumarins in host hemolymph. Assays of xanthotoxin metabolism in C. sosares failed to show any ability to metabolize this compound. Parasitized webworms, collected from populations of Heracleum sphondylium in the Netherlands in 2004, were on average 55 % larger by weight than unparasitized individuals. This weight is inclusive of host and parasitoid masses. Absolute rates of detoxification (nmoles min^−1) of five different Furanocoumarins were indistinguishable between parasitized and unparasitized ultimate instars, suggesting that the intrinsic rates of metabolism are fixed. Thus, although parasitized larvae are larger, detoxification rates are not commensurate with size; rates in parasitized larvae expressed per gram of larval mass were 25 % lower than in unparasitized larvae.
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Cytochrome P450-Mediated Metabolism of Xanthotoxin by Papilio multicaudatus
Journal of Chemical Ecology, 2006Co-Authors: Mark A. Berhow, Arthur R. Zangerl, Jennifer Mcgovern, May R BerenbaumAbstract:Within the genus Papilio , the P. glaucus group contains the most polyphagous Papilio species within the Papilionidae. The majority of Papilio species are associated with hostplants in the families Rutaceae and Apiaceae, and characterizing most are secondary metabolites called Furanocoumarins. Recent phylogenetic studies suggest that Furanocoumarin metabolism is an ancestral trait, with the glaucus group derived from ancestors associated with Furanocoumarin-containing Rutaceae. In this study, we examined this relationship by conducting a gravimetric analysis of growth that used various concentrations of the Furanocoumarin xanthotoxin. Papilio multicaudatus , the putative ancestor of the glaucus group, includes at least one Furanocoumarin-containing rutaceous species among its hostplants; this species can consume leaf tissue containing up to 0.3% xanthotoxin with no detectable effect on relative growth rate, relative consumption rate, or efficiency of conversion of ingested food. As is the case for other Papilio species, xanthotoxin metabolism is mediated by cytochrome P450 monooxygenases (P450s). Ingestion of xanthotoxin by ultimate instar P. multicaudatus increases activity up to 30-fold in a dose-dependent fashion. Midguts of induced larvae can also effectively metabolize six other Furanocoumarins, including both linear (bergapten, isopimpinellin, imperatorin) and angular (angelicin, sphondin) forms. A metabolite of xanthotoxin in the frass from xanthotoxin-treated larvae, identified as 6-(7-hydroxy-8-methoxycoumaryl)-acetic acid by MS–MS and NMR analyses, is identical to one from the frass of P. polyxenes . The occurrence of this metabolite in two swallowtails and the presence of a second metabolite of xanthotoxin, 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol in the frass of both P. polyxenes and Depressaria pastinacella are consistent with the suggestion that lepidopterans share as the first step of xanthotoxin metabolism the P450-mediated epoxidation of the furan ring 2′–3′ double bond.
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characterization and evolution of Furanocoumarin inducible cytochrome p450s in the parsnip webworm depressaria pastinacella
Insect Molecular Biology, 2004Co-Authors: W. Li, Mary A Schuler, Arthur R. Zangerl, May R BerenbaumAbstract:: Depressaria pastinacella, the parsnip webworm, a specialist on two genera in the Apiaceae, routinely consumes plant tissues high in Furanocoumarin content and is capable of rapid cytochrome P450-mediated detoxification of these compounds. In this study, four cDNAs were cloned from the larval midgut of this insect: two full-length CYP6AB3 and CYP6AE1 cDNAs are closely related to members of the Furanocoumarin-metabolizing CYP6B subfamily and two partial CYP9A6 and CYP9A7 cDNAs are related to members of the CYP9A subfamily that have also been linked to the detoxification of xenobiotics. At least one of these P450s (CYP6AB3) is inducible by dietary Furanocoumarins, indicating its potential involvement in Furanocoumarin metabolism. A homology model of CYP6AB3 was constructed and compared to models of CYP6B1 from the specialist species, Papilio polyxenes, and CYP6B4 from the generalist species, P. glaucus. Structural superpositioning of these models has revealed very high spatial similarity of elements, including the B helix, B'-C loop, I helix and C-terminal domain, within the catalytic sites of these proteins. Most importantly, key amino acid residues that can potentially come into contact with Furanocoumarin substrates display conservation in their spatial positioning and side chain polarities. Three of these residues, Val103, Leu113 and Phe118 (numbered according to CYP6AB3), are conserved in all three of these proteins, further implicating CYP6AB3 in Furanocoumarin metabolism by parsnip webworms. Characterization of these P450 cDNAs will allow for functional analyses aimed at elucidating the molecular mechanisms underlying the coevolutionary interactions between this herbivore and its principal host plant.
