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...
Glyphosate, the world's most used herbicide, is a massive success because it enables efficient weed control with minimal animal and environmental toxicity. The molecular target of glyphosate is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the sixth step of the shikimate pathway in plants and microorganisms. Glyphosate-tolerant variants of EPSPS constitute the basis of genetically engineered herbicide-tolerant crops. A single-site mutation of Pro 101 in EPSPS (numbering according to the enzyme from Escherichia coli) has been implicated in glyphosate-resistant weeds, but this residue is not directly involved in glyphosate binding, and the basis for this phenomenon has remained unclear in the absence of further kinetic and structural characterization. To probe the effects of mutations at this site, E. coli EPSPS enzymes were produced with glycine, alanine, serine, or leucine substituted for Pro 101. These mutant enzymes were analyzed by steady-state kinetics, and the crystal structures of the substrate binary and substrate⅐glyphosate ternary complexes of P101S and P101L EPSPS were determined to between 1.5-and 1.6-Å resolution. It appears that residues smaller than leucine may be substituted for Pro 101 without decreasing catalytic efficiency. Any mutation at this site results in a structural change in the glyphosate-binding site, shifting Thr 97 and Gly 96 toward the inhibitor molecule. We conclude that the decreased inhibitory potency observed for glyphosate is a result of these mutation-induced longrange structural changes. The implications of our findings concerning the development and spread of glyphosate-resistant weeds are discussed.Glyphosate (N-phosphonomethylglycine) inhibits the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS 2 ; EC 2.5.1.19) (1), which is essential for the biosynthesis of aromatic compounds in plants, fungi, bacteria, and apicomplexan parasites (2-5). Glyphosate, the active ingredient in Roundup, exhibits broad-spectrum herbicidal activity, yet is essentially nontoxic to animals and does not persist in the environment. These characteristics have made it the world's most popular herbicide, and usage continues to increase with the adoption of glyphosate-dependent technologies, including herbicide-tolerant crops and minimal tillage (no-till) agriculture. The enormous reliance on glyphosate and the absence of suitably safe alternative herbicides mean that the widespread emergence of glyphosatetolerant weeds would have devastating agricultural and environmental consequences. EPSPS, the molecular target of glyphosate, catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (P-enolpyruvate) to the 5-hydroxy position of shikimate 3-phosphate (S3P) (see Fig. 1). The structure of the glyphosate-inhibited complex shows that glyphosate binds to the P-enolpyruvate-binding site of EPSPS (6 -8), corroborating early kinetic data demonstrating that glyphosate binding is competitive with respect to P-enolpyruvate (1, 9, 10). Before bacte...
The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway, and is the target of the broad-spectrum herbicide glyphosate. Kinetic analysis of the cloned EPSPS from Staphylococcus aureus revealed that this enzyme exerts a high tolerance to glyphosate, while maintaining a high affinity for its substrate phosphoenolpyruvate. Enzymatic activity is markedly influenced by monovalent cations such as potassium or ammonium, which is due to an increase in catalytic turnover. However, insensitivity to glyphosate appears to be independent from the presence of cations. Therefore, we propose that the Staphylococcus aureus EPSPS should be classified as a class II EPSPS. This research illustrates a critical mechanism of glyphosate resistance naturally occurring in certain pathogenic bacteria.
The natriuretic peptides (NPs), 2 mainly atrial (ANP), Btype (BNP), and C-type natriuretic peptides (CNP), play key roles in many cardiovascular functions (1). Their diuretic, natriuretic, and vasodilatory properties have been developed as therapeutic strategies for human cardiovascular diseases (2). The actions of NPs are mediated through binding and signal transduction of three natriuretic peptide receptors (NPR). NPR-A and NPR-B have guanylyl cyclase activity that raises intracellular cGMP levels. Effects of these receptors are mediated by the preferential binding of ANP and BNP to NPR-A and by that of CNP to NPR-B. NPR-C is a non-guanylyl cyclase receptor. In addition to its role in the clearance of NPs, NPR-C also can transmit signals via heterotrimeric G protein, G i (3, 4).NPs have a short half-life and their circulation levels are tightly controlled (5, 6). In addition to the regulation of NPs by gene transcription, secretion, and NPR-C mediated clearance, NP maturation and breakdown by multiple proteases are also key in NP regulation. For example, active NPs are converted from pro-NPs by furin, corin, and likely, by other proteases (7,8). Active NPs are postulated to be proteolytically inactivated by a membrane-bound metalloprotease, neprilysin (NEP) (9 -12). However, growing evidence propose the role of other proteases in the clearance of NPs. Meprin A is shown to be involved in the initial N-terminal cleavage of BNP and meprin A and NEP are thought to work together in the clearance of BNP (13). In addition, insulin-degrading enzyme (IDE) and DPP-IV have been shown to cleave NPs in vitro (14 -16) However, the functional consequence in the cleavage of NPs by these two proteases in the cellular setting remains unknown.IDE is a ubiquitously expressed zinc-metalloprotease that is involved in the clearance of insulin and amyloid- (A), peptides implicated in the pathogenesis of diabetes and Alzheimer's disease, respectively (17-19). We have recently solved the structure of human IDE in complex with various substrates and elucidated the molecular basis for the recognition and selective degradation of substrates by IDE (20 -22). Our structures reveal that IDE uses a sizable catalytic chamber to entrap, unfold, and degrade insulin, A, and other substrates. IDE recognizes its substrates mainly based on their tertiary structures. The size, dipole moment, structure flexibility, and location of the N-terminal end of the substrates are key factors for the selectivity of IDE.IDE was shown to degrade ANP and BNP (15,16), however it was never established whether the cleavages of ANP and * This work was supported, in whole or in part, by National Institutes of Health Grants GM81539 (to W.-J. T.), F32 GM87093 (to L. A. R.), 5T32HL07237-33 (to T. F.), and R21HL093402-01 (to L. R. P.). The atomic coordinates and structure factors (codes 3N56 and 3N57) CNP, C-type natriuretic peptide; DNP, dendroaspis natriuretic peptide; fsANP, frameshift mutant atrial natriuretic peptide; NPR-A, natriuretic peptide receptor-A; NPR-B,...
The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase or EPSPS) is best known as the target of the herbicide glyphosate. EPSPS is also considered an attractive target for the development of novel antibiotics since the pathogenicity of many microorganisms depends on the functionality of the shikimate pathway. Here, we have investigated the inhibitory potency of stable fluorinated or phosphonate-based analogues of the tetrahedral reaction intermediate (TI) in a parallel study utilizing class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSPS. The (R)-difluoromethyl and (R)-phosphonate analogues of the TI are the most potent inhibitors of EPSPS described to date. However, we found that class II EPSPS are up to 400 times less sensitive to inhibition by these TI analogues. X-ray crystallographic data revealed that the conformational changes of active site residues observed upon inhibitor binding to the representative class I EPSPS from Escherichia coli do not occur in the prototypical class II enzyme from Agrobacterium sp. strain CP4. It appears that because the active sites of class II EPSPS do not possess the flexibility to accommodate these TI analogues, the analogues themselves undergo conformational changes, resulting in less favorable inhibitory properties. Since pathogenic microorganisms such as Staphylococcus aureus utilize class II EPSPS, we conclude that the rational design of novel EPSPS inhibitors with potential as broad-spectrum antibiotics should be based on the active site structures of class II EPSP synthases.
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 Å 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.
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