EPSP Synthase (PDB ID: 2GGA) from Agrobacterium sp. CP4

Created by: Annie Yang

CP4 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (PDB ID: 2GGA) is a critical enzyme found in Agrobacterium sp. CP4 that catalyzes the sixth step of the shikimate pathway. The shikimate pathway is a metabolic pathway that allows plants and bacteria to synthesize their own aromatic amino acids and other aromatic compounds from phosphoenolpyruvate (PEP) and erythrose-4-phosphate through a series of seven enzyme catalyzed steps. Chorismate is the final product of the shikimate pathway and is the precursor to the aromatic amino acids (1). Unlike animals, which must get their aromatic amino acids from their diet, plants often must direct over 30% of the carbon fixed through photosynthesis to the shikimate pathway (2). Because it is the only way for microorganisms and plants to obtain the essential aromatic amino acids, inhibition of this pathway leads to organism death. EPSP synthase (EPSPS) is the main commercial target for disruption of the shikimate pathway because it can be selectively inhibited by the molecule N-[phosphonomythyl]glycine (glyphosate). Glyphosate acts as a competitive inhibitor for one of the reactants, PEP. Normally, EPSP synthase catalyzes the reaction between shikimate-3-phosphate (S3P) and PEP to produce enolpyruvylshikimate-3-phosphate (EPSP), as seen in Figure 1. 

 

Glyphosate is a potent broad spectrum herbicide that is the active ingredient in Roundup® (6). Sensitivity towards glyphosate divides the EPSP synthases into two classes, class I and class II (7). CP4 EPSP synthase is a characteristic class II EPSPS because it is not sensitive to glyphosate and still retains its catalytic efficency, making it a useful gene in the genetically modified Roundup Ready® crops (5,6). Research into the function of CP4 EPSP synthase helps scientists to design herbicides effective at targetting other class II EPSPS for agricultural and antibiotic uses (8). Because the shikimate pathway does not exist in animals and because glyphosate does not bind to other enzymes that interact with PEP, it is generally found to have no negative health effects (6,9). 

The synthesis of EPSP occurs through an addition-elimination reaction  in which the hydroxyl group at the five position of S3P undergoes nucleophilic attack on the sp3 carbon of PEP to form a tetrahedral intermediate and an oxocarbenium ion (3). In the elimination step, a C—O bond to the phosphate group is cleaved to give off inorganic phosphate and EPSP (10). Functional theoretical calculations have shown that the rate determining step of the EPSP synthase reaction is the formation of the tetrahedral intermediate and that the tetrahedral intermediate is always in the S-configuration (3). CP4 EPSP synthase catalyzes this step of the reaction through its Lys28 and Glu354 residues. In the addition step of the reaction, the amine on Lys28 acts as a base to deprotonate the hydroxyl group on the five position of S3P, making it more negatively charged and a better nucleophile. The proximity of the resulting cation and anion also serve to stabilize the reactants before reaction occurs. Glu354 acts as an acid in the addition step by donating a proton to the methylene group on PEP to stabilize the oxocarbenium ion that forms when the tetrahedral intermediate forms (3). During the elimination step, the glutamate acts as a base to deprotonate the carbon and allows the tetrahedral intermediate to reform. 

While most EPSPS are inhibited by glyphosate, CP4 EPSPS, which is glyphosate resistant, has a unique mutation at the conserved Gly100 residue in the active site to an alanine. This mutation is found to be the major determinant in the glyphosate resistant properties of CP4 EPSPS. The methyl group on alanine sterically interferes with conformation of glyphosate and forces it to adopt a higher energy condensed conformation which has reduced inhibitory capabilities (5). Mutation of the Ala100 residue back to glycine in CP4 EPSPS has been shown to restore the sensitivity of the enzyme to glyphosate (5). The Ala100Gly CP4 EPSPS mutant (PDB ID: 2GGD) has been shown to bind glyphosate in the extended conformation and to have decreased catalytic activity (5). Mutation of Gly100 to alanine in other glyphosate sensitive proteins has been shown to confer glyphosate resistance (11). Other critical residues in glyphosate resistance include Glu354. When glyphosate binds to the active site in CP4 EPSPS, the Glu354 does not rotate away as it does in many other proteins, resulting in unfavorable steric clash (11). CP4 EPSP synthase has been found to be more resistant to competitive inhibitors, including glyphosate, than most other EPSPSs (8). The prevailing reason for this resilience is dependent on the structure of CP4 EPSPS’s active site, which is found to be more rigid and strongly stabilized by intermolecular forces (8). This stability may be linked to the hydrophobic residues in the hydrophobic pocket of the molecule (11). Because of its rigidity, CP4 EPSP synthase will not adapt its active site as much in order to fit ligands that are not its natural substrate. Instead, it will force these competitive ligands into energetically unfavorable conformations that reduce their ability to compete with the natural ligand for the binding site and therefore reduce their ability to inhibit enzymatic activity. 

