Molecular basis for the herbicide resistance of Roundup Ready crops

Abstract

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.

Fig. 1.

Key kinetic properties of CP4 EPSP synthase. (A) The reaction catalyzed by EPSP synthase. (B) The activity of CP4 EPSP synthase depends strongly on the presence of cations, such as NH₄⁺, Rb⁺, and K⁺. (Inset) Activation by K⁺ is saturable with an apparent dissociation constant of 25 mM. (C) IC₅₀ studies with wild-type CP4 EPSP synthase (■), Ala-100–Gly CP4 EPSP synthase (▴), and wild-type E. coli EPSP synthase (●) reveal that CP4 EPSP synthase is inhibited only by high millimolar concentrations of glyphosate (IC₅₀ = 11 mM). The Ala-100–Gly mutant CP4 EPSP synthase is approximately two orders of magnitude more sensitive to glyphosate (IC₅₀ = 160 μM). The E. coli enzyme is inhibited by even lower glyphosate concentrations (IC₅₀ = 2.5 μM).

Fig. 2.

Three-dimensional structure of CP4 EPSP synthase. (A) (Left) Unliganded CP4 EPSP synthase exists in an open conformation. (Right) Upon interaction with S3P, the enzyme undergoes a large conformational change to a closed state. Shown in orange is a loop spanning residues 347–358, which is highly flexible in the open conformation but becomes ordered in the closed conformation. This loop contains the strictly conserved EPSP synthase residues Glu-354 and Arg-357, which are involved in PEP/glyphosate binding. Monovalent cations may influence the conformation of this loop and facilitate binding of PEP. (B) Stereoview showing that, in the binary complex, S3P (yellow) binds to the enzyme residues shown in magenta through multiple hydrogen-bonding/electrostatic interactions (black dotted lines). In addition, the cyclohexene moiety of S3P is sandwiched between Arg-200 and Gln-175. Residues shown in light blue constitute the PEP/glyphosate binding site. Attracted by the accumulation of positive charges, a sulfate ion (shown in green) from the crystallization solution binds to the space occupied by the phosphate moiety of PEP or the phosphonate moiety of glyphosate in either ternary complex. Water molecules are shown as cyan spheres.

Fig. 3.

Interaction of glyphosate with CP4 EPSP synthase (stereoview). (A) Glyphosate (shown in green) binds to the active site of CP4 EPSP synthase adjacent to S3P (shown in yellow) in a condensed conformation different from that observed in E. coli or Str. pneumoniae EPSP synthase, as the result of a clash between the Ala-100 side chain and oxygen atoms of glyphosate’s phosphonate group. Notably, in this shortened conformation, the carbon atom of the phosphonate group clashes with the side chain of Glu-354 (red dotted line). (B) Replacing Ala-100 with a Gly allows glyphosate to bind in its extended conformation, positioning glyphosate’s nitrogen atom midway between the target hydroxyl of S3P and Glu-354. The view is ≈90° clockwise from that of Fig. 2B. Hydrogen bonding/electrostatic interactions are indicated by black dotted lines.

Fig. 4.

Two distinct conformations of glyphosate. Displayed are the electron densities, contoured at 3σ, derived from 1F_o − 1F_c Fourier syntheses to 1.7-Å resolution, omitting the model of glyphosate during the refinement of the ternary complexes of CP4 EPSP synthase (Left) and Ala-100–Gly CP4 EPSP synthase (Right). (Right) The conformation of glyphosate upon interaction with the Ala-100–Gly CP4 EPSP synthase is identical to the one observed in the E. coli or Str. pneumoniae enzymes. (Left) With an Ala residue in position 100, the glyphosate molecule is ≈0.6 Å shorter, mainly because of a rotation around the CN bond next to the carboxyl group.