 Introduction History Gene and Protein Isolation Structure and Mechanism Biological Significance Summary References BCMB 8010 website |
Please note these web pages are part of an assignment for a graduate course in Advanced Biochemistry and Molecular Biology BCMB8010 at the University of Georgia. Questions should be directed to Gina Pries (gpries@uga.edu).
EPSP Synthase
Structure and Mechanism
EPSP synthase is an essential element in the shikimate pathway, transferring the enolypyruvyl group of phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P) to form 5-enolpyruvyl-3-shikimate phosphate (EPSP) and inorganic phosphate (Pi) (Fig. 1). This mechanism of enolpyruvyl transfer is shared only by MurA, an enzyme involved in synthesis of the bacterial cell wall. The structures and mechanisms of these two closely related enzymes are similar. The two domains are each composed of three copies of ßaßaßß-folding units (1) (Fig. 3). In between are two crossover chain segments that hinge the nearly topologically symmetrical domains (14) together and allow conformational changes necessary for substrate conversion. Each domain structure has been described as a "mushroom button" where the inner helices form the stem and the outer beta strands act as the cap (14). Each stem base faces the other, and the parallel helices extend with the positive end of their macrodipoles situated at this interface. It has been postulated that part of the binding mechanism is facilitated by a helical macrodipole effect (24). Since the substrates are heavily anionic, it is reasonable to assume that the localized positive core of the active site would assist in anion binding.
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Figure 3 a. Top: One ßaßaßß-folding unit. Bottom: N-terminal domain from bottom with lines separating the three folding units.
b. Stallings' nomenclature for subdomain tertiary structures. Yellow arrows represent beta-strands, with open arrows indicating a strand that is not directly connected to other secondary structures within its own folding unit, and magenta rectangles represent alpha-helices. Domain 1 is composed of folding units 1, 2, and 6, and Domain 2 is composed of folding units 3, 4, and 5. Both carboxy and amino termini are contained within folding unit 1. Letter assignments for each secondary structure within a folding unit begin at the n-terminal end of the folding unit and are lettered in alphabetical order consecutively to the c-terminal end. (adapted from Stallings et al. (14)).
Induced fit mechanism
Two EPSP synthase structures were resolved by Schönbrunn et al. (1), the first with S3P and glyphosate bound to the active site (PDBID 1G6S), and the second with S3P and a formate and phosphate ion (mimicking the side groups of PEP) bound to the active site (PDBID 1G6T). It is reasonable to assume that glyphosate occupies the PEP-binding site since the formate and phosphate ions in 1G6T are coordinated by the same goups as glyphosate in G6S (1). Also, the phosphate and formate ions mimic the active groups of the PEP oxocarbenium ion, and the two structures are virtually identical (1). The Schönbrunn structures now conclusively confirm the induced fit mechanism, especially when compared to the EPSP backbone structure (PDBID 1EPS) resolved by Stallings in 1991 (Fig. 4) (14).
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It has been proposed that the EPSP synthase reaction is ordered with S3P binding first, followed by PEP with subsequent release of phosphate followed by EPSP (25). Alanine substitution of Arg-27, which binds exclusively to S3P, blocks both S3P and glyphosate binding (26), thus indicating that S3P binding is necessary for glyphosate (and presumably PEP) binding. Ligand binding induces the closure of the two domains to form the active site in the interdomain cleft (1). It is somewhat controversial whether binding of S3P alone stimulates complete conversion to the closed state; some data suggests that this is not the case. Mutagenesis on residues not involved with substrate binding decrease EPSP synthase activity, thereby implicating a possible hindrance of domain closure (26, 1). This would indicate that more than just S3P binding is needed to convert the enzyme from open to closed state. If S3P did initiate domain closure, how then could glyphosate or PEP enter the active site (27)? Other experiments involving fluorescence spectroscopy have shown that the binary EPSPS-S3P complex conformation changes upon glyphosate binding (3). These results and others (28,29) suggest that conformational change and stabilization requires the binding of both S3P and glyphosate, implying that the same would hold true for PEP. The crystal structures of the binary EPSPS-S3P and ternary EPSPS-S3P-glyphosate complexes reported by Schönbrunn and coworkers are nearly alike; however, it is possible that the formate and phosphate ions used in the glyphosate-free experiment to simulate PEP binding could have played a role in inducing the closed conformation (27). Regardless of order of closure, it is readily apparent that there is a drastic conformational change which distinguishes the inactive from the active form of the enzyme.
Stabilizing Interactions
Sequence analysis of EPSP synthase isolated from various organisms has revealed a conserved region consisting of a histidine, an aspartate, and an arginine (30). Mutagenesis experiments by Shuttleworth and Evans (31) concluded that His-385 may be responsible for H-bond stabilization of residues at or near the active site. This postulation was confirmed by the Schönbrunn structure which also indicates that His-385 may possibly serve as a proton donor to Glu-341 (at 2.85Å away) (1). Glu-341 is also held in place by interactions with its own backbone nitrogen and Lys-411, and it appears to aid in stabilization of the oxocarbenium ion of PEP (1). Mutations in both His-385 and Lys-411 drastically decrease the catalytic activity of EPSP synthase (26). Other residues which upon mutational analysis have decreased catalytic activity are Arg-100, Asp-242, and Asp-384 (26) and appear to be involved in the conformational change necessary for formation of the active site since they are not involved in substrate binding.
Substrate and inhibitor binding
Mutagenesis analysis, structural revelation, and MurA comparisons have all aided in the finding of residues which play direct roles in substrate and glyphosate binding. The recent X-ray structure of the EPSP synthase in the closed conformation has aided tremendously in isolating specific associations made by substrates in the active site.
