The Experts below are selected from a list of 255 Experts worldwide ranked by ideXlab platform
Wolfgang Wohlleben - One of the best experts on this subject based on the ideXlab platform.
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Phosphinothricin tripeptide biosynthesis an original version of bacterial secondary metabolism
Phytochemistry, 2009Co-Authors: Eva Schinko, Klaus Schad, Sema Eys, Ullrich Keller, Wolfgang WohllebenAbstract:Streptomyces viridochromogenes Tu494 produces the herbicide phosphinothricyl-alanyl-alanine (Phosphinothricin-tripeptide=PTT; bialaphos). Its bioactive moiety Phosphinothricin competitively inhibits bacterial and plant glutamine synthetases. The biosynthesis of PTT includes the synthesis of the unusual amino acid N-acetyl-demethyl-Phosphinothricin and a three step condensation via non-ribosomal peptide synthetases. Two characteristics within the PTT biosynthesis make it suitable to study the evolution of secondary metabolism biosynthesis. First, PTT biosynthesis represents the only known system where all peptide synthetase modules are located on separate proteins. This 'single enzyme system' might be an archetype of the multimodular and multienzymatic non-ribosomal peptide synthetases in evolutionary terms. The second interesting feature of PTT biosynthesis is the pathway-specific aconitase Pmi that is involved in the supply of N-acetyl-demethyl-Phosphinothricin. Pmi is highly similar to the tricarboxylic acid aconitase AcnA. They share 64% identity at the DNA level and both belong to the Iron-Regulatory-Protein/AcnA family. Despite their high sequence similarity, AcnA and Pmi catalyze different reactions and are not able to substitute for each other. Thus, the enzyme pair AcnA/Pmi presents an example of the evolution of a secondary metabolite-specific enzyme from a primary metabolism enzyme.
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Inactivation of the tricarboxylic acid cycle aconitase gene from Streptomyces viridochromogenes Tu494 impairs morphological and physiological
1999Co-Authors: D. Schwartz, S. Kaspar, G. Kienzlen, K. Muschko, Wolfgang WohllebenAbstract:The tricarboxylic acid (TCA) cycle aconitase gene acnA from Streptomyces viridochromogenes Tü494 was cloned and analyzed. AcnA catalyzes the isomerization of citrate to isocitrate in the TCA cycle, as indicated by the ability of acnA to complement the aconitase-deficient Escherichia coli mutant JRG3259. An acnA mutant was unable to develop aerial mycelium and to sporulate, resulting in a bald phenotype. Furthermore, the mutant did not produce the antibiotic Phosphinothricin tripeptide, demonstrating that AcnA also affects physiological differentiation. The structurally identical antibiotics Phosphinothricin tripeptide (PTT) and bialaphos are produced by Streptomyces viridochromogenes Tü494 and S. hygroscopicus (1, 9), respectively. They consist of two molecules of L-alanine and one molecule of the unusual amino acid Phosphinothricin. In both organisms, several proteins and genes involved in PTT biosynthesis have been characterized (4, 15, 16, 18). By analyzing bialaphosnonproducing mutants of S. hygroscopicus, a putative biosynthetic pathway was postulated consisting of at least 13 biosyntheti
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Isolation and characterization of the PEP-phosphomutase and the phosphonopyruvate decarboxylase genes from the Phosphinothricin tripeptide producer Streptomyces viridochromogenes Tü494.
FEMS microbiology letters, 1998Co-Authors: D. Schwartz, J. Recktenwald, Stefan Pelzer, Wolfgang WohllebenAbstract:The previously isolated non-Phosphinothricin tripeptide producing Streptomyces viridochromogenes gene disruption mutant SP62/2 was used to identify and analyze genes encoding early steps of the Phosphinothricin tripeptide biosynthesis. Cross-feeding and bioconversion experiments between SP62/2 and known non-Phosphinothricin tripeptide producing mutants or presumptive Phosphinothricin tripeptide precursors revealed that SP62/2 was blocked in step one or two of the Phosphinothricin tripeptide biosynthesis. It was shown that the block in the biosynthesis is due to the integration of a temperature-sensitive plasmid by illegitimate recombination into the Phosphinothricin tripeptide biosynthetic gene cluster. The corresponding region was isolated from the wild-type. A 2.7-kb DNA fragment was analyzed comprising three ORFs (ppm, ppd, orfX) which are probably translationally coupled. The ppm gene encodes a protein which is similar to PEP-phosphomutases and the deduced Ppd product shows similarity to the phosphonopyruvate decarboxylase from Streptomyces wedmorensis.
