The enzyme 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19) is essential for the biosynthesis of aromatic compounds in plants and microbes and is the unique target of the herbicide glyphosate. One of the first glyphosate-insensitive enzymes reported was a Gly96Ala mutant of EPSP synthase from Klebsiella pneumoniae. We have introduced this single-site mutation into the highly homologous EPSP synthase from Escherichia coli. The mutant enzyme is insensitive to glyphosate with unaltered affinity for its first substrate, shikimate-3-phosphate (S3P), but displays a 30-fold lower affinity for its second substrate, phosphoenolpyruvate (PEP). Using X-ray crystallography, we solved the structure of Gly96Ala-EPSP synthase liganded with S3P to 0.17 nm resolution. The crystal structure shows that the additional methyl group from Ala96 protrudes into the active site of the enzyme. While the interactions between enzyme and S3P remain unaffected, the accessible volume for glyphosate binding is substantially reduced. Exploiting the crystallographic results for molecular modeling, we demonstrate that PEP but not glyphosate can be docked in the Gly96Ala-modified binding site. The predicted PEP binding site satisfies the earlier proposed interaction pattern for PEP with EPSP synthase and corroborates the assumption that glyphosate and PEP target the same binding site.

MurA (UDP-N

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 spatial and temporal organization of biological systems offers a level of complexity that is challenging to probe with conventional reagents. Photoactivatable (caged) compounds represent one strategy by which spatiotemporal organizational complexities can be addressed. However, since the vast majority of caged species are triggered by UV light, it is not feasible to orthogonally control two or more spatiotemporal elements of the phenomenon under investigation. For example, the cGMP-and cAMP-dependent protein kinases are highly homologous enzymes, separated in time and space, which mediate the phosphorylation of both distinct and common protein substrates. However, current technology is unable to discriminate, in a temporally or spatially selective fashion, between these enzymes and/or the pathways they influence. We describe herein the intracellular triggering of a cGMP-mediated pathway with 360 nm light and the corresponding cAMP-mediated pathway with 440 nm light. Dual wavelength photoactivation was assessed in A10 cells by monitoring the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a known substrate for both the cAMP-and cGMP-dependent protein kinases. Illumination at 440 nm elicits a cAMP-dependent phosphorylation of VASP at Ser157 whereas 360 nm exposure triggers the phosphorylation of both Ser157 and Ser239. This is the first example of wavelength-distinct activation of two separate nodes of a common signaling pathway.Biological behavior is inherently dynamic, be it at the cellular, tissue, organ, or organismal level.(1) The response of living organisms and their component parts to environmental stimuli often reflects the spatiotemporal elements associated with that stimulus. For example, metastasis is driven by the directed migration of tumor cells toward spatially focused chemoattractant gradients.(2) However, spatiotemporal control of biological behavior is not limited to just environmental stimuli. As a result of evolution's efficiency, individual proteins, and the biochemical pathways they inhabit, are often used for more than one purpose. The activation of a specific protein can have a multitude of biological consequences yet, within the appropriate spatiotemporal context, elicits only a single response. Cytochrome c, as a key member of the mitochondrial electron transport chain, is essential for life in eukaryotes. However, upon release from the mitochondrion, it serves as a purveyor of death.(3) Only a micron separates life from death, a fact which highlights the control exerted by the spatiotemporal context under which the biological event occurs. Unfortunately, conventional probes of cellular biochemistry, such as inhibitors, activators, or sensors, typically lack the temporal or spatial resolution to adequately address biochemically-driven behaviors, especially those that transpire during a short time frame or occur within a restricted spatial environment.A wide variety of photoactivatable (caged) compounds and biomolecules have been described since their first in...

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.