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Jacob Schaefer - One of the best experts on this subject based on the ideXlab platform.

  • Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate Synthase
    Journal of Biomolecular NMR, 2004
    Co-Authors: Lynda M. Mcdowell, Daniel R. Studelska, Robert D. O'connor, Barbara Poliks, Denise D. Beusen, Jacob Schaefer
    Abstract:

    The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) Synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP Synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP Synthase–S3P–Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP Synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native ^31P in S3P and Glp, biosynthetically ^13C-labeled S3P, specifically ^13C and ^15N labeled Glp, and a variety of protein-^15N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP Synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.

  • LIGAND GEOMETRY OF THE TERNARY COMPLEX OF 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE Synthase FROM ROTATIONAL-ECHO DOUBLE-RESONANCE NMR
    Biochemistry, 1996
    Co-Authors: Lynda M. Mcdowell, Christopher A. Klug, Denise D. Beusen, Jacob Schaefer
    Abstract:

    The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) Synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP. The reaction is inhibited by N-(phosphonomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP Synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) to determine intra- and interligand internuclear distances. DRAMA was used to determine the single 31P-31P distance, while REDOR was used to determine one 31P-15N distance and five 31P-13C distances. These experimental distances were used as restraints in molecular dynamics simulations of an S3P-Glp complex to examine the geometry of the two ligands relative to one another in the ternary complex. The simulations were compared to unrestrained simulations of the EPSP Synthase tetrahedral intermediate and its phosphonate analog. The results suggest that Glp is unlikely to bind in the same fashion as PEP, a conclusion that is consistent with recent studies that have questioned the role of Glp as a transition-state or intermediate analog.

  • Structural Constraints on the Ternary Complex of 5-Enolpyruvylshikimate-3-phosphate Synthase from Rotational-echo Double-resonance NMR
    Journal of molecular biology, 1996
    Co-Authors: Lynda M. Mcdowell, Daniel R. Studelska, Asher Schmidt, Eric R. Cohen, Jacob Schaefer
    Abstract:

    Abstract The 46 kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) Synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phospho enolpyruvate to form EPSP. The reaction is inhibited by N -(phosphono- methyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP Synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, 15 N labels were introduced selectively into the lysine, arginine and histidine residues of EPSP Synthase and distances to a 13 C label in Glp and to the 31 P in S3P and Glp were measured by rotational-echo double-resonance NMR. Three lysine and four arginine residues are in the proximity of the phosphate group of S3P and the carboxyl and phosphonate groups of Glp. A single histidine residue is in the vicinity of the binding site (closer to Glp than to S3P) but is more distant than the lysine and arginine residues.

Celso Omoto - One of the best experts on this subject based on the ideXlab platform.

  • high levels of biological activity of cry1ac protein expressed on mon 87701 mon 89788 soybean against heliothis virescens lepidoptera noctuidae
    Pest Management Science, 2014
    Co-Authors: Oderlei Bernardi, Geraldo Ubirajara Berger, Patrick Marques Dourado, Renato Assis De Carvalho, Samuel Martinelli, Graham Phillip Head, Celso Omoto
    Abstract:

    Background Genetically modified MON 87701 × MON 89788 soybean (Glycine max) that expresses the Cry1Ac and EPSP-Synthase proteins is a new Integrated Pest Management (IPM) tool for controlling Heliothis virescens in Brazil. To Support Insect Resistance Management (IRM) programs and understand the value of this event for IPM, we conducted laboratory and field studies to assess the biological activity of Cry1Ac protein expressed on Bt soybean against this insect pest. Results Heliothis virescens was highly susceptible to purified Cry1Ac protein [LC50 (FL 95%) = 0.026 (0.021–0.033) µg Cry1Ac mL–1 diet]. In bioassays with freeze-dried MON 87701 × MON 89788 soybean tissue diluted 25 times in an artificial diet, there was 100% mortality of H. virescens. In bioassays with leaf disc, young trifoliate, flower buds, pods and high artificial infestation under greenhouse condition, MON 87701 × MON 89788 soybean showed a high level of control against H. virescens. Moreover, larvae from first through fifth instar were highly susceptible to MON 87701 × MON 89788 soybean. Conclusions The MON 87701 × MON 89788 soybean provides an effective alternative in controlling Heliothis virescens and complies with the concept of high-dose for IRM programs. © 2013 Society of Chemical Industry

