Molecular basis for the herbicide resistance of Roundup Ready crops

Abstract

The engineering of transgenic crops resistant to the broad-spectrum herbicide glyphosate has greatly improved agricultural efficiency worldwide. Glyphosate-based herbicides, such as Roundup, target the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, the functionality of which is absolutely required for the survival of plants. Roundup Ready plants carry the gene coding for a glyphosate-insensitive form of this enzyme, obtained from Agrobacterium sp. strain CP4. Once incorporated into the plant genome, the gene product, CP4 EPSP synthase, confers crop resistance to glyphosate. Although widely used, the molecular basis for this glyphosate-resistance has remained obscure. We generated a synthetic gene coding for CP4 EPSP synthase and characterized the enzyme using kinetics and crystallography. The CP4 enzyme has unexpected kinetic and structural properties that render it unique among the known EPSP synthases. Glyphosate binds to the CP4 EPSP synthase in a condensed, noninhibitory conformation. Glyphosate sensitivity can be restored through a single-site mutation in the active site (Ala-100 -Gly), allowing glyphosate to bind in its extended, inhibitory conformation.conformational change ͉ crystal structure ͉ genetic modification ͉ mutation T he broad-spectrum herbicide glyphosate, the active ingredient of Roundup, inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the enzyme catalyzing the penultimate step of the shikimate pathway toward the biosynthesis of aromatic amino acids. Roundup Ready crop lines contain a gene derived from Agrobacterium sp. strain CP4, encoding a glyphosate-tolerant enzyme, the so-called CP4 EPSP synthase (1, 2). Expression of CP4 EPSP synthase results in glyphosate-tolerant crops, enabling more effective weed control by allowing postemergent herbicide application. The substantial advantages of glyphosate-tolerant crops have resulted in rapid adoption: 87% of soybeans, 61% of cotton, and 26% of corn planted in the United States in 2005 were glyphosate-tolerant varieties (3). However, lingering concerns about the potential health and environmental effects of genetically modified organisms have limited the acceptance of such seed lines and food products, particularly in Europe and Japan.EPSP synthase catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P) (Fig. 1A). Beginning in the early 1980s, researchers sought to identify glyphosate-insensitive EPSP synthases that could be introduced into crops to provide herbicide resistance. A number of promising enzymes were identified through selective evolution, site-directed mutagenesis, and microbial screens (4-7). However, an increased tolerance for glyphosate in EPSP synthase was often accompanied by a concomitant decrease in the enzyme's affinity for PEP, resulting in decreased catalytic efficiency. More favorable kinetic characteristics were observed in some enzymes with substitutions including Pro-101-Ser and Thr-97-Ile (nu...

Comparative Studies Involving Transgenic and Non-Transgenic Soybean: What is Going On?

“…Due to these genetic modifications, the proteins composition and profile can be affected, causing changes in the species proteome [91][92]. As previously described, the knowledge of the soybean genotype alone does not show enough information about the protein modifications due to environmental interactions.…”

Inhibition of Shikimate Kinase and Type II Dehydroquinase for Antibiotic Discovery: Structure-Based Design and Simulation Studies

“…Moreover, the (3R)- (15) and (3S)-3-hydroxyshikimic acids (16) are also good scaffolds for the future generation of inhibitors.…”

“…Structural studies have shown that glyphosate occupies the phosphoenol pyruvate binding site in the enzyme, mimicking an intermediate state of the ternary enzyme substrate complex [15,16]. The shikimic acid pathway starts with the condensation of erythrose-4-phosphate (1) and phosphoenol pyruvate (2) catalyzed by DAHP synthase (7-phosphono-2-dehydro-3-deoxyheptonate aldolase, EC 4.1.2.15) to give DAHP (3, 3-deoxy-D-arabinoheptulosonate-7-phosphate), which is converted into 3-dehydroquinic acid (4) by dehydroquinate synthase (EC 4.6.1.3) (Scheme 1).…”

“…The (3R)- (15) and (3S)-3-aminoshikimic acids (16) were also designed as mimetics of the natural substrate. Both compounds proved to be reversible competitive inhibitors of the Mt-SK enzyme with K i values of 62 and 65 µM, respectively.…”

Transgenics: A new breed

“…For example, all plants have an analogue of the bacterial EPSPS gene that is inserted into Monsanto's Roundup Ready crops. It should be possible to create similar herbicide resistance by editing a plant's own version, rather than bringing in an external gene 4 .…”