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Effects of Furanocoumarins on feeding behavior of parsnip webworms Depressaria pastinacella
Journal of Chemical Ecology, 2002Co-Authors: J. A. Cianfrogna, Arthur R. Zangerl, May R BerenbaumAbstract:The parsnip webworm, Depressaria pastinacella, exhibits limited physiological resistance to Furanocoumarin toxins in its principal host, the wild parsnip, Pastinaca sativa. These insects are typically found attacking individual plants low in Furanocoumarins, relative to others within populations. They also feed preferentially on parthenocarpic fruits, which are lower in Furanocoumarin content than are normal fruits. However, in a previous study with artificial diets, they did not appear to discriminate between high and low concentrations of Furanocoumarins. In this study, the ability of webworms to distinguish between diets differing in Furanocoumarin content was examined with an artificial diet containing wild parsnip and in green parsnip fruits with and without supplemental Furanocoumarins. Larvae showed no preference for high or low Furanocoumarin diets containing equal amounts of freeze-dried parsnip fruit powder. When given a choice between otherwise similar wild parsnip fruits, webworms strongly preferred fruits that were not augmented with Furanocoumarins in one plant but showed no preference or only a weak preference for nonaugmented fruits in four other plants. In order to identify chemical constituents other than Furanocoumarins that might determine feeding preferences, we compared the chemical profile of parthenocarpic fruits (which are preferred) to that of normal fruit. Octyl butyrate, a known deterrent to webworms, is highly correlated with Furanocoumarin content, occurs in all plants, and differs significantly among normal and parthenocarpic fruit, suggesting that webworms may be able to avoid Furanocoumarins by virtue of their behavioral response to octyl butyrate.
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expression of cyp6b1 and cyp6b3 cytochrome p450 monooxygenases and Furanocoumarin metabolism in different tissues of papilio polyxenes lepidoptera papilionidae
Insect Biochemistry and Molecular Biology, 2001Co-Authors: Rebecca A Petersen, May R Berenbaum, Arthur R. Zangerl, Mary A SchulerAbstract:Abstract The CYP6B1 and CYP6B3 cytochrome P450 monooxygenases in the midgut of the black swallowtail participate in the metabolism of toxic Furanocoumarins present in its host plants. In this study, biochemical analyses indicate that the fat body metabolizes significant amounts of the linear Furanocoumarins bergapten and xanthotoxin after larvae feed on xanthotoxin. Northern analyses of the combined CYP6B1/3 transcript expression patterns indicate that transcripts in this P450 subfamily are induced in the midgut and fat body by xanthotoxin. Semi-quantitative RT–PCR analyses of individual CYP6B1/CYP6B3 mRNAs indicate that CYP6B1 transcripts are induced by xanthotoxin in all tissues examined and that CYP6B3 transcripts are induced in the fat body only. These results indicate that the fat body participates in the P450-mediated metabolism of excess Furanocoumarins unmetabolized by the midgut. Although transcripts of both genes were detected and CYP6B1 transcripts were induced by xanthotoxin in the integument, Furanocoumarin metabolism was not detected. Comparison of these P450 promoters with the promoters of alcohol dehydrogenase genes expressed in the fat bodies of several Drosophila species suggest that the xanthotoxin inducibilities of these P450 genes in fat bodies are regulated by elements other than those modulating expression of Adh genes.
Paul B Watkins - One of the best experts on this subject based on the ideXlab platform.
