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Daniel Wiegmann - One of the best experts on this subject based on the ideXlab platform.
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pyridoxal 5 phosphate dependent alkyl transfer in nucleoside antibiotic biosynthesis
Nature Chemical Biology, 2020Co-Authors: Zheng Cui, Jonathan Overbay, Xiachang Wang, Xiaodong Liu, Yinan Zhang, Minakshi Bhardwaj, Anke Lemke, Daniel WiegmannAbstract:Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5′S,6′S)-5′-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR–GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-l-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5′-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway. Rather than a typical S-adenosylmethionine-dependent alkyltransferase, the installation of the N-alkylamine linker in several nucleoside antibiotics is catalyzed via γ-replacement by a pyridoxal-5′-phosphate-dependent aminobutyryltransferase.
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pyridoxal 5 phosphate dependent alkyl transfer in nucleoside antibiotic biosynthesis
Nature Chemical Biology, 2020Co-Authors: Zheng Cui, Jonathan Overbay, Xiachang Wang, Xiaodong Liu, Yinan Zhang, Minakshi Bhardwaj, Anke Lemke, Daniel WiegmannAbstract:Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5'S,6'S)-5'-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-L-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5'-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.
Xiachang Wang - One of the best experts on this subject based on the ideXlab platform.
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pyridoxal 5 phosphate dependent alkyl transfer in nucleoside antibiotic biosynthesis
Nature Chemical Biology, 2020Co-Authors: Zheng Cui, Jonathan Overbay, Xiachang Wang, Xiaodong Liu, Yinan Zhang, Minakshi Bhardwaj, Anke Lemke, Daniel WiegmannAbstract:Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5′S,6′S)-5′-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR–GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-l-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5′-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway. Rather than a typical S-adenosylmethionine-dependent alkyltransferase, the installation of the N-alkylamine linker in several nucleoside antibiotics is catalyzed via γ-replacement by a pyridoxal-5′-phosphate-dependent aminobutyryltransferase.
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pyridoxal 5 phosphate dependent alkyl transfer in nucleoside antibiotic biosynthesis
Nature Chemical Biology, 2020Co-Authors: Zheng Cui, Jonathan Overbay, Xiachang Wang, Xiaodong Liu, Yinan Zhang, Minakshi Bhardwaj, Anke Lemke, Daniel WiegmannAbstract:Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5'S,6'S)-5'-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-L-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5'-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.
Yinan Zhang - One of the best experts on this subject based on the ideXlab platform.
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pyridoxal 5 phosphate dependent alkyl transfer in nucleoside antibiotic biosynthesis
Nature Chemical Biology, 2020Co-Authors: Zheng Cui, Jonathan Overbay, Xiachang Wang, Xiaodong Liu, Yinan Zhang, Minakshi Bhardwaj, Anke Lemke, Daniel WiegmannAbstract:Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5′S,6′S)-5′-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR–GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-l-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5′-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway. Rather than a typical S-adenosylmethionine-dependent alkyltransferase, the installation of the N-alkylamine linker in several nucleoside antibiotics is catalyzed via γ-replacement by a pyridoxal-5′-phosphate-dependent aminobutyryltransferase.
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pyridoxal 5 phosphate dependent alkyl transfer in nucleoside antibiotic biosynthesis
Nature Chemical Biology, 2020Co-Authors: Zheng Cui, Jonathan Overbay, Xiachang Wang, Xiaodong Liu, Yinan Zhang, Minakshi Bhardwaj, Anke Lemke, Daniel WiegmannAbstract:Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5'S,6'S)-5'-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-L-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5'-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.
Scott M Laster - One of the best experts on this subject based on the ideXlab platform.
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mast cell degranulation and calcium influx are inhibited by an echinacea purpurea extract and the Alkylamide dodeca 2e 4e dienoic acid isobutylamide
Journal of Ethnopharmacology, 2018Co-Authors: Travis V. Gulledge, Nadja B Cech, Daniel A. Todd, Nicholas Collette, Emily Mackey, Stephanie E Johnstone, Yasamin Moazami, Adam J Moeser, Joshua G Pierce, Scott M LasterAbstract:Abstract Ethnopharmacological relevance Native Americans used plants from the genus Echinacea to treat a variety of different inflammatory conditions including swollen gums, sore throats, skin inflammation, and gastrointestinal disorders. Today, various Echinacea spp. preparations are used primarily to treat upper respiratory infections. Aim of the study The goal of this study was to evaluate the effects of an ethanolic E. purpurea (L) Moench root extract and the Alkylamide dodeca-2E,4E-dienoic acid isobutylamide (A15) on mast cells, which are important mediators of allergic and inflammatory responses. Inhibition of mast cell activation may help explain the traditional use of Echinacea. Materials and methods A15 was evaluated for its effects on degranulation, calcium influx, cytokine and lipid mediator production using bone marrow derived mast cells (BMMCs) and the transformed rat basophilic leukemia mast cell line RBL-2H3. Methods included enzymatic assays, fluorimetry, ELISAs, and microscopy. A root extract of E. purpurea, and low and high Alkylamide-containing fractions prepared from this extract, were also tested for effects on mast cell function. Finally, we tested A15 for effects on calcium responses in RAW 264.7 macrophage and Jurkat T cell lines. Results A15 inhibited s-hexosaminidase release from BMMCs and RBL-2H3 cells after treatment with the calcium ionophore A23187 by 83.5% and 48.4% at 100 µM, respectively. Inhibition also occurred following stimulation with IgE anti-DNP/DNP-HSA. In addition, A15 inhibited 47% of histamine release from A23187-treated RBL-2H3 cells. A15 prevented the rapid rise in intracellular calcium following FceRI crosslinking and A23187 treatment suggesting it acts on the signals controlling granule release. An E. purpurea root extract and a fraction with high Alkylamide content derived from this extract also displayed these activities while fractions with little to no detectable amounts of Alkylamide did not. A15 mediated inhibition of calcium influx was not limited to mast cells as A23187-stimulated calcium influx was blocked in both RAW 264.7 and Jurkat cell lines with 60.2% and 43.6% inhibition at 1 min post-stimulation, respectively. A15 also inhibited the release of TNF-α, and PGE2 to a lesser degree, following A23187 stimulation indicating its broad activity on mast cell mediator production. Conclusions These findings suggest that Echinacea extracts and Alkylamides may be useful for treating allergic and inflammatory responses mediated by mast cells. More broadly, since calcium is a critical second messenger, the inhibitory effects of Alkylamides on calcium uptake would be predicted to dampen a variety of pathological responses, suggesting new uses for this plant and its constituents.
