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...
The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (K i ؍ 2.4 mM) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (K m ؍ 0.1 mM). The crystal structure at 1.7-Å resolution revealed that the dual mutation causes a shift of residue Gly 96 toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile 97 points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr 97 and Pro 101 induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS.Glyphosate (N-phosphonomethylglycine) is a potent inhibitor of the shikimate pathway in plants, specifically targeting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, 3 EC 2.5.1.19) (1). Glyphosate-based formulations exhibit broad spectrum herbicidal activity with minimal human and environmental toxicity (2, 3). The safety and efficacy of glyphosate, together with the existence of genetically modified, glyphosate-resistant crop varieties (4, 5), have combined to make glyphosate the most used herbicide in the world. Enzymes of the shikimate pathway are also regarded as attractive antimicrobial targets (6 -9).EPSPS catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 5-hydroxy position of shikimate-3-phosphate (S3P) (Fig. 1). Binding of the first substrate, S3P, to the enzyme triggers a global conformational change from an "open" to a "closed" conformation. PEP and glyphosate bind in the active site, formed at the interface between the Nand C-terminal globular domains. Glyphosate inhibition is competitive with respect to PEP (10, 11), and structural studies confirmed that glyphosate occupies the PEP-binding site (12-15).EPSPS from different organisms have been divided into two classes according to intrinsic glyphosate sensitivity: in Class I enzymes, found in all plants and i...
Pseudomonas aeruginosa secretes a 205 residue long hemophore (full-length HasAp) that is subsequently cleaved at the C'-terminal domain to produce mainly a 184 residue long truncated HasAp that scavenges heme [Letoffé, S., Redeker, V., and Wandersman, C. (1998) Mol. Microbiol. 28, 1223-1234. HasAp has been characterized by X-ray crystallography and in solution by NMR spectroscopy. The X-ray crystal structure of truncated HasAp revealed a polypeptide αβ fold and a ferriheme coordinated axially by His32 and Tyr75, with the side chain of His83 poised to accept a hydrogen bond from the Tyr75 phenolic acid group. NMR investigations conducted with full-length HasAp showed that the carboxyl terminal tail (21 residues) is disordered and conformationally flexible. NMR spectroscopic investigations aimed at studying a complex between apo-HasAp and human met-hemoglobin were stymied by the rapid heme capture by the hemophore. In an effort to circumvent this problem NMR spectroscopy was used to monitor the titration of 15 Nlabeled holo-HasAp with hemoglobin. These studies allowed identification of a specific area on the surface of truncated HasAp, encompassing the axial ligand His32 loop that serves as a transient site of interaction with hemoglobin. These findings are discussed in the context of a putative encounter complex between apo-HasAp and hemoglobin that leads to efficient hemoglobin-heme capture by the hemophore. Similar experiments conducted with full-length 15 N-labeled HasAp and hemoglobin revealed a transient interaction site in full-length HasAp similar to that observed in the truncated hemophore. The spectral perturbations observed while investigating these interactions, however, are weaker than those observed for the interactions between hemoglobin and truncated HasAp, † This work was supported by grants from the National Institute of Health, GM-50503 (M.R.), NSF-MCB-0818488 (M.R.) and NSF-MCB-0811888 (P.M.L.). ‡ Coordinates and crystallographic structure factors for HasAp have been deposited in the protein data bank under accession code 3ELL.Backbone resonance assignments for truncated and full-length holo-HasAp have been deposited in the BMRB under access code 15962 and 15963, respectively. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 January 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptsuggesting that the disordered tail in the full-length HasAp must be proteolyzed in the extracellular milieu to make HasAp a more efficient hemophore.The preferred aerobic metabolism of Pseudomonas aeruginosa requires respiratory enzymes that need iron or iron-containing cofactors for their function. The extremely low concentrations of free iron in mammalian hosts trigger a stress response in the opportunistic P. aeruginosa (and in many other pathogens) that involves the deployment of several iron-acquisition systems (1-4). The systems involved in the capture of iron typically fall in two categories, (i) excretion of low-molecular weight i...
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