Chemical Education Today
Reports from Other Journals
Research Advances by Angela G. King
Roundup Shutdown Due to its low cost, low environmental hazards, and effectiveness, glyphosate has become the world’s most popular herbicide. Discovered in 1971 by a scientist working for Monsanto Corp., glyphosate is now the active ingredient in a number of water-soluble herbicides such as Roundup and Glyphomax. Glyphosate works by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme in the shikimic acid pathway. It also inhibits the synthesis of aminolevulinic acid (ALA), which halts porphyrin ring synthesis and the production of all compounds containing porphyrin rings such as chlorophyll. The herbicide is readily absorbed after foliar application and translocated throughout the plant. In the 1990s scientists developed commercial glyphosate-tolerant crops through genetic modifications that inserted microbial EPSPS variants that are not inhibited by glyphosate into plants such as corn and soybeans. Marketed as Roundup Ready, these crops occupy the greatest acreage of any transgenic trait. However, in these genetically modified (GM) plants, the herbicide remains in the plant and as glyphosate accumulates it may interfere with reproductive development and cause lower crop yields. In contrast to the build-up of glyphosate seen in Roundup Ready plants, GM crops exhibiting tolerance for other herbicides have a mechanism for metabolic detoxification, which may employ hydrolysis, acetylation, and/or oxidative cleavage. In all cases, the mechanism employs an enzyme of microbial origin. The desire for a similar mechanism by which to eliminate glyphosate build-up in crops led scientists at Verdia, Inc. and Pioneer Hi-Bred Intl. Inc. to search for a microbial enzyme that would carry out an N-acetylation of glyphosate, thus converting the herbicide
O C −O
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H Acetyl CoA CoASH
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Structure 1. Enzymatic N-acetylation of glyphosate, provided by L. Castle.
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Enzymatic N-acetylation of glyphosate. 10 days after 1x (26 oz/ ac Roundup UltraMAX) glyphosate spray: (A) Untransformed maize plants. (B–D) Untreated plants on the left, plants 10 days after glyphosate spray on the right; (B) maize plants expressing a fifthiteration gat variant; (C) maize plants expressing tenth- and eleventh-iteration gat variant 10 days after 6x glyphosate spray; (D) maize plants expressing a seventh-iteration gat variant. Reprinted with permission of Castle, L.; Siehl, D.; Gorton, R.; Patten, P.; Chen, Y.; Bertain, S.; Cho, H.-J.; Duck, N.; Wong, J.; Liu, D.; Lassner, M. Discovery and Directed Evolution of a Glyphosate Tolerance Gene. Science 2004, 304, 1151–1154. Copyright 2004 AAAS.
to N-acetylglyphosate, which does not act as an EPSPS inhibitor and is not an herbicide. Employing a mass spectrometry method to detect N-acetylglyphosate, researchers were able to identify glyphosate-N-acetyltransferase (GAT) from a strain of Bacillus licheniformis. GAT is similar to enzymes of the GNAT superfamily. Those enzymes are present in mammals, plants, fungi, and bacteria. However GAT is unusual among characterized acetyltransferases, since most will not acetylate a secondary amine such as glyphosate. Another protein, YITI, was found to be capable of acetylating glyphosate but with lower efficiency than GAT. Iterative fragmentation-based multi-gene shuffling of gat genes was used to create libraries of new enzymes that displayed higher efficiency and greater specificity for glyphosate. Screening demonstrated that after the eleventh iteration, the most promising GAT had a kcat of 416 min⫺1 and KM of 0.05 mM glyphosate and was 76–79% identical to the original GAT enzymes from Bacillus licheniformis. At the same time scientists used the multi-gene shuffling approach to improve the natural enzyme, other researchers were making specific changes to the protein’s sequence based on sequence-function relationships, but the DNA shuffling coupled with functional selection provided faster and superior results. The genes for the optimized GAT protein were inserted into the nuclear genome of tobacco, maize, and Arabidopsis. Most transformed plants using genes from the 10th and 11th iterations were tolerant to glyphosate spray and showed no adverse symptoms, demonstrating the potential of directed evolution to increase activity of useful enzymes.