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synthesis and biological evaluation of a series of fatty acid amides from echinacea
Bioorganic & Medicinal Chemistry Letters, 2015Co-Authors: Yasamin Moazami, Travis V. Gulledge, Scott M Laster, Joshua G PierceAbstract:Abstract Alkylamides are lipophilic constituents of Echinacea and possess numerous biological activities. Although significant effort has been focused on the study of crude Echinacea extracts, very little is known regarding the activities of the individual constituents that make up these herbal treatments. Herein we explore the SAR of simple Alkylamides found in Echinacea extracts with respect to their ability to decrease the production of the pro-inflammatory mediator TNF-α. Our results have revealed the key structural requirements for activity and provide lead compounds for further investigation of these poorly understood molecules.
Travis V. Gulledge - One of the best experts on this subject based on the ideXlab platform.
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mast cell degranulation and calcium influx are inhibited by an echinacea purpurea extract and the Alkylamide dodeca 2e 4e dienoic acid isobutylamide
Journal of Ethnopharmacology, 2018Co-Authors: Travis V. Gulledge, Nadja B Cech, Daniel A. Todd, Nicholas Collette, Emily Mackey, Stephanie E Johnstone, Yasamin Moazami, Adam J Moeser, Joshua G Pierce, Scott M LasterAbstract:Abstract Ethnopharmacological relevance Native Americans used plants from the genus Echinacea to treat a variety of different inflammatory conditions including swollen gums, sore throats, skin inflammation, and gastrointestinal disorders. Today, various Echinacea spp. preparations are used primarily to treat upper respiratory infections. Aim of the study The goal of this study was to evaluate the effects of an ethanolic E. purpurea (L) Moench root extract and the Alkylamide dodeca-2E,4E-dienoic acid isobutylamide (A15) on mast cells, which are important mediators of allergic and inflammatory responses. Inhibition of mast cell activation may help explain the traditional use of Echinacea. Materials and methods A15 was evaluated for its effects on degranulation, calcium influx, cytokine and lipid mediator production using bone marrow derived mast cells (BMMCs) and the transformed rat basophilic leukemia mast cell line RBL-2H3. Methods included enzymatic assays, fluorimetry, ELISAs, and microscopy. A root extract of E. purpurea, and low and high Alkylamide-containing fractions prepared from this extract, were also tested for effects on mast cell function. Finally, we tested A15 for effects on calcium responses in RAW 264.7 macrophage and Jurkat T cell lines. Results A15 inhibited s-hexosaminidase release from BMMCs and RBL-2H3 cells after treatment with the calcium ionophore A23187 by 83.5% and 48.4% at 100 µM, respectively. Inhibition also occurred following stimulation with IgE anti-DNP/DNP-HSA. In addition, A15 inhibited 47% of histamine release from A23187-treated RBL-2H3 cells. A15 prevented the rapid rise in intracellular calcium following FceRI crosslinking and A23187 treatment suggesting it acts on the signals controlling granule release. An E. purpurea root extract and a fraction with high Alkylamide content derived from this extract also displayed these activities while fractions with little to no detectable amounts of Alkylamide did not. A15 mediated inhibition of calcium influx was not limited to mast cells as A23187-stimulated calcium influx was blocked in both RAW 264.7 and Jurkat cell lines with 60.2% and 43.6% inhibition at 1 min post-stimulation, respectively. A15 also inhibited the release of TNF-α, and PGE2 to a lesser degree, following A23187 stimulation indicating its broad activity on mast cell mediator production. Conclusions These findings suggest that Echinacea extracts and Alkylamides may be useful for treating allergic and inflammatory responses mediated by mast cells. More broadly, since calcium is a critical second messenger, the inhibitory effects of Alkylamides on calcium uptake would be predicted to dampen a variety of pathological responses, suggesting new uses for this plant and its constituents.