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further characterization of a Furanocoumarin free grapefruit juice on drug disposition studies with cyclosporine
The American Journal of Clinical Nutrition, 2008Co-Authors: Mary F Paine, Wilbur W Widmer, Susan N Pusek, Kimberly L Beavers, Anne B Criss, Jennifer A Snyder, Paul B WatkinsAbstract:Background: We previously established Furanocoumarins as mediators of the interaction between grapefruit juice (GFJ) and the model CYP3A4 substrate felodipine in healthy volunteers using a GFJ devoid of Furanocoumarins. It remains unclear whether Furanocoumarins mediate drug-GFJ interactions involving CYP3A4 substrates that are also P-glycoprotein substrates. Objective: The effects of Furanocoumarin-free GFJ on drug disposition were further characterized by using the dual CYP3A4/P-glycoprotein substrate cyclosporine. Design: By randomized crossover design, 18 healthy volunteers received cyclosporine (5 mg/kg) with 240 mL orange juice (control), GFJ, or Furanocoumarin-free GFJ. Blood was collected over 24 h. Juice treatments were separated by ≥1 wk. The effects of diluted extracts of each juice and of purified Furanocoumarins on [ 3 H]cyclosporine translocation in Caco-2 cells were then compared. Results: The median (range) dose-corrected cyclosporine area under the curve and the maximum concentration with GFJ (P ≤ 0.007), but not with Furanocoumarin-free GFJ (P ≥ 0.50), were significantly higher than those with orange juice [15.6 (6.7-33.5) compared with 11.3 (4.8-22.0) X 10 -3 h/L and 3.0 (1.6-5.8) compared with 2.4 (1.1-3.1) mL- respectively]. The median time to reach maximum concentration and terminal elimination half-life were not significantly different between the juices (2-3 and 7- 8 h, respectively; P ≥ 0.08). Relative to vehicle, the GFJ extract, orange juice extract, and purified Furanocoumarins partially increased apical-to-basolateral and decreased basolateral-to-apical [ 3 H]cydosporine translocation in Caco-2 cells, whereas the Furanocoumarin-free GFJ extract had negligible effects. Reanalysis of the clinical juices identified polymethoxyflavones as candidate P-glycoprotein inhibitors in orange juice but not in GFJ. Conclusions: Furanocoumarins mediate, at least partially, the cyclosporine-GFJ interaction in vivo. A plausible mechanism involves the combined inhibition of enteric CYP3A4 and P-glycoprotein.
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a Furanocoumarin free grapefruit juice establishes Furanocoumarins as the mediators of the grapefruit juice felodipine interaction
The American Journal of Clinical Nutrition, 2006Co-Authors: Mary F Paine, Wilbur W Widmer, Susan N Pusek, Kimberly L Beavers, Anne B Criss, Heather L Hart, Sherri S Brown, Brian F Thomas, Paul B WatkinsAbstract:Background: Grapefruit juice (GFJ) enhances the systemic exposureofnumerousCYP3A4drugsubstrates,includingfelodipine,by inhibitingintestinal(butnothepatic)first-passmetabolism.Furanocoumarins have been identified as major CYP3A4 inhibitors contained in the juice, but their contribution to the GFJ effect in vivo remains unclear. Objective:ToascertainwhetherFuranocoumarinsmediatetheGFJfelodipine interaction, a Furanocoumarin-free GFJ was created and tested against orange juice and the original GFJ with respect to the oral pharmacokinetics of felodipine. Design: With the use of food-grade solvents and absorption resins, Furanocoumarins were removed (99%) from whole GFJ, whereas other major ingredients (flavonoids) were retained. In an open, 3-way,randomizedcrossoverdesign,18healthyvolunteersingested felodipine (10 mg) with 1 of the 3 juices (240 mL). Blood was collected over 24 h. At least 1 wk elapsed between juice treatments. Results: The median and range of the area under the curve and the maximum concentration of felodipine were significantly (P 0.001) greater with consumption of GFJ [110 (range: 58–270) nmol · h/L and 21 (7.6–50) nmol/L, respectively] than with that of orange juice [54 (29–150) nmol · h/L and 7.6 (3.4–13.9) nmol/L, respectively] or Furanocoumarin-free GFJ [48 (23–120) nmol · h/L and 8.3 (3.0–16.6) nmol/L, respectively]. GFJ, orange juice, and Furanocoumarin-free GFJ did not differ significantly (P 0.09) in median time to reach maximum plasma concentration [2.5 (1.5–6), 2.8 (1.5–4), and 2.5 (2–6) h, respectively] or terminal half-life [6.6 (4.2–13.6), 7.8 (4.4–13.2), and 6.8 (2.6–14.4) h, respectively]. Conclusion: Furanocoumarins are the active ingredients in GFJ responsible for enhancing the systemic exposure of felodipine and probablyotherCYP3A4substratesthatundergoextensiveintestinal first-pass metabolism. Am J Clin Nutr 2006;83:1097–105.