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Chemical Education Today
Dendrobates pumilio from the Bocas del Toro region of Panama. Reprinted with permission from Smith, S. A.; Jones, T. H. PNAS, 2004, 101, 7841–7842. Copyright 2004 National Academy of Sciences, U.S.A.
Pumiliotoxin-containing ants also from the Bocas del Toro region of Panama. Reprinted with permission from Saporito, R.; Garraffo, H. M.; Donnelly, M.; Edwards, A.; Longino, J.; Daly, J. Formicine Ants: An Arthropod Source for Pumiliotoxin Alkaloids of Dendrobatid Poison Frogs. PNAS, 2004, 101, 8045–9050. Copyright 2004 National Academy of Sciences, U.S.A.
More Information 1. Castle, L.; Siehl, D.; Gorton, R.; Patten, P.; Chen, Y.; Bertain, S.; Cho, H.-J.; Duck, N.; Wong, J.; Liu, D.; Lassner, M. Discovery and Directed Evolution of a Glyphosate Tolerance Gene. Science 2004, 304, 1151–1154. 2. Fact sheets on glyphosate are available from the EPA and the National Pesticide Telecommunications Network at http:// www.epa.gov/OGWDW/dwh/t-soc/glyphosa.html and http:// www.npic.orst.edu/factsheets/glyphogen.pdf (accessed Aug 2004). 3. An article on genetically modified foods, with an emphasis on Roundup Ready crops, is available: Pöpping, B. Are You Ready for [a] Roundup?—What Chemistry Has To Do with Genetic Modifications. J. Chem. Educ. 2001, 78, 752–756.
Dietary Source of Poison Frog Toxins Frogs and toads use more than 500 identified alkaloids, from 24 different structural classes, as a passive means of defense against predators and harmful microorganisms. Pumiliotoxins are one class of defensive alkaloids and are found in virtually all anurans employing lipophilic alkaloids as a means of defense. Scientists originally thought the anurans produced the defensive alkaloids through metabolism, but research has now shown that the alkaloids are produced by arthropods on which the toads and frogs feed and simply accumulate in the animals’ skin after ingestion. The defensive alkaloids cannot be detected in captive dendrobatid and mantellid frogs fed a diet free of these arthropods, but the same animals accumulate them if the alkaloids are added to their diet.
OH OH
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The structures of pumiliotoxins A (top) and B (bottom).
OH OH OH N
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The dietary sources of most defensive anuran alkaloids are known. For instance, myrmicine ants are the likely source of all alkaloids with unbranched carbon skeletons, such as 3,5-disubstituted indolizidines found in skin extracts of poison frogs and toads. Coccinellines and related tricyclic compounds accumulate in anurans after the ingestion of coccinellid beetles. Millipedes are the reported source of spiropyrrolizidine oximes and nitropolyzonamines. However, no known dietary source for the pumiliotoxin class was known before recent breakthroughs by John Daly and colleagues. This team made extensive arthropod collections in a region of Panama where the population of Dendrobates pumilio was known to have high concentrations of pumiliotoxins A and B. During two different seasons, arthropods small enough to be eaten by the frogs were collected by forceps from leaf litter and plants. Live frogs were collected simultaneously for analysis of skin alkaloids, while other frogs were stomach-flushed to verify their diets. GC–MS of methanol extracts from more than 500 arthropod samples collected during wet and dry seasons compared the GC mass spectra of major and minor alkaloids to those of known compounds. Pumiliotoxins A and B were detected in formicine ants from two different genera, but the alkaloids were not present in all ant samples. Frogs collected at the same sites as ants in which the pumiliotoxins were detected also had the alkaloids in their skin, and ants of this type were identified in the stomach contents of studied frogs. The presence of pumiliotoxins A and B in the ants as well as frog skin extracts, coupled with the presence of such ants in the frogs’ digestive system, strongly supports the hypothesis that these ants are the dietary source of the defensive alkaloids for this population of frogs.