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synthesis and biological evaluation of a series of fatty acid amides from echinacea
Bioorganic & Medicinal Chemistry Letters, 2015Co-Authors: Yasamin Moazami, Travis V. Gulledge, Scott M Laster, Joshua G PierceAbstract:Abstract Alkylamides are lipophilic constituents of Echinacea and possess numerous biological activities. Although significant effort has been focused on the study of crude Echinacea extracts, very little is known regarding the activities of the individual constituents that make up these herbal treatments. Herein we explore the SAR of simple Alkylamides found in Echinacea extracts with respect to their ability to decrease the production of the pro-inflammatory mediator TNF-α. Our results have revealed the key structural requirements for activity and provide lead compounds for further investigation of these poorly understood molecules.
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ethanolic echinacea purpurea extracts contain a mixture of cytokine suppressive and cytokine inducing compounds including some that originate from endophytic bacteria
PLOS ONE, 2015Co-Authors: Daniel A. Todd, Travis V. Gulledge, Emily R. Britton, Martina Oberhofer, Ashley N. Moody, Tatsiana Shymanovich, Laura F. Grubbs, Monika Juzumaite, Martha Leytelugo, Tyler N. GrafAbstract:Echinacea preparations, which are used for the prevention and treatment of upper respiratory infections, account for 10% of the dietary supplement market in the U.S., with sales totaling more than $100 million annually. In an attempt to shed light on Echinacea's mechanism of action, we evaluated the effects of a 75% ethanolic root extract of Echinacea purpurea, prepared in accord with industry methods, on cytokine and chemokine production from RAW 264.7 macrophage-like cells. We found that the extract displayed dual activities; the extract could itself stimulate production of the cytokine TNF-α, and also suppress production of TNF-α in response to stimulation with exogenous LPS. Liquid:liquid partitioning followed by normal-phase flash chromatography resulted in separation of the stimulatory and inhibitory activities into different fractions, confirming the complex nature of this extract. We also studied the role of Alkylamides in the suppressive activity of this E. purpurea extract. Our fractionation method concentrated the Alkylamides into a single fraction, which suppressed production of TNF-α, CCL3, and CCL5; however fractions that did not contain detectable Alkylamides also displayed similar suppressive effects. Alkylamides, therefore, likely contribute to the suppressive activity of the extract but are not solely responsible for that activity. From the fractions without detectable Alkylamides, we purified xanthienopyran, a compound not previously known to be a constituent of the Echinacea genus. Xanthienopyran suppressed production of TNF-α suggesting that it may contribute to the suppressive activity of the crude ethanolic extract. Finally, we show that ethanolic extracts prepared from E. purpurea plants grown under sterile conditions and from sterilized seeds, do not contain LPS and do not stimulate macrophage production of TNF-α, supporting the hypothesis that the macrophage-stimulating activity in E. purpurea extracts can originate from endophytic bacteria. Together, our findings indicate that ethanolic E. purpurea extracts contain multiple constituents that differentially regulate cytokine production by macrophages.
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Alkylamide content of an Echinacea purpurea extract, chloroform layer, and column fractions from the chloroform layer.
2015Co-Authors: Daniel A. Todd, Travis V. Gulledge, Emily R. Britton, Martina Oberhofer, Martha Leyte-lugo, Ashley N. Moody, Tatsiana Shymanovich, Laura F. Grubbs, Monika Juzumaite, Tyler N. GrafAbstract:a. Concentration is reported for one of the major Alkylamides in the extract, Alkylamide 5 in Fig 3 (dodeca-2E,4Z-diene-8,10-diynoic acid isobutylamide). The total Alkylamide concentration was estimated using the calibration curve for this compound. Reported concentrations are means+/- SD from triplicate measurements by LC-MS as described in the Materials and Methods.b. Below LOQ (limit of quantification) indicates that alkyamides were detected but were present at concentrations below the limit of quantitation for the method.c. ND indicates that Alkylamides were not detected, i.e. not present at levels above the limit of detection (LOD).Alkylamide content of an Echinacea purpurea extract, chloroform layer, and column fractions from the chloroform layer.
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Alkylamides from Echinacea purpurea.
2015Co-Authors: Daniel A. Todd, Travis V. Gulledge, Emily R. Britton, Martina Oberhofer, Martha Leyte-lugo, Ashley N. Moody, Tatsiana Shymanovich, Laura F. Grubbs, Monika Juzumaite, Tyler N. GrafAbstract:Chromatogram (A) of the crude chloroform layer (CL) from the E. purpurea extract showing the presence of a series of Alkylamides (3,4,5,8,9,10,11A,11B,14,15) which were identified according to molecular weight and retention time. The relative amounts of these Alkylamides in the silica gel column pooled fractions (1–13) are indicated by the peak areas shown in panel B. The numbering system used for these Alkylamides is consistent with that used in a previous report [18]. * indicates compounds detected, but not identified. The structures for seven Alkylamides selected to represent the Alkylamide content are shown in panel C.