  • assessment of the high dose concept and level of control provided by mon 87701 mon 89788 soybean against anticarsia gemmatalis and pseudoplusia includens lepidoptera noctuidae in brazil
    Pest Management Science, 2012
    Co-Authors: Oderlei Bernardi, Patrick Marques Dourado, Samuel Martinelli, Graham Phillip Head, Glaucia Sossai Malvestiti, Wladecir Salles De Oliveira, Geraldo Ubrajara Berger, Celso Omoto
    Abstract:

    BACKGROUND: Genetically modified MON 87701 × MON 89788 soybean (Glycine max), which expresses the Cry1Ac and EPSP-Synthase proteins, has been registered for commercial use in Brazil. To develop an Insect Resistance Management (IRM) program for this event, laboratory and field studies were conducted to assess the high-dose concept and level of control it provides against Anticarsia gemmatalis and Pseudoplusia includens. RESULTS: The purified Cry1Ac protein was more active against A. gemmatalis [LC50 (FL 95%) = 0.23 (0.15–0.34) µg Cry1Ac mL−1 diet] than P. includens [LC50 (FL 95%) = 3.72 (2.65–4.86) µg Cry1Ac mL−1 diet]. In bioassays with freeze-dried MON 87701 × MON 89788 soybean tissue diluted 25 times in an artificial diet, there was 100% mortality of A. gemmatalis and up to 95.79% mortality for P. includens. In leaf-disc bioassays and under conditions of high artificial infestation in the greenhouse and natural infestation in the field, MON 87701 × MON 89788 soybean showed a high level of efficacy against both target pests. CONCLUSIONS: The MON 87701 × MON 89788 soybean provides a high level of control against A. gemmatalis and P. includes, but a high-dose event only to A. gemmatalis. Copyright © 2012 Society of Chemical Industry

Lynda M. Mcdowell - One of the best experts on this subject based on the ideXlab platform.

  • Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate Synthase
    Journal of Biomolecular NMR, 2004
    Co-Authors: Lynda M. Mcdowell, Daniel R. Studelska, Robert D. O'connor, Barbara Poliks, Denise D. Beusen, Jacob Schaefer
    Abstract:

    The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) Synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP Synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP Synthase–S3P–Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP Synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native ^31P in S3P and Glp, biosynthetically ^13C-labeled S3P, specifically ^13C and ^15N labeled Glp, and a variety of protein-^15N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP Synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.

  • LIGAND GEOMETRY OF THE TERNARY COMPLEX OF 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE Synthase FROM ROTATIONAL-ECHO DOUBLE-RESONANCE NMR
    Biochemistry, 1996
    Co-Authors: Lynda M. Mcdowell, Christopher A. Klug, Denise D. Beusen, Jacob Schaefer
    Abstract:

    The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) Synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP. The reaction is inhibited by N-(phosphonomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP Synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) to determine intra- and interligand internuclear distances. DRAMA was used to determine the single 31P-31P distance, while REDOR was used to determine one 31P-15N distance and five 31P-13C distances. These experimental distances were used as restraints in molecular dynamics simulations of an S3P-Glp complex to examine the geometry of the two ligands relative to one another in the ternary complex. The simulations were compared to unrestrained simulations of the EPSP Synthase tetrahedral intermediate and its phosphonate analog. The results suggest that Glp is unlikely to bind in the same fashion as PEP, a conclusion that is consistent with recent studies that have questioned the role of Glp as a transition-state or intermediate analog.