The Schönbrunn and coworkers' structures indicate a close proximity of S3P to glyphosate, and solid-state NMR data (32,33) is in agreement with the reported distances between glyphosate and S3P (1). Hydrogen-bonding appears to exist between the 5-hydroxyl of S3P and nitrogen of glyphosate as well as other interactions involving Lys-22 and a water molecule (W-2) (Figure 5) (1).
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Figure 5 Left: S3P and glyphosate orientation in the active site. Right: Residues involved in PEP binding, shown here with glyphosate.
Since the same salt bridges coordintate the anionic centers of glyphosate and the formate and phosphate ions, it is presumed that glyphosate occupies the same binding site as PEP (1). This is confirmed by kinetic data supporting glyphosate as a competive inhibitor with respect to PEP and an uncompetitve inhibitor with respect to S3P (34). Further clarification of the residues that coordinate glyphosate and PEP can be gained by comparison analysis with the PEP binding site of MurA. The residues in EPSP synthase corresponding to the PEP binding residues of MurA are Lys-22, Arg-124 (hydrogen-bonding to the phosphoryl group), Asp-313, Arg-344, Arg-386, and Lys-411 (Fig. 6) (1). The PEP binding site of MurA is located mainly on the C-terminal domain while the UDP-NAG binding site, corresponding to the S3P binding site in EPSP synthase, involves primarily N-terminal domain residues (2).
Figure 6 Conserved active site residues. S3P and glyphosate are rendered in CPK as are atoms within 3.0 Angstroms of S3P or glyphosate. Conserved residue Lys22 not shown.
With MurA comparisons in mind, Stauffer et al. (15,2) employed NMR spectroscopy to study S3P binding to the isolated N-terminal domain which by itself maintains the same overall tertiary structure as in the full-length enzyme. Other studies have also reported S3P-binding residues all located in the N-terminal domain of EPSP synthase (35) and that the N-terminal domain is the major contributor of binding energy of S3P (E). S3P binding was found to be specific to subdomains 3 and 5 (Fig. 7) (Stalling nomenclature (14), Fig. 3b) (15). The residues located on the inner face of the N-terminal domain postulated to be involved in S3P binding are Ser23, Arg27, Ser197, and Tyr200 (Fig. 8) (15). Stuaffer et al. propose a partial conformational change in EPSP synthase upon S3P binding which involves a shift in the last two beta strands of subdomain 4 in response to S3P binding to subdomain 5. This theory is supported by the findings (36) that monoclonal antibodies raised against the binary EPSPS-S3P complex would not recognize the enzyme alone. Furthermore, monoclonal antibodies raised against EPSP synthase had no change in their dissociation constant upon binding S3P, suggesting that S3P did not induce the complete conformational change from the open to the closed state (37).
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Figure 8 Residues involved in S3P binding, all in N-terminal domain. Blue: Subdomain 5d. Green: Subdomain 3b.
Catalytic Mechanism
The enzymatic mechanism proceeds through an addition-elimination reaction with the formation of a tetrahedral intermediate between S3P and PEP. EPSP synthase also catalyzes via an ordered mechanism, binding S3P first followed by PEP with subsequent release of phosphate and lastly EPSP (25). There has been conflicting work supporting both ordered and a random mechanisms; however, Lewis et al. showed by substrate trapping experiments that the kinetically preferred pathway was ordered (38). This enzyme is unusual among those that involve PEP since the C-O bond of PEP is cleaved rather than the high-energy P-O bond. Only three other enzymes share this method of PEP-catalysis: MurA, 3-deoxy-D-manno-2-octulosonate 8-phosphate (Kdo8P) synthase, and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (38). A cyclic ketal side product is also formed spontaneously from the intermediate (Fig. 9), but at a very significantly slower rate than the reaction proceeds (38). The cyclic ketal most likely forms through an attack of the benzyl hydroxyl on the vinylic carbon (38).
Figure 9
Diagram of reaction pathway with both tetrahedral intermediate and cyclic byproduct shown. Structures adapted from Lewis et al. (38) [Reaction generated with ISIS Draw 2.4 in TGF format]
The Schönbrunn structures have given better understanding to the details of the enzymatic mechanism involved in the enolpyruvyl transfer reaction of EPSP synthase. The following mechanism was proposed by Schönbrunn et al. based on studies for active site residues and the proximity of these residues to substrates in the solved structures. Glu341 acts as a proton donor to the methylene group of PEP, after which it is reprotonated by His385 which is 2.85 Angstroms away (1). The 5' hydroxyl of S3P undergoes deprotonation by Asp313, priming it for attack on the C2 of PEP oxocarbenium ion (1). The tetrahedral intermediate is formed, and Lys22 protonates the oxygen, cleaving the scissile bond to release inorganic phosphate. Subsequently the product, EPSP, is released (Fig. 10) (1).
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Figure 10 Active site residues as they would act on PEP according to hypothesis by Schönbrunn et al (1). In place of PEP is glyphosate, shown here in magenta. S3P is shown in green. Left: Glu341 donates a proton to the methylene group of PEP with His385 serving as a proton source. Middle: Asp313 abstracts and electron from the 5' hydroxyl of S3P in preparation for attack on PEP. Right: S3P 5' oxygen attacks oxocarbenium ion of PEP, forming the tetrahedral intermediate. Not shown: Lys22 protonating scissile bond to release inorganic phosphate with subsequent EPSP release.
Please note that all EPSP synthase structure figures displayed were generated in RasMol using PDB structures 1EPS (open conformation) and 1G6S (closed conformation with S3P and glyphosate bound). Figures were imported to MS Paint and labeled.
Updated on 12/9/02
Author: Gina Pries (gpries@uga.edu)