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Dieter Spiteller - One of the best experts on this subject based on the ideXlab platform.
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identification of the biosynthetic gene cluster for 3 Methylarginine a toxin produced by pseudomonas syringae pv syringae 22d 93
Applied and Environmental Microbiology, 2010Co-Authors: Sascha D Braun, Beate Volksch, J Hofmann, Annette Wensing, Matthias S Ullrich, Helge Weingart, Dieter SpitellerAbstract:The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-Methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km, 7 mM; kcat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.
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Identification of the biosynthetic gene cluster for 3-Methylarginine, a toxin produced by Pseudomonas syringae pv. syringae 22d/93
Applied and Environmental Microbiology, 2010Co-Authors: Sascha D Braun, Beate Volksch, J Hofmann, Annette Wensing, Matthias S Ullrich, Helge Weingart, Dieter SpitellerAbstract:The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-Methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km, 7 mM; kcat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.
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3 Methylarginine from pseudomonas syringae pv syringae 22d 93 suppresses the bacterial blight caused by its close relative pseudomonas syringae pv glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-Methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-Methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-Methylarginine biosynthesis to be identified.
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3-Methylarginine from Pseudomonas syringae pv. syringae 22d/93 suppresses the bacterial blight caused by its close relative Pseudomonas syringae pv. glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-Methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-Methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-Methylarginine biosynthesis to be identified.
Sascha D Braun - One of the best experts on this subject based on the ideXlab platform.
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identification of the biosynthetic gene cluster for 3 Methylarginine a toxin produced by pseudomonas syringae pv syringae 22d 93
Applied and Environmental Microbiology, 2010Co-Authors: Sascha D Braun, Beate Volksch, J Hofmann, Annette Wensing, Matthias S Ullrich, Helge Weingart, Dieter SpitellerAbstract:The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-Methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km, 7 mM; kcat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.
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Identification of the biosynthetic gene cluster for 3-Methylarginine, a toxin produced by Pseudomonas syringae pv. syringae 22d/93
Applied and Environmental Microbiology, 2010Co-Authors: Sascha D Braun, Beate Volksch, J Hofmann, Annette Wensing, Matthias S Ullrich, Helge Weingart, Dieter SpitellerAbstract:The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-Methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km, 7 mM; kcat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.
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3 Methylarginine from pseudomonas syringae pv syringae 22d 93 suppresses the bacterial blight caused by its close relative pseudomonas syringae pv glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-Methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-Methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-Methylarginine biosynthesis to be identified.
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3-Methylarginine from Pseudomonas syringae pv. syringae 22d/93 suppresses the bacterial blight caused by its close relative Pseudomonas syringae pv. glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-Methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-Methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-Methylarginine biosynthesis to be identified.
Beate Volksch - One of the best experts on this subject based on the ideXlab platform.
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identification of the biosynthetic gene cluster for 3 Methylarginine a toxin produced by pseudomonas syringae pv syringae 22d 93
Applied and Environmental Microbiology, 2010Co-Authors: Sascha D Braun, Beate Volksch, J Hofmann, Annette Wensing, Matthias S Ullrich, Helge Weingart, Dieter SpitellerAbstract:The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-Methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km, 7 mM; kcat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.
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Identification of the biosynthetic gene cluster for 3-Methylarginine, a toxin produced by Pseudomonas syringae pv. syringae 22d/93
Applied and Environmental Microbiology, 2010Co-Authors: Sascha D Braun, Beate Volksch, J Hofmann, Annette Wensing, Matthias S Ullrich, Helge Weingart, Dieter SpitellerAbstract:The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-Methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km, 7 mM; kcat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.
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3 Methylarginine from pseudomonas syringae pv syringae 22d 93 suppresses the bacterial blight caused by its close relative pseudomonas syringae pv glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-Methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-Methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-Methylarginine biosynthesis to be identified.
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3-Methylarginine from Pseudomonas syringae pv. syringae 22d/93 suppresses the bacterial blight caused by its close relative Pseudomonas syringae pv. glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-Methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-Methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-Methylarginine biosynthesis to be identified.
Marc R Wilkins - One of the best experts on this subject based on the ideXlab platform.