  • Structural Constraints on the Ternary Complex of 5-Enolpyruvylshikimate-3-phosphate Synthase from Rotational-echo Double-resonance NMR
    Journal of molecular biology, 1996
    Co-Authors: Lynda M. Mcdowell, Daniel R. Studelska, Asher Schmidt, Eric R. Cohen, Jacob Schaefer
    Abstract:

    Abstract The 46 kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) Synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phospho enolpyruvate to form EPSP. The reaction is inhibited by N -(phosphono- methyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP Synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, 15 N labels were introduced selectively into the lysine, arginine and histidine residues of EPSP Synthase and distances to a 13 C label in Glp and to the 31 P in S3P and Glp were measured by rotational-echo double-resonance NMR. Three lysine and four arginine residues are in the proximity of the phosphate group of S3P and the carboxyl and phosphonate groups of Glp. A single histidine residue is in the vicinity of the binding site (closer to Glp than to S3P) but is more distant than the lysine and arginine residues.

Oderlei Bernardi - One of the best experts on this subject based on the ideXlab platform.

  • high levels of biological activity of cry1ac protein expressed on mon 87701 mon 89788 soybean against heliothis virescens lepidoptera noctuidae
    Pest Management Science, 2014
    Co-Authors: Oderlei Bernardi, Geraldo Ubirajara Berger, Patrick Marques Dourado, Renato Assis De Carvalho, Samuel Martinelli, Graham Phillip Head, Celso Omoto
    Abstract:

    Background Genetically modified MON 87701 × MON 89788 soybean (Glycine max) that expresses the Cry1Ac and EPSP-Synthase proteins is a new Integrated Pest Management (IPM) tool for controlling Heliothis virescens in Brazil. To Support Insect Resistance Management (IRM) programs and understand the value of this event for IPM, we conducted laboratory and field studies to assess the biological activity of Cry1Ac protein expressed on Bt soybean against this insect pest. Results Heliothis virescens was highly susceptible to purified Cry1Ac protein [LC50 (FL 95%) = 0.026 (0.021–0.033) µg Cry1Ac mL–1 diet]. In bioassays with freeze-dried MON 87701 × MON 89788 soybean tissue diluted 25 times in an artificial diet, there was 100% mortality of H. virescens. In bioassays with leaf disc, young trifoliate, flower buds, pods and high artificial infestation under greenhouse condition, MON 87701 × MON 89788 soybean showed a high level of control against H. virescens. Moreover, larvae from first through fifth instar were highly susceptible to MON 87701 × MON 89788 soybean. Conclusions The MON 87701 × MON 89788 soybean provides an effective alternative in controlling Heliothis virescens and complies with the concept of high-dose for IRM programs. © 2013 Society of Chemical Industry

  • assessment of the high dose concept and level of control provided by mon 87701 mon 89788 soybean against anticarsia gemmatalis and pseudoplusia includens lepidoptera noctuidae in brazil
    Pest Management Science, 2012
    Co-Authors: Oderlei Bernardi, Patrick Marques Dourado, Samuel Martinelli, Graham Phillip Head, Glaucia Sossai Malvestiti, Wladecir Salles De Oliveira, Geraldo Ubrajara Berger, Celso Omoto
    Abstract:

    BACKGROUND: Genetically modified MON 87701 × MON 89788 soybean (Glycine max), which expresses the Cry1Ac and EPSP-Synthase proteins, has been registered for commercial use in Brazil. To develop an Insect Resistance Management (IRM) program for this event, laboratory and field studies were conducted to assess the high-dose concept and level of control it provides against Anticarsia gemmatalis and Pseudoplusia includens. RESULTS: The purified Cry1Ac protein was more active against A. gemmatalis [LC50 (FL 95%) = 0.23 (0.15–0.34) µg Cry1Ac mL−1 diet] than P. includens [LC50 (FL 95%) = 3.72 (2.65–4.86) µg Cry1Ac mL−1 diet]. In bioassays with freeze-dried MON 87701 × MON 89788 soybean tissue diluted 25 times in an artificial diet, there was 100% mortality of A. gemmatalis and up to 95.79% mortality for P. includens. In leaf-disc bioassays and under conditions of high artificial infestation in the greenhouse and natural infestation in the field, MON 87701 × MON 89788 soybean showed a high level of efficacy against both target pests. CONCLUSIONS: The MON 87701 × MON 89788 soybean provides a high level of control against A. gemmatalis and P. includes, but a high-dose event only to A. gemmatalis. Copyright © 2012 Society of Chemical Industry

