See the article"Among enzyme inhibitors
used in agriculture, glyphosate (N-phosphomethyl glycine) is
remarkable. A nonselective herbicide discovered in 1970 by a group of
scientists at Monsanto led by Dr. John Franz (1), glyphosate, since
first commercialization under the trade name Roundup, has been used
globally as a safe and effective means of weed control. The discovery
of glyphosate's herbicidal activity was not quite serendipity, but
instead resulted from a synthetic strategy based on the hypothesis that
the weak herbicidal activities of related compounds derived from the
possibility of their similar metabolic fate (2). Nevertheless, the
initial discovery by greenhouse screening has been followed by
intensive biochemical studies that have now led to nearly complete
understanding of glyphosate's mode of action. In 1972, scientists at
Monsanto led by Dr. E. Jaworski observed (3) that application of
glyphosate resulted in the inhibition of aromatic amino acid
biosynthesis in plants. In 1980, Professor N. Amrhein and coworkers (4)
identified its target enzyme from the shikimate pathway (4):
5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS; EC
2.5.1.19). EPSPS is a key enzyme involved in aromatic amino acid
biosynthesis (5). The enzyme catalyzes an unusual reaction, wherein the
enolpyruvoyl group from phosphoenol pyruvate (PEP) is transferred to
the 5-hydroxyl of shikimate-3-phosphate (S3P) to form the products
5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic
phosphate (Pi). The only other enzyme known to catalyze carboxyvinyl
transfer by using PEP is UDP-N-acetylglucosamine enolpyruvyl
transferase (MurA), which catalyzes the first committed step in the
biosynthesis of the peptidoglycan layer of the bacterial cell. In the
case of EPSPS, the reaction proceeds through a tetrahedral intermediate
(Scheme (SchemeS1)S1) formed from S3P and PEP (6). Inhibition
of EPSPS by glyphosate has been shown to proceed through the
formation of an EPSPS-S3P-glyphosate ternary complex and the
binding is ordered with glyphosate binding to the enzyme only after the
formation of a binary EPSPS-S3P complex. Binding of glyphosate to EPSPS
has been shown to be competitive with PEP and uncompetitive with
respect to S3P (7). What makes glyphosate a remarkable inhibitor
and herbicide? Glyphosate is a relatively simple molecule—an
N-methyl phosphonate derivative of glycine with a chemical
structure not unlike that of the universal high energy
phosphoryl-transfer agent PEP. Despite this, glyphosate retains
exquisite specificity for EPSPS and is not known to appreciatively
inhibit any other enzyme, even MurA (8).
The EPSPS reaction is the penultimate step in the shikimic acid pathway
for the biosynthesis of aromatic amino acids (Phe, Tyr, and Trp) and
many secondary metabolites, including tetrahydrofolate, ubiquinone, and
vitamin K (9). The shikimic acid pathway, present in plants and
microorganisms, is completely absent in mammals, fish, birds, reptiles,
and insects. The importance of the shikimate pathway in plants is
further substantiated by the estimation that up to 35% or more of the
ultimate plant mass in dry weight is represented by aromatic molecules
derived from the shikimate pathway (2). On the basis of this
information, it is readily apparent why EPSPS is a good target for
novel antibiotics and herbicides.
The crystal structure of the ternary EPSPS-S3P-glyphosate complex was
reported in PNAS at a stunning resolution of 1.5 Å and allows detailed
visualization of the complete set of interactions of the ligands with
their target (10). Using formate and phosphate as part of their
crystallization protocol, the authors also report the high-resolution
structure of the quaternary EPSPS-S3P-Pi-formate complex and suggest
that the structure of this complex is representative of the true binary
EPSPS-S3P complex. The new structures advance our understanding of the
interaction of the widely used, broad spectrum herbicide glyphosate
with EPSPS and provide an important opportunity for designing new and
unique inhibitors for use as herbicides as well as antibiotic and
antiparasitic drugs.
In recent years significant progress has been made toward
understanding how glyphosate inhibits EPSPS. The first
three-dimensional x-ray crystal structure of native Escherichia
coli EPSPS (Fig. (Fig.1)1) showed that the
enzyme consists of two domains of nearly equal size and that each
domain contains six parallel α-helices aligned in a way that their
macrodipoles create a significant electropositive attraction for the
anionic ligands at the interface between the two domains (11). The
EPSPS structure was found to be remarkably similar to the x-ray crystal
structure of MurA (12), the closely related enolpyruvyl transferase
whose structure was reported by some of the same investigators who now
describe the new EPSPS structures. Binding of substrate
(UDP-N-acetylglucosamine) and inhibitor (fosfomycin) to MurA
was shown in these studies to induce a dramatic conformational change.
On ligand binding, the MurA tertiary structure changes from the
“open” form of the enzyme to the “closed” form, in which
the two domains rearrange substantially into a tightly packed
conformation (13). Using limited tryptic digestion and matrix-assisted
laser desorption ionization–time-of-flight mass spectroscopy, Krekel
et al. (14) have suggested that binding of S3P in
combination with glyphosate also stabilizes EPSPS in a closed
conformation. In addition, fluorescence spectroscopy studies imply that
the association of glyphosate with the EPSPS-S3P binary complex induces
a substantial conformational change in the enzyme (15). In a separate
study using radiolabeled plant PreEPSPS (mature enzyme with a short
chloroplast transit peptide), Della-Cioppa and Kishore (16) have shown
that the native precursor protein is transported into chloroplasts,
whereas a PreEPSPS-S3P-glyphosate ternary complex was not readily
transported into the chloroplast stroma, suggesting that the ternary
complex in its closed form is probably hindered in temporarily
undergoing a conformational change to facilitate the process of
importation across the chloroplast membrane. Collectively, these
results have suggested that EPSPS undergoes a macroconformational
change on the binding of S3P and glyphosate.
 | Figure 1 Folding and topological symmetry of EPSPS (adapted from ref. 11).