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Genetic Analysis of Different Resistance Mechanisms Against the Herbicidal Antibiotic Phosphinothricyl-Alanyl-Alanine
Genetics and Product Formation in Streptomyces, 1991Co-Authors: Wolfgang Wohlleben, Alfred Pühler, Walter Arnold, Iris Behrmann, Inge Broer, Doris Hillemann, Eckhard StrauchAbstract:Streptomyces viridochromogenes Tu494, the producer of phosphinothricyl-alanyl-alanine (Ptt), is sensitive to its own antibiotic. Two phenotypically discernible Ptt-resistant S. viridochromogenes mutants, ES1 and ES2, were isolated and employed to clone resistance genes. Thus, two different DNA fragments both conferring Ptt resistance could be detected. The DNA regions including the resistance genes were sequenced and the gene products were investigated. The first gene (pat) encodes a Phosphinothricin N-acetyltransferase which inactivates the antibiotically effective component Phosphinothricin. Following modification of the 5’ region, the pat gene was transferred into plants and Phosphinothricin-resistant transgenic plants were obtained. The second gene, a glutamine synthetase (GS) gene mediated Ptt resistance in multi-copy state only. The gene product is heat-labile, and the deduced amino acid sequence was shown to be highly homologous to eucaryotic and to Rhizobiaceae GSII-type enzymes. Therefore the gene was named glnII. Southern hybridizations with different Streptomyces strains suggest that they all carry two types of GS genes, g1nA and g1nII.
William W. Metcalf - One of the best experts on this subject based on the ideXlab platform.
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An unusual carbon–carbon bond cleavage reaction during Phosphinothricin biosynthesis
Nature, 2009Co-Authors: Robert M. Cicchillo, Houjin Zhang, Joshua A. V. Blodgett, John T. Whitteck, Satish K. Nair, Wilfred A. Van Der Donk, William W. MetcalfAbstract:Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture. One such compound, Phosphinothricin tripeptide, contains the unusual amino acid Phosphinothricin attached to two alanine residues. Synthetic Phosphinothricin (glufosinate) is a component of two top-selling herbicides (Basta and Liberty), and is widely used with resistant transgenic crops including corn, cotton and canola. Recent genetic and biochemical studies showed that during Phosphinothricin tripeptide biosynthesis 2-hydroxyethylphosphonate (HEP) is converted to hydroxymethylphosphonate (HMP). Here we report the in vitro reconstitution of this unprecedented C(sp(3))-C(sp(3)) bond cleavage reaction and X-ray crystal structures of the enzyme. The protein is a mononuclear non-haem iron(ii)-dependent dioxygenase that converts HEP to HMP and formate. In contrast to most other members of this family, the oxidative consumption of HEP does not require additional cofactors or the input of exogenous electrons. The current study expands the scope of reactions catalysed by the 2-His-1-carboxylate mononuclear non-haem iron family of enzymes.
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an unusual carbon carbon bond cleavage reaction during Phosphinothricin biosynthesis
Nature, 2009Co-Authors: Robert M. Cicchillo, Houjin Zhang, Joshua A. V. Blodgett, John T. Whitteck, Satish K. Nair, Wilfred A. Van Der Donk, William W. MetcalfAbstract:Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture. One such compound, Phosphinothricin tripeptide, contains the unusual amino acid Phosphinothricin attached to two alanine residues. Synthetic Phosphinothricin (glufosinate) is a component of two top-selling herbicides (Basta and Liberty), and is widely used with resistant transgenic crops including corn, cotton and canola. Recent genetic and biochemical studies showed that during Phosphinothricin tripeptide biosynthesis 2-hydroxyethylphosphonate (HEP) is converted to hydroxymethylphosphonate (HMP). Here we report the in vitro reconstitution of this unprecedented C(sp(3))-C(sp(3)) bond cleavage reaction and X-ray crystal structures of the enzyme. The protein is a mononuclear non-haem iron(ii)-dependent dioxygenase that converts HEP to HMP and formate. In contrast to most other members of this family, the oxidative consumption of HEP does not require additional cofactors or the input of exogenous electrons. The current study expands the scope of reactions catalysed by the 2-His-1-carboxylate mononuclear non-haem iron family of enzymes.