Ernst Schönbrunn - One of the best experts on this subject based on the ideXlab platform.

  • Molecular basis for the glyphosate-insensitivity of the reaction of 5-enolpyruvylshikimate 3-phosphate Synthase with shikimate
    FEBS Letters, 2005
    Co-Authors: Melanie A. Priestman, M.l. Healy, T. Funke, A. Becker, Ernst Schönbrunn
    Abstract:

    The shikimate pathway enzyme 5-enolpyruvyl shikimate-3-phosphate Synthase (EPSP Synthase) has received attention in the past because it is the target of the broad-spectrum herbicide glyphosate. The natural substrate of EPSP Synthase is shikimate-3-phosphate. However, this enzyme can also utilize shikimate as substrate. Remarkably, this reaction is insensitive to inhibition by glyphosate. Crystallographic analysis of EPSP Synthase from Escherichia coli, in complex with shikimate/glyphosate at 1.5 A resolution, revealed that binding of shikimate induces changes around the backbone of the active site, which in turn impact the efficient binding of glyphosate. The implications from these findings with respect to the design of novel glyphosate-insensitive EPSP Synthase enzymes are discussed.

  • how the mutation glycine96 to alanine confers glyphosate insensitivity to 5 enolpyruvyl shikimate 3 phosphate Synthase from escherichia coli
    Planta, 2002
    Co-Authors: Susanne Eschenburg, Melanie A. Priestman, M.l. Healy, Gerald H Lushington, Ernst Schönbrunn
    Abstract:

    The enzyme 5-enolpyruvyl shikimate-3-phosphate (EPSP) Synthase (EC 2.5.1.19) is essential for the biosynthesis of aromatic compounds in plants and microbes and is the unique target of the herbicide glyphosate. One of the first glyphosate-insensitive enzymes reported was a Gly96Ala mutant of EPSP Synthase from Klebsiella pneumoniae. We have introduced this single-site mutation into the highly homologous EPSP Synthase from Escherichia coli. The mutant enzyme is insensitive to glyphosate with unaltered affinity for its first substrate, shikimate-3-phosphate (S3P), but displays a 30-fold lower affinity for its second substrate, phosphoenolpyruvate (PEP). Using X-ray crystallography, we solved the structure of Gly96Ala-EPSP Synthase liganded with S3P to 0.17 nm resolution. The crystal structure shows that the additional methyl group from Ala96 protrudes into the active site of the enzyme. While the interactions between enzyme and S3P remain unaffected, the accessible volume for glyphosate binding is substantially reduced. Exploiting the crystallographic results for molecular modeling, we demonstrate that PEP but not glyphosate can be docked in the Gly96Ala-modified binding site. The predicted PEP binding site satisfies the earlier proposed interaction pattern for PEP with EPSP Synthase and corroborates the assumption that glyphosate and PEP target the same binding site.

  • interaction of the herbicide glyphosate with its target enzyme 5 enolpyruvylshikimate 3 phosphate Synthase in atomic detail
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Ernst Schönbrunn, Susanne Eschenburg, Wendy A Shuttleworth, John V Schloss, Nikolaus Amrhein, Jeremy N S Evans, Wolfgang Kabsch
    Abstract:

    Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) Synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP Synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP Synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme⋅substrates complex. The elucidation of the active site of EPSP Synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.