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A Furanocoumarin-free grapefruit juice establishes Furanocoumarins as the mediators of the grapefruit juice–felodipine interaction
The American journal of clinical nutrition, 2006Co-Authors: Mary F Paine, Wilbur W Widmer, Susan N Pusek, Kimberly L Beavers, Anne B Criss, Heather L Hart, Sherri S Brown, Brian F Thomas, Paul B WatkinsAbstract:Background: Grapefruit juice (GFJ) enhances the systemic exposureofnumerousCYP3A4drugsubstrates,includingfelodipine,by inhibitingintestinal(butnothepatic)first-passmetabolism.Furanocoumarins have been identified as major CYP3A4 inhibitors contained in the juice, but their contribution to the GFJ effect in vivo remains unclear. Objective:ToascertainwhetherFuranocoumarinsmediatetheGFJfelodipine interaction, a Furanocoumarin-free GFJ was created and tested against orange juice and the original GFJ with respect to the oral pharmacokinetics of felodipine. Design: With the use of food-grade solvents and absorption resins, Furanocoumarins were removed (99%) from whole GFJ, whereas other major ingredients (flavonoids) were retained. In an open, 3-way,randomizedcrossoverdesign,18healthyvolunteersingested felodipine (10 mg) with 1 of the 3 juices (240 mL). Blood was collected over 24 h. At least 1 wk elapsed between juice treatments. Results: The median and range of the area under the curve and the maximum concentration of felodipine were significantly (P 0.001) greater with consumption of GFJ [110 (range: 58–270) nmol · h/L and 21 (7.6–50) nmol/L, respectively] than with that of orange juice [54 (29–150) nmol · h/L and 7.6 (3.4–13.9) nmol/L, respectively] or Furanocoumarin-free GFJ [48 (23–120) nmol · h/L and 8.3 (3.0–16.6) nmol/L, respectively]. GFJ, orange juice, and Furanocoumarin-free GFJ did not differ significantly (P 0.09) in median time to reach maximum plasma concentration [2.5 (1.5–6), 2.8 (1.5–4), and 2.5 (2–6) h, respectively] or terminal half-life [6.6 (4.2–13.6), 7.8 (4.4–13.2), and 6.8 (2.6–14.4) h, respectively]. Conclusion: Furanocoumarins are the active ingredients in GFJ responsible for enhancing the systemic exposure of felodipine and probablyotherCYP3A4substratesthatundergoextensiveintestinal first-pass metabolism. Am J Clin Nutr 2006;83:1097–105.
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A Furanocoumarin-free grapefruit juice establishes Furanocoumarins as the mediators of the grapefruit juice-felodipine interaction [Erratum: 2006 July, v. 84, no. 1, p. 264.]
The American Journal of Clinical Nutrition, 2006Co-Authors: Mary F Paine, Wilbur W Widmer, Susan N Pusek, Kimberly L Beavers, Anne B Criss, Heather L Hart, Sherri S Brown, Brian F Thomas, Paul B WatkinsAbstract:BACKGROUND: Grapefruit juice (GFJ) enhances the systemic exposure of numerous CYP3A4 drug substrates, including felodipine, by inhibiting intestinal (but not hepatic) first-pass metabolism. Furanocoumarins have been identified as major CYP3A4 inhibitors contained in the juice, but their contribution to the GFJ effect in vivo remains unclear. OBJECTIVE: To ascertain whether Furanocoumarins mediate the GFJ-felodipine interaction, a Furanocoumarin-free GFJ was created and tested against orange juice and the original GFJ with respect to the oral pharmacokinetics of felodipine. DESIGN: With the use of food-grade solvents and absorption resins, Furanocoumarins were removed ([approximate]99%) from whole GFJ, whereas other major ingredients (flavonoids) were retained. In an open, 3-way, randomized crossover design, 18 healthy volunteers ingested felodipine (10 mg) with 1 of the 3 juices (240 mL). Blood was collected over 24 h. At least 1 wk elapsed between juice treatments. RESULTS: The median and range of the area under the curve and the maximum concentration of felodipine were significantly (P 0.09) in median time to reach maximum plasma concentration [2.5 (1.5-6), 2.8 (1.5-4), and 2.5 (2-6) h, respectively] or terminal half-life [6.6 (4.2-13.6), 7.8 (4.4-13.2), and 6.8 (2.6-14.4) h, respectively]. CONCLUSION: Furanocoumarins are the active ingredients in GFJ responsible for enhancing the systemic exposure of felodipine and probably other CYP3A4 substrates that undergo extensive intestinal first-pass metabolism.