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An unusual carbon–carbon bond cleavage reaction during Phosphinothricin biosynthesis
Nature, 2009Co-Authors: Robert M. Cicchillo, Houjin Zhang, Joshua A. V. Blodgett, John T. Whitteck, Satish K. Nair, Gongyong Li, Wilfred A. Van Der Donk, William W. MetcalfAbstract:Natural products containing phosphorus–carbon bonds have found widespread use in medicine and agriculture^ 1 . One such compound, Phosphinothricin tripeptide, contains the unusual amino acid Phosphinothricin attached to two alanine residues. Synthetic Phosphinothricin (glufosinate) is a component of two top-selling herbicides (Basta and Liberty), and is widely used with resistant transgenic crops including corn, cotton and canola. Recent genetic and biochemical studies showed that during Phosphinothricin tripeptide biosynthesis 2-hydroxyethylphosphonate (HEP) is converted to hydroxymethylphosphonate (HMP)^ 2 . Here we report the in vitro reconstitution of this unprecedented C( sp ^3)–C( sp ^3) bond cleavage reaction and X-ray crystal structures of the enzyme. The protein is a mononuclear non-haem iron( ii )-dependent dioxygenase that converts HEP to HMP and formate. In contrast to most other members of this family, the oxidative consumption of HEP does not require additional cofactors or the input of exogenous electrons. The current study expands the scope of reactions catalysed by the 2-His–1-carboxylate mononuclear non-haem iron family of enzymes. The Phosphinothricin tripeptide (PTT) is a naturally occurring compound that contains a phosphorous-carbon bond. The biosynthesis of PTT involves the conversion of hydroxyethylphosphonate (HEP) to hydroxymethylphosphonate (HMP). The enzyme responsible for this chemical transformation (hydroxyethylphosphonate dioxygenase or HEPD) is a mononuclear non-haem iron(II)-dependent dioxygenase, and it catalyses the cleavage of a C( sp ^3)–C( sp ^3) bond. Here Robert Cicchillo and colleagues solve the X-ray crystal structure of HEPD and biochemically explore the mechanism of this remarkable transformation. The biosynthesis of the naturally occurring compound Phosphinothricin tripeptide (PTT) involves the conversion of 2-hydroxyethylphosphonate (HEP) to hydroxymethylphosphonate (HMP). This requires the cleavage of a C( sp ^3)–C( sp ^3) bond. Here, the X-ray crystal structure of the enzyme that catalyzes this reaction (hydroxyethylphosphonate dioxygenase, HEPD) is solved, and the mechanism of this remarkable transformation is explored.
D. Schwartz - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of the tricarboxylic acid cycle aconitase gene from Streptomyces viridochromogenes Tu494 impairs morphological and physiological
1999Co-Authors: D. Schwartz, S. Kaspar, G. Kienzlen, K. Muschko, Wolfgang WohllebenAbstract:The tricarboxylic acid (TCA) cycle aconitase gene acnA from Streptomyces viridochromogenes Tü494 was cloned and analyzed. AcnA catalyzes the isomerization of citrate to isocitrate in the TCA cycle, as indicated by the ability of acnA to complement the aconitase-deficient Escherichia coli mutant JRG3259. An acnA mutant was unable to develop aerial mycelium and to sporulate, resulting in a bald phenotype. Furthermore, the mutant did not produce the antibiotic Phosphinothricin tripeptide, demonstrating that AcnA also affects physiological differentiation. The structurally identical antibiotics Phosphinothricin tripeptide (PTT) and bialaphos are produced by Streptomyces viridochromogenes Tü494 and S. hygroscopicus (1, 9), respectively. They consist of two molecules of L-alanine and one molecule of the unusual amino acid Phosphinothricin. In both organisms, several proteins and genes involved in PTT biosynthesis have been characterized (4, 15, 16, 18). By analyzing bialaphosnonproducing mutants of S. hygroscopicus, a putative biosynthetic pathway was postulated consisting of at least 13 biosyntheti
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Isolation and characterization of the PEP-phosphomutase and the phosphonopyruvate decarboxylase genes from the Phosphinothricin tripeptide producer Streptomyces viridochromogenes Tü494.
FEMS microbiology letters, 1998Co-Authors: D. Schwartz, J. Recktenwald, Stefan Pelzer, Wolfgang WohllebenAbstract:The previously isolated non-Phosphinothricin tripeptide producing Streptomyces viridochromogenes gene disruption mutant SP62/2 was used to identify and analyze genes encoding early steps of the Phosphinothricin tripeptide biosynthesis. Cross-feeding and bioconversion experiments between SP62/2 and known non-Phosphinothricin tripeptide producing mutants or presumptive Phosphinothricin tripeptide precursors revealed that SP62/2 was blocked in step one or two of the Phosphinothricin tripeptide biosynthesis. It was shown that the block in the biosynthesis is due to the integration of a temperature-sensitive plasmid by illegitimate recombination into the Phosphinothricin tripeptide biosynthetic gene cluster. The corresponding region was isolated from the wild-type. A 2.7-kb DNA fragment was analyzed comprising three ORFs (ppm, ppd, orfX) which are probably translationally coupled. The ppm gene encodes a protein which is similar to PEP-phosphomutases and the deduced Ppd product shows similarity to the phosphonopyruvate decarboxylase from Streptomyces wedmorensis.