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1. Saporito, R.; Garraffo, H. M.; Donnelly, M.; Edwards, A.; Longino, J.; Daly, J. Formicine Ants: An Arthropod Source for Pumiliotoxin Alkaloids of Dendrobatid Poison Frogs. PNAS 2004, 101, 8045–8050. 2. Information on poison dart frogs and their alkaloids is available online at http://www.amnh.org/exhibitions/expeditions/ treasure_fossil/Treasures/Poison_Dart_Frogs/frogs.html?50 (accessed Aug 2004).
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Reports from Other Journals Generation of Copper(II) Oxide “Dandelions” Technical applications have created a high demand for curved atomic-scale structures such as tubes and spheres. The production of such molecular architecture is a challenge for material scientists striving to control molecular self-assembly into desired building components. Two approaches to controlling the organization of building units into curved surfaces have been developed in the past. The first method utilized emulsified droplets as templates for the self-assembly of polymeric beads into spherical shells. The second method organized one-dimensional molecular rods into curved superstructures by controlling hydrophobic attraction. Researchers in Singapore now report a third method for generating curved architectures based on the geometric constraints of building units. Their one-pot method employs two steps. First, CuO nanoribbons spontaneously form rhombic crystal strips; in the second step, the rhombic strips selfassemble into hollow core structures resembling dandelions. The “dandelions” were prepared by mixing a solution of Cu(NO3)2⭈3H2O in ethanol with ammonia, followed by sodium hydroxide solution. After solid NaNO3 was added the reaction mixed was heated in an electric oven, cooled, and the CuO products were washed by centrifugation. Powder X-ray diffraction peaks obtained from the resulting CuO samples were broad, indicating their nanocrystalline nature; monoclinic symmetry was also evidenced. The atomic ratio of copper to oxygen in the samples was 1:1. The “dandelions” formed by the self-assembly were all of consistent morphology with diameters of 4–8 mm and a shell wall thickness of 25–33% the sphere’s diameter. The puffy appearance was due to the crystal strips, formed from nanoribbons in step 1 of the synthesis, all aligning perpendicular to the hollow sphere’s surface. An extension of this work indicates that other metal oxides might adopt similar morphology when exposed to the new two-step method of: (1) mesoscale formation of rhombic crystalline building units from the oriented aggregation of smaller nanoribbons, and (2) macroscopic organization of the resulting building blocks into hollow microspheres. Exploitation of geometric constraints in synthesized building blocks
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SEM image of CuO microspheres prepared by new methodology. Reprinted with permission from Liu, B.; Zeng, C. Mesoscale Organization of CuO Nanoribbons: Formation of “Dandelions”. J. Am. Chem. Soc. 2004, 126, 8124. Copyright 2004 American Chemical Society.
may soon allow materials scientists to meet technology’s demand for curved architecture in new materials.
More Information 1. Liu, B.; Zeng, C. Mesoscale Organization of CuO Nanoribbons: Formation of “Dandelions”. J. Am. Chem. Soc. 2004, 126, 8124–8125. 2. Several procedures for high school stoichiometry labs using CuO as the starting material are available. See Zidick, C.; Weismann, T. The Reduction of CuO with Burner Gas and without a Fume Hood. A High School Chemistry Experiment. J. Chem. Educ. 1973, 50, 717 or Sanger, M.; Geer, K. Determination of the Empirical Formula of a Copper Oxide Salt Using Two Different Methods. J. Chem. Educ. 2002, 79, 994. 3. The Exploring the Nanoworld Web site introduces students and teachers to the tools that let scientists see, manipulate, and create nano-architectural wonders. Available online at http:/ /mrsec.wisc.edu/edetc/index.html (accessed Aug 2004).
Angela G. King is Senior Lecturer in Chemistry at Wake Forest University, P. O. Box 7486, Winston-Salem, NC 27109;
[email protected].
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