Frédéric Bourgaud - One of the best experts on this subject based on the ideXlab platform.
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The distribution of coumarins and Furanocoumarins in Citrus species closely matches Citrus phylogeny and reflects the organization of biosynthetic pathways
2015Co-Authors: Audray Dugrand, Alexandre Olry, Alain Hehn, Yann Froelicher, Frédéric BourgaudAbstract:In 2011, citrus crops represented one of the most important fruit productions in the world with more than 131 million tons. Citrus belong to the Rutaceae family able to produce coumarins which constitute a class of secondary metabolites commonly found in higher plants. In these plants, 7-hydroxycoumarin can undergo a subsequent two step enzymatic modification corresponding firstly to the prenylation at C6 and secondly to the closure of a furan ring, leading to Furanocoumarins. In parallel to ecological functions in plants, these molecules can be deleterious for humans. They are potential photosensitizers that can cause photophytodermatitis either after skin contact or ingestion followed by UV A exposure. This photosensitization property is a notable problem with Citrus essential oils, such as bergamot oil, since they are extensively used in perfumes. In this study, coumarin and Furanocoumarin contents found in 61 citrus varieties are compared and the chemical diversity is discussed with respect to the genetic diversity. Based on hierarchical ascendant classification, coumarins and Furanocoumarins can be separated into 4 groups. Each of these 4 groups is logically organized with respect to the already described plant biosynthetic pathways and allows drawing hypotheses on the doubtful biosynthetic origin of compounds. With reference to Citrus diversity obtained from molecular markers, we highlight the genetic crossings which may have resulted in low coumarin and Furanocoumarin content varieties. Citrus varieties identified in this study with low coumarin and Furanocoumarin content will constitute invaluable genetic resources to breeding programs, promoting citrus species devoid of these toxic molecules.
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Recent Advances in Molecular Genetics of Furanocoumarin Synthesis in Higher Plants
Recent Advances in Redox Active Plant and Microbial Products, 2014Co-Authors: Frédéric Bourgaud, Alexandre Olry, Alain HehnAbstract:Furanocoumarins are important plant defense compounds, which are mainly found in four higher plant families: Apiaceae, Moraceae, Fabaceae, and Rutaceae. These molecules constitute a subclass of polyphenolic compounds for which the biogenesis in plants has remained elusive until recently at the molecular level. Over the last decade, the first genes and enzymes involved in Furanocoumarin synthesis have been described. As regards the linear Furanocoumarin bergapten, starting from the precursor molecule p-coumaroyl CoA, we now have three different enzymatic steps (2 cytochrome P450 enzymes and one dioxygenase) out of a total of five that have been unraveled so far. With the exception of one enzyme that was characterized in Rutaceae, all other enzymes have been isolated from Apiaceae plants. The isolation of orthologous genes from other plant families is now under way and will likely reveal whether Furanocoumarins appeared in an ancestor phylum or if they emerged independently over different plant taxonomical groups.
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A coumarin-specific prenyltransferase catalyzes the crucial biosynthetic reaction for Furanocoumarin formation in parsley.
The Plant journal : for cell and molecular biology, 2014Co-Authors: Fazeelat Karamat, Alexandre Olry, Ryosuke Munakata, Takao Koeduka, Akifumi Sugiyama, Cédric Paris, Alain Hehn, Frédéric Bourgaud, Kazufumi YazakiAbstract:Summary Furanocoumarins constitute a sub-family of coumarin compounds with important defense properties against pathogens and insects, as well as allelopathic functions in plants. Furanocoumarins are divided into two sub-groups according to the alignment of the furan ring with the lactone structure: linear psoralen and angular angelicin derivatives. Determination of Furanocoumarin type is based on the prenylation position of the common precursor of all Furanocoumarins, umbelliferone, at C6 or C8, which gives rise to the psoralen or angelicin derivatives, respectively. Here, we identified a membrane-bound prenyltransferase PcPT from parsley (Petroselinum crispum), and characterized the properties of the gene product. PcPT expression in various parsley tissues is increased by UV irradiation, with a concomitant increase in Furanocoumarin production. This enzyme has strict substrate specificity towards umbelliferone and dimethylallyl diphosphate, and a strong preference for the C6 position of the prenylated product (demethylsuberosin), leading to linear Furanocoumarins. The C8-prenylated derivative (osthenol) is also formed, but to a much lesser extent. The PcPT protein is targeted to the plastids in planta. Introduction of this PcPT into the coumarin-producing plant Ruta graveolens showed increased consumption of endogenous umbelliferone. Expression of PcPT and a 4–coumaroyl CoA 2'–hydroxylase gene in Nicotiana benthamiana, which does not produce Furanocoumarins, resulted in formation of demethylsuberosin, indicating that Furanocoumarin production may be reconstructed by a metabolic engineering approach. The results demonstrate that a single prenyltransferase, such as PcPT, opens the pathway to linear Furanocoumarins in parsley, but may also catalyze the synthesis of osthenol, the first intermediate committed to the angular Furanocoumarin pathway, in other plants.
