Molecular basis of glyphosate resistance-different approaches through protein engineering

Abstract

Glyphosate (N-phosphonomethyl-glycine) is the most widely used herbicide in the world: glyphosate-based formulations exhibit broad-spectrum herbicidal activity with minimal human and environmental toxicity. The extraordinary success of this simple, small molecule is mainly attributable to the high specificity of glyphosate for the plant enzyme enolpyruvyl shikimate-3-phosphate synthase in the shikimate pathway, leading to the biosynthesis of aromatic amino acids. Starting in 1996, transgenic glyphosate-resistant plants were introduced, thus allowing application of the herbicide to the crop (post-emergence) to remove emerged weeds without crop damage. This review focuses on mechanisms of resistance to glyphosate as obtained through natural diversity, the gene-shuffling approach to molecular evolution, and a rational, structure-based approach to protein engineering. In addition, we offer a rationale for the means by which the modifications made have had their intended effect.

Fig. 1

Shikimate pathway that leads to the biosynthesis of aromatic amino acids and mode of action of glyphosate on the reaction catalyzed by EPSPS.

Fig. 2

Molecular mode of action of glyphosate and the structural basis for glyphosate resistance. A) In its ligand-free state, EPSPS exists in the open conformation (left; PDB: 1eps). Binding of S3P induces large conformational in the enzyme to the closed state to which glyphosate or substrate PEP bind (PDB: 1g6s). Shown are the respective crystal structures of the E. coli enzyme, with the N-terminal globular domain colored in palegreen and the C-terminal domain colored in wheat. The helix containing P101 is indicated in magenta and the S3P and glyphosate molecules in green and yellow, respectively. B) Schematic representation of potential hydrogen bonding and electrostatic interactions between glyphosate and active site residues including bridging water molecules in EPSPS from E. coli (PDB: 1g6s). C) The glyphosate binding site in EPSPS from E. coli (PDB: 1g6s). Water molecules are shown as cyan spheres and the residues known to confer glyphosate resistance upon mutation are indicated in magenta. D) The glyphosate binding site in CP4 EPSPS (PDB: 2gga). The spatial arrangement of the highly conserved active site residues is almost identical for class I (E. coli) and class II (CP4) enzymes, with the exception of an alanine residue in position 100 (G96 in E. coli). Another significant difference is the replacement of P101 (E. coli) by a leucine (L105) in the CP4 enzyme. Note the markedly different, condensed conformation of glyphosate as a result of the reduced space provided for binding in the CP4 enzyme.

Fig. 3

Microbial mechanisms of glyphosate degradation. A) Two principal pathways of glyphosate degradation are known: top) cleavage of the carbon-phosphorus bond yielding phosphate and sarcosine (the C-P lyase pathway); bottom) cleavage to yield the formation of aminomethylphosphonic acid (AMPA) and glyoxylate (the AMPA pathway), referred to as the glyphosate oxidase (GOX) pathway. B) Reaction catalyzed by GO on glyphosate [49], an alternative of the AMPA pathway as catalyzed by GOX (panel B, bottom).

Fig. 4

The superposition of wild-type (PDB: 1rhl, green) and G51S/A54R/H244A GO (PDB: 3if9, blue) structures shows the different conformation of the main chain of α2–α3 loop, see arrows [49]. For the sake of clarity, only the FAD and the ligand belonging to the wild-type GO structure are shown, and Arg329 was omitted.

Fig. 5

Substrates of acetyltransferase reactions mentioned in the text [53,55].

Fig. 6

R7 GLYAT ligated with glyphosate and acetyl coenzyme A (Z. Hou, Pioneer Hi-Bred, unpublished, based on PDB: 2jdd). The altered residues (R7 vs. native) and ligands are shown with ball-and-sticks.

Fig. 7

GLYAT reaction mechanism [55]. Glyphosate, whose nitrogen pK is 10.3, enters the active site as the protonated form and binds with its phosphonate group ligated by charge interactions with Arg21 and Arg111, and its carboxyl group in contact with Arg73. The shortness of the hydrogen bond between the N-ε of His138 and a phosphonate oxygen of glyphosate suggests a specific mechanism in which a proton from the secondary amino group of glyphosate is stabilized on a phosphonate oxygen atom, resulting in formation of the strong hydrogen bond between His-138 and glyphosate and activation of the substrate amine. This substrate-assisted proton transfer mechanism is consistent with the observed pH dependence of k_cat and explains the dual role of His138 in substrate binding and as a catalytic base. To complete the reaction, attack by the lone pair of the glyphosate nitrogen on the carbonyl carbon of AcCoA results in a tetrahedral intermediate. Tyr118 is perfectly positioned to protonate the sulfur atom of coenzyme A as the tetrahedral intermediate breaks down to yield the products. This research was originally published in [55].