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analysis of the proteome of saccharomyces cerevisiae for Methylarginine
Journal of Proteome Research, 2013Co-Authors: Gene Hartsmith, Melissa A Erce, Marc R WilkinsAbstract:Arginine methylation is a post-translational modification that has been implicated in a plethora of cellular processes. In the present manuscript, using two antiMethylarginine antibodies and combinatorial deletion mutants of arginine methyltransferases, we found evidence of widespread arginine methylation in the Saccharomyces cerevisiae proteome. Immunoprecipitation was used for enrichment of Methylarginine-containing proteins, which were identified via tandem mass spectrometry. From this, we identified a total of 90 proteins, of which 5 were previously known to be methylated. The proteins identified were involved in known Methylarginine-associated biological functions such as RNA processing, nuclear transport, carbohydrate metabolic process, GMP biosynthetic process and protein folding. Through in vivo methylation by the incorporation of [3H]-methyl groups, we validated the methylation of 7 proteins (Ded1, Imd4, Lhp1, Nop1, Cdc11, Gus1, Pob3). By LC–MS/MS, we then confirmed a total of 15 novel methylargi...
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Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry
Journal of The American Society for Mass Spectrometry, 2012Co-Authors: Gene Hart-smith, Melissa A Erce, Marc R WilkinsAbstract:When localizing protein post-translational modifications (PTMs) using liquid-chromatography (LC)-tandem mass spectrometry (MS/MS), existing implementations are limited by inefficient selection of PTM-carrying peptides for MS/MS, particularly when PTM site occupancy is sub-stoichiometric. The present contribution describes a method by which peptides carrying specific PTMs of interest—in this study, Methylarginines—may be selectively targeted for MS/MS: peptide features are extracted from high mass accuracy single-stage MS data, searched against theoretical PTM-carrying peptide masses, and matching features are subjected to targeted data acquisition LC-MS/MS. Using trypsin digested Saccharomyces cerevisiae Npl3, in which evidence is presented for 18 Methylarginine sites—17 of which fall within a glycine-arginine-rich (GAR) domain spanning
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enhanced Methylarginine characterization by post translational modification specific targeted data acquisition and electron transfer dissociation mass spectrometry
Journal of the American Society for Mass Spectrometry, 2012Co-Authors: Gene Hartsmith, Melissa A Erce, Marc R WilkinsAbstract:When localizing protein post-translational modifications (PTMs) using liquid-chromatography (LC)-tandem mass spectrometry (MS/MS), existing implementations are limited by inefficient selection of PTM-carrying peptides for MS/MS, particularly when PTM site occupancy is sub-stoichiometric. The present contribution describes a method by which peptides carrying specific PTMs of interest—in this study, Methylarginines—may be selectively targeted for MS/MS: peptide features are extracted from high mass accuracy single-stage MS data, searched against theoretical PTM-carrying peptide masses, and matching features are subjected to targeted data acquisition LC-MS/MS. Using trypsin digested Saccharomyces cerevisiae Npl3, in which evidence is presented for 18 Methylarginine sites—17 of which fall within a glycine-arginine-rich (GAR) domain spanning <120 amino acids—it is shown that this approach outperforms conventional data dependent acquisition (DDA): when applied to a complex protein mixture featuring in vivo methylated Npl3, 95 % more (P = 0.030) Methylarginine-carrying peptides are selected for MS/MS than DDA, leading to an 86 % increase (P = 0.044) in the number of methylated peptides producing Mascot ion scores ≥20 following electron-transfer dissociation (ETD). Notably, significantly more low abundance arginine methylated peptides (maximum ion intensities <6 × 104 cps) are selected for MS/MS using this approach relative to DDA (50 % more in a digest of purified in vitro methylated Npl3). It is also demonstrated that relative to collision-induced dissociation (CID), ETD facilitates a 586 % increase (P = 0.016) in average Mascot ion scores of Methylarginine-carrying peptides. The present PTM-specific targeted data acquisition approach, though described using Methylarginine, is applicable to any ionizable PTM of known mass.
Jorg Nuske - One of the best experts on this subject based on the ideXlab platform.
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3 Methylarginine from pseudomonas syringae pv syringae 22d 93 suppresses the bacterial blight caused by its close relative pseudomonas syringae pv glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-Methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-Methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-Methylarginine biosynthesis to be identified.
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3-Methylarginine from Pseudomonas syringae pv. syringae 22d/93 suppresses the bacterial blight caused by its close relative Pseudomonas syringae pv. glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-Methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-Methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-Methylarginine biosynthesis to be identified.