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Coumarin and Furanocoumarin Quantitation in Citrus Peel via Ultraperformance Liquid Chromatography Coupled with Mass Spectrometry (UPLC-MS)
Journal of Agricultural and Food Chemistry, 2013Co-Authors: Audray Dugrand, Alexandre Olry, Alain Hehn, Thibault Duval, Yann Froelicher, Frédéric BourgaudAbstract:Coumarins and Furanocoumarins are secondary metabolites commonly found in citrus plants. These molecules are allelochemical compounds in plants that have controversial effects on humans, such as phototoxicity and the commonly described interactions with drugs, referred to as the ``grapefruit juice effect''. Thus, it is important to develop a reliable method to identify and quantitate the coumarins and Furanocoumarins in citrus extracts. For this purpose, we herein describe an ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS)-based method. We first developed a rapid UPLC method (20 min) to separate the isomers of each Furanocoumarin. A subsequent single ion monitoring MS detection method was performed to distinguish between the molecules, which were possibly coeluting but had different molecular weights. The method was successfully used to separate and quantitate 6 coumarins and 21 Furanocoumarins in variable amounts within peel extracts (flayed and albedo) of 6 varieties of Citrus (sweet orange, lemon, grapefruit, bergamot, pummelo, and clementine). This method combines high selectivity and sensitivity in a rapid analysis and is useful for fingerprinting Citrus species via their coumarin and Furanocoumarin contents.
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CYP98A22, a phenolic ester 3'-hydroxylase specialized in the synthesis of chlorogenic acid, as a new tool for enhancing the Furanocoumarin concentration in Ruta graveolens
BMC Plant Biology, 2012Co-Authors: Fazeelat Karamat, Alexandre Olry, Frédéric Bourgaud, Sebastien Doerper, Guilhem Vialart, Pascaline Ullmann, D. Werck-reichhart, Alain HehnAbstract:Background: Furanocoumarins are molecules with proven therapeutic properties and are produced in only a small number of medicinal plant species such as Ruta graveolens. In vivo, these molecules play a protective role against phytophageous insect attack. Furanocoumarins are members of the phenylpropanoids family, and their biosynthetic pathway is initiated from p-coumaroyl coA. The enzymes belonging to the CYP98A cytochrome P450 family have been widely described as being aromatic meta-hydroxylases of various substrates, such as p-coumaroyl ester derivatives, and are involved in the synthesis of coumarins such as scopoletin. In Furanocoumarin-producing plants, these enzymes catalyze the step directly downstream of the junction with the Furanocoumarin biosynthetic pathway and might indirectly impact their synthesis. Results: In this work, we describe the cloning and functional characterization of the first CYP98A encoding gene isolated from R. graveolens. Using Nicotiana benthamiana as a heterologous expression system, we have demonstrated that this enzyme adds a 3-OH to p-coumaroyl ester derivatives but is more efficient to convert p-coumaroyl quinate into chlorogenic acid than to metabolize p-coumaroyl shikimate. Plants exposed to UV-B stress showed an enhanced expression level of the corresponding gene. The R. graveolens cyp98a22 open reading frame and the orthologous Arabidopsis thaliana cyp98a3 open reading frame were overexpressed in stable transgenic Ruta plants. Both plant series were analyzed for their production of scopoletin and Furanocoumarin. A detailed analysis indicates that both genes enhance the production of Furanocoumarins but that CYP98A22, unlike CYP98A3, doesn't affect the synthesis of scopoletin.