Lirong Yang - One of the best experts on this subject based on the ideXlab platform.
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solubility of l Phosphinothricin andits hydrochloride in water methanol and ethanol binary solventmixtures from 278 15 to 318 15 k
Journal of Chemical & Engineering Data, 2019Co-Authors: Yayun Liu, Lijun Meng, Xinjian Yin, Haisheng Zhou, Lirong YangAbstract:The solubility of l-Phosphinothricin (l-PPT) and its hydrochloride in water + (methanol and ethanol) binary solvent mixtures was experimentally measured from 278.15 to 318.15 K by a static analytic...
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Solubility of l‑Phosphinothricin andIts Hydrochloride in Water + (Methanol and Ethanol) Binary SolventMixtures from 278.15 to 318.15 K
Journal of Chemical & Engineering Data, 2019Co-Authors: Liu Yayun, Lijun Meng, Yin Xinjian, Zhou Haisheng, Lirong YangAbstract:The solubility of l-Phosphinothricin (l-PPT) and its hydrochloride in water + (methanol and ethanol) binary solvent mixtures was experimentally measured from 278.15 to 318.15 K by a static analytic...
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efficient reductive amination process for enantioselective synthesis of l Phosphinothricin applying engineered glutamate dehydrogenase
Applied Microbiology and Biotechnology, 2018Co-Authors: Jianping Wu, Lirong YangAbstract:The objective of this study was to identify and exploit a robust biocatalyst that can be applied in reductive amination for enantioselective synthesis of the competitive herbicide L-Phosphinothricin. Applying a genome mining-based library construction strategy, eight NADPH-specific glutamate dehydrogenases (GluDHs) were identified for reductively aminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) to L-Phosphinothricin. Among them, the glutamate dehydrogenase cloned from Pseudomonas putida (PpGluDH) exhibited relatively high catalytic activity and favorable soluble expression. This enzyme was purified to homogeneity for further characterization. The specific activity of PpGluDH was 296.1 U/g-protein, which is significantly higher than the reported value for a GluDH. To the best of our knowledge, there has not been any report on protein engineering of GluDH for PPO-oriented activity. Taking full advantage of the available information and the diverse characteristics of the enzymes in the enzyme library, PpGluDH was engineered by site-directed mutation based on multiple sequence alignment. The mutant I170M, which had 2.1-fold enhanced activity, was successfully produced. When the I170M mutant was applied in the batch production of L-Phosphinothricin, it showed markedly improved catalytic efficiency compared with the wild type enzyme. The conversion reached 99% (0.1 M PPO) with an L-Phosphinothricin productivity of 1.35 g/h·L, which far surpassed the previously reported level. These results show that PpGluDH I170M is a promising biocatalyst for highly enantioselective synthesis of L-Phosphinothricin by reductive amination.
German Spangenberg - One of the best experts on this subject based on the ideXlab platform.
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transgenic plants of tall fescue festuca arundinacea schreb obtained by direct gene transfer to protoplasts
Nature Biotechnology, 1992Co-Authors: Z Y Wang, Tadashi Takamizo, Victor A Iglesias, Martin Osusky, J Nagel, Ingo Potrykus, German SpangenbergAbstract:Chimeric hygromycin phosphotransferase (hph) and Phosphinothricin acetyltransferase (bar) genes were introduced, using polyethylene glycol treatment, into protoplasts isolated from embryogenic cell suspension cultures of tall fescue (Festuca arundinacea Schreb.), a graminaceous plant that is an important forage crop in temperate pastures. Colonies resistant to either 200 mg/l hygromycin or 100 mg/l Phosphinothricin, respectively, were recovered upon selection using bead-type culture systems. Stable integration of the transgenes in the genomes of plants regenerated from resistant callus clones was shown by Southern hybridization analysis. In situ hybridization of a labeled transgene-probe to metaphase chromosomes is shown for one transgenic primary regenerant. Expression of the transgenes in mature plants was demonstrated by HPH enzyme assay or by Phosphinothricin-herbicide spraying.