Other critical residues in the active site that are involved in the catalytic activity and stabilization of the substrate molecules and include Arg128, Arg357, Arg405, Lys28, Asp326 and Glu354 (5,8). Glyphosate shares a similar but non-identical binding site with PEP, which allows CP4 EPSP synthase to be glyphosate resistant yet still bind PEP and be enzymatically active (12). The charged critical residues interact with PEP and S3P via electrostatic interactions to stabilize the reactant molecules via hydrogen bonding and electrostatic interactions (12). These residues are also positioned in a way that only allows weak interactions with the inhibitor, promoting enzymatic activity and decreasing glyphosate sensitivity. 

The tertiary and secondary structures of CP4 EPSP synthase are also critical to the enzymatic and glyphosate resistant properties of the molecule. CP4 EPSPS is a monomeric unit that weighs 47588.49Da and has a pI of 5.13 (13). It contains two globular domains connected by a two-stranded hinge region. The hinge motif of this protein is important, because it facilitates the induced-fit mechanism by which EPSP synthase binds its substrates. EPSP synthase is usually in an open conformation when unbound (PDB ID: 2GG4) and shifts to the closed conformation after it is bound to S3P (PDB ID: 2GG6). Once the protein is in the closed conformation, the critical residues form a pocket adjacent to S3P where PEP or glyphosate can bind (10). 

The secondary structure of the protein consists of alternating alpha helices and beta sheets arranged in an inside-out α/β barrel with the beta sheets on the outside of the barrel (14). This arrangement is optimal for EPSP synthase because the active site is within the hydrophobic pocket that lines the interior surface of the globular domains which are composed of alpha helices (4). Cys103 is an important residue in CP4 EPSPS located in this pocket that both promotes glyphosate resistance and maintains the integrity of the hydrophobic pocket (11). Mutation of this residue in other species to cysteine reduces the binding efficiency of glyphosate. A notable secondary structure is the third alpha helix, because it houses residues important for glyphosate resistance including Ala100 and Cys103. Other residues in this helix are also known to be highly conserved and factorial in glyphosate sensitivity (11). 

CP4 EPSP synthase’s catalytic activity also depends on the concentration of monovalent cations such as K+ and NH4+. These cations are suggested to help organize a twelve residue loop in the C-terminal domain containing the critical Glu354 residue when the enzyme is not bound to a substrate. However, crystallization with these cations was not possible so their exact effect on protein structure was not determinable (5). 

The structural significance of CP4 EPSP synthase becomes most apparent when compared to Escherichia. coli EPSP synthase (PDB ID: 1G6T), a class I EPSP synthase that has a similar tertiary structure to that of CP4 EPSP synthase. Structural comparisons were made using the Dali and BLAST servers. The Dali server uses a sum-of-pairs method with respect to intramolecular distances to calculate the similarities between the tertiary structures of proteins. The Z-score is the result of the server calculations and is a reflection based on the relative similarities between the query and subject proteins. A Z-score of 2 or greater indicates a significantly similar folding pattern (15). The Blast server compares proteins based on their primary structures and calculates an E-score based on gaps and distances in the amino acid sequences. An E-score of less than 0.05 indicates a significant degree of primary structure similarity between proteins (16).  E. coli EPSP synthase has a Z-score of 46.7 and an E-score of 2x10-3 indicating similar tertiary and primary structures. However, like all class I and class II EPSP synthases, the sequence of E. coli EPSPS shares a low percentage of amino acid similarity of only 27%. Regardless, the tertiary structures of the two proteins are remarkably similar as shown by the Z-score that is much higher than 2, the fact that both consist of two globular domains connected by a two-stranded hinge region, and possess an inside-out α/β-barrel arrangement of secondary structures. Some significant similarities between the two enzymes is that when the EPSPS protein is bound to S3P and in its closed position, the binding pocket for glyphosate and PEP in E. coli  EPSP synthase is lined with the positively charged residues lysine and arginine, resembling the positively charged pocket in CP4 EPSP synthase. That this feature is shared between EPSP synthase classes, suggests that the arginine and lysine residues in this region are required for enzymatic activity (4,7).  The presence of glycine in the 96 position instead of alanine and the binding of glyphosate in the extended conformation indicates that E. coli EPSPS is sensitivite to glyphosate. However, mutation of this residue to alanine has shown to confer glyphosate resistance onto E. coli (7). Unlike in CP4 EPSPS, this change also results in a decreased binding affinity and catalytic efficiency for PEP, suggesting that the active site in E. coli EPSPS contains more overlap between PEP and glyphosate binding regions than does CP4 EPSP synthase (7). 

Compared to other EPSP synthases, CP4 EPSP synthase is a protein that has its primary structure altered to be glyphosate resistant and remain catalytically active in glyphosate saturated environments. Because of the interest in developing drugs to control class II EPSP synthase containing organisms, study of CP4 EPSP synthase could lead to novel forms of weed control as well as low-side effect antibiotics. Already, glyphosate has proven to be an effective drug against a variety of microorganisms such as parasites and class I bacteria, but discovery of a new drug that can target class II EPSPS will have added benefits to the biomedical community.