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The only known mode of action of glyphosate in plants is inhibition of EPSPS (Fig 3). The monomeric enzyme is one of two enzymes in the class of enolpyruvyltransferases. This class shares a unique structure containing two globular domains composed of beta sheets and alpha helices, which form something like an inverse alpha/beta barrel. The two domains are connected by two strands which act like a hinge to bring the upper and lower domains together, sandwiching the substrates in the active site. Ligand binding converts the enzyme from an open state to a tightly-packed closed state, following the pattern of an induced-fit mechanism (see http://www.biochem.arizona.edu/classes/bioc462/462bH2002/462bHonorsProjects/462bHonors1999/oks/Enzym.htm#5-enolpyruvyl%20shikimate%203-phosphate%20(EPSP) and http://www.arches.uga.edu/~gpries/bcmb8010/structure.htm ). The fact that this enzyme does not occur in mammals, fish, reptiles, birds and insects makes it a good target for antibiotics and herbicides.
The agricultural benefit comes with genetically engineered plants which are resistant to the herbicide; Roundup-ready crops may be treated to eliminate weeds and other problem plants while leaving the engineered plants unharmed. Research indicates that glyphosate acts as transition-state analogue, effectively shutting down the shikimate pathway. In this way, glyphosate deregulates feedback inhibition of the first enzyme in the pathway by a near-end product of the pathway, and as a result, there is an unregulated flow of carbon into the pathway causing high levels of S3P to accumulate. Glyphosate displays high specificity for EPSP synthase, not even binding to UDP-N-acetylglucosamine enolpyruvyltransferase (MurA), the only other enzyme in the enolpyruvyltransferase class.
Despite its usefulness, the drawback of glyphosate is that besides killing the weed it also harms the crop plants at the same time. However, genetic engineering enables biotechnology companies such as Monsanto to develop crops resistant to the herbicide. Indeed seeds of crops resistant to glyphosate such as canola, soybean and cotton have been introduced for commercial growth since the early of 1980's. Although glyphosate is very effective in killing plants, some bacteria are able to tolerate it. For instance, the Agrobacterium sp. Strain CP4 is resistant to glyphosate. In Round-up Ready soybeans developed by Monsanto, the CP4 EPSPS genes were introduced to wild soybeans by transformation. The novel CP4 EPSPS gene allows the transfected plants to continued producing amino acids in the presence of glyphosate.
Buchanan, Gruissem and Jones (2000) Biochemistry and Molecular Biology of Plants. (581.192 BIO, short loan), chapter 8.
Schonbrunn et al (2001) Proc Natl Acad Sci 98: 1376-1380.
Using web sites such as those above and others listed below, you need to answer the following questions. Prepare your report as a Word file, with your name, student number and date on it. Return it to me by e mail (p.Bramley@rhul.ac.uk) by the hand-in date. If you paste diagrams, figures etc into it, you must quote the URL or publication from which it was obtained.
This practical is almost exclusively web-based and you will need to use several sites simultaneously. The main site is NCBI, at http://www.ncbi.nlm.nih.gov/. When you first go to this site, read what NCBI does and note the variety of searches that are possible.
What is the reaction catalysed by EPSP synthase? To what class of enzymes does it belong?
What is the mode of action of glyphosate? Include a diagram of the enzyme with and without the inhibitor in your answer.
What are the amino acid sequences of three (Arabidopsis, tomato and tobacco) higher plant EPSP synthases? Use the NCBI site (http://www.ncbi.nlm.nih.gov/) and the 'Protein' search for these. Type in the enzyme name and the species. Quote the accession numbers when you find the sequences in your report next to the amino acid sequences.
What are the molecular masses of these proteins? Can you detect any transit peptide sequences for these proteins?
What is the amino acid sequence of EPSP synthase from Agrobacterium strain CP4 (accession number Q9R4E4)? What is its molecular mass? Use NCBI for this.
Compare the amino acid sequences of the higher plant and bacterial EPSP synthases. To do this, you need to convert the amino acid sequences into a 'Fasta' format. Go to NCBI, choose the FASTA format from 'display'. Then paste in each sequence, including the part starting with '>', into CLUSTALW (http://clustalw.genome.ad.jp/ ) one at a time. Press 'execute multiple alignment' and you can now compare the 4 sequences.
What are the differences? Differences in the amino acid sequences must account for resistance to glyphosate. You will find several differences, but we know that the key changes are around amino acid 96. Look at this region to see what differences in amino acids are there. How may these account for the bacterial enzyme being resistant to glyphosate inhibition?
What other genetic manipulations are used to engineer glyphosate resistance into crops? (See the abstract of Klee et al (1987) Mol Gen Genet 210: 437-442).
PMB Oct 2010