The two domain structure is formed by 6-fold replication of a protein
folding unit (Upper) comprising two parallel helices and
a four-stranded β-sheet. Each domain is formed (more ...) |
The results presented in this issue of PNAS clearly show that the
two domains of EPSPS converge on ligand binding (Fig.
(Fig.2).2). The x-ray structures also indicate that
residues required for ligand binding are found in both globular domains
and this effective interaction likely helps exclude bulk solvent from
the active site during catalysis. The results further demonstrate that
the x-ray structure of EPSPS bound with S3P, but not glyphosate, is
virtually identical to that of EPSPS-S3P-glyphosate complex. Because
S3P is necessary for crystal formation, the authors suggest that it is
S3P alone that triggers the conformational change of the enzyme from
the open to the closed state. It should be noted that there are
potential alternative explanations for this observation. Studies using
fluorescence spectroscopy have shown that there is no fluorescence
change on the binding of S3P or glyphosate alone, but that formation of
the ternary enzyme complex is signaled by a fluorescence decrease (15).
Studies using native, open-form E. coli enzyme crystals
soaked in buffered 100 mM S3P solution have shown that EPSPS-S3P binary
complex, like the native enzyme, contains two widely separated globular
domains (2). Another point to note would be that, if indeed the enzyme
is in a closed state after S3P binding and formation of the binary
complex, how then does glyphosate enter the solvent-inaccessible active
site? Channels or interactions that might mediate this process are not
identified by the authors in their analysis of the structures. The
possibility that the phosphate and formate ions occupying the
glyphosate binding site in the glyphosate-free structure are partly
responsible for inducing the macroconformational change from the open
to the closed form of the ligand-bound enzyme is also not
considered.
 | Figure 2 (Left) Space-filling model of E. coli
EPSPS in the open form. (Right) Model of an EPSPS
molecule in the closed, ligand-bound form. Comparisons of the figures
illustrate the dramatic conformational change that attends ligand
binding. |
By using the x-ray crystal structure of MurA bound with a
fluorinated analog of its tetrahedral intermediate, the authors have
identified residues involved in the interaction with the PEP moiety.
Comparisons of this structure with that of the newly observed binding
mode for glyphosate to EPSPS reveal that homologous residues
stabilize the binding of glyphosate in the ternary complex. Based on
this and the observation that the formate and phosphate ions in the
inhibitor free structure bind in the same anionic sites as the
corresponding groups in glyphosate, the authors suggest that PEP
occupies the glyphosate binding site in the EPSPS enzyme. This
suggestion is at odds with kinetic and direct binding data that have
shown that glyphosate can bind to an EPSPS-EPSP binary complex forming
an EPSPS-EPSP-glyphosate ternary complex (17). Furthermore, glyphosate
was found to be an uncompetitive inhibitor versus EPSP in the reverse
reaction (17). These results would suggest that the glyphosate and PEP
binding sites are not completely superimposable in their ternary EPSPS
complexes. More studies will be required to fully detail the
interactions of PEP and possible binding differences between PEP and
glyphosate in the EPSPS active site.
Regardless of whether the true binary complex with S3P is open or
closed, or whether the PEP and glyphosate sites partly overlap, the new
EPSPS structures are significant new tools for the rational design and
discovery of novel inhibitors. Quite recently, the growth of several
pathogenic parasites such as Plasmodium falciparum,
Taxoplasma gondii, and Cryptosporidium parvum was
shown to be inhibited by glyphosate (18). Thus, the new EPSPS
structures come at an especially opportune time for impact on new
infectious diseases programs. Studies on the binding of the tetrahedral
reaction intermediate to EPSPS have demonstrated that tapping into the
structural determinants involved in S3P and glyphosate recognition
could lead to inhibitors of picomolar affinity (19). However, though
the detailed description of the active site can be used for virtual
screening or even de novo ligand design, the catalytic site
has been designed by nature to bind multiply charged anionic ligands
like S3P, PEP, glyphosate, and products, EPSP and Pi. Given that
interactions with these anionic ligands are stabilized by an array of
basic EPSPS side chains and helix macrodipoles that, on ligand binding,
converge by a massive conformationally induced fit mechanism, new
ligands complementary to this site would also be expected to be highly
anionic. Such molecules might be unlikely drugable leads unless
facilitated transport mechanisms were available for membrane passage.
With some wisdom, the authors therefore propose an alternate
strategy for structure-based inhibitor design. The results presented in
the paper clearly demonstrate what other studies have predicted: the
binding of ligands to EPSPS causes a macroconformational change from an
open form to a closed form of the enzyme. Now, using studies that
spatially identify residues responsible for the conformational change
in MurA and mapping them in the EPSPS crystal structure (20–22), the
authors have identified a new array of residues common to both enzymes
(Fig. (Fig.3)3) that might be important for the
conformational change and thereby provide new templates for future
efforts that target the design of novel antimicrobial and herbicidal
agents that block closure of the enzymes and formation of the catalytic
sites.
 | Figure 3 Crystal structure of the open form of EPSPS. Schönbrunn et
al. (10) identify residues in MurA and their homologs in
EPSPS that are determinants in the control of domain closure, and
suggest that inhibitors that bind to these residues will interfere
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