Structural Basis of Glyphosate Resistance Resulting from the Double Mutation Thr97 → Ile and Pro101 → Ser in 5-Enolpyruvylshikimate-3-phosphate Synthase from Escherichia coli

Abstract

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (K i ‫؍‬ 2.4 mM) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (K m ‫؍‬ 0.1 mM). The crystal structure at 1.7-Å resolution revealed that the dual mutation causes a shift of residue Gly 96 toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile 97 points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr 97 and Pro 101 induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS.Glyphosate (N-phosphonomethylglycine) is a potent inhibitor of the shikimate pathway in plants, specifically targeting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, 3 EC 2.5.1.19) (1). Glyphosate-based formulations exhibit broad spectrum herbicidal activity with minimal human and environmental toxicity (2, 3). The safety and efficacy of glyphosate, together with the existence of genetically modified, glyphosate-resistant crop varieties (4, 5), have combined to make glyphosate the most used herbicide in the world. Enzymes of the shikimate pathway are also regarded as attractive antimicrobial targets (6 -9).EPSPS catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 5-hydroxy position of shikimate-3-phosphate (S3P) (Fig. 1). Binding of the first substrate, S3P, to the enzyme triggers a global conformational change from an "open" to a "closed" conformation. PEP and glyphosate bind in the active site, formed at the interface between the Nand C-terminal globular domains. Glyphosate inhibition is competitive with respect to PEP (10, 11), and structural studies confirmed that glyphosate occupies the PEP-binding site (12-15).EPSPS from different organisms have been divided into two classes according to intrinsic glyphosate sensitivity: in Class I enzymes, found in all plants and i...

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase

“…The other is the third helix of the core of the N-terminal domain. Given that this helix is a universal mutation hot spot for glyphosate resistance, the Thr97Ala Pro101Ser double mutation in class I EPSPS from E. coli produced the first commercial varieties of glyphosate-tolerant maize (10). Three new residues-N267S, P318R, and M425T-that also affect glyphosate resistance have been identified in our study.…”

“…Numerous amino acid residues that affect EPSPS glyphosate resistance have already been identified (1,8,10,13,15,19,20,24,25,31,35,40,50). Notably, these residues are mostly distributed in two regions according to their 3D structure.…”

Evolution of a Double Amino Acid Substitution in the 5-Enolpyruvylshikimate-3-Phosphate Synthase in Eleusine indica Conferring High-Level Glyphosate Resistance

“…The transition state inhibitor designation for glyphosate comes from the observation that PEP contacts 17 amino acids responsible for catalysis, which necessarily prevents any substitutions of these essential amino acids (Schönbrunn et al, 2001). The P106S EPSPS provides relatively low glyphosate resistance (Arnaud et al, 1998), whereas the T102I EPSPS alone endows high-level resistance, but with drastically decreased affinity for the second substrate, PEP (Kishore et al, 1992;Funke et al, 2009). The concomitant mutations at the 106 and 102 codons are merely adjacent to the active site and together make very small fractional Ångstrom modifications structurally to the EPSPS active site, therefore selectively impacting glyphosate binding more than PEP (Funke et al, 2009).…”

“…Indeed, highly glyphosateresistant EPSPS variants (e.g. mutants at G101 or T102) have greatly increased K m values (decreased affinity) for PEP when expressed in Escherichia coli (Eschenburg et al, 2002;Funke et al, 2009; for review, see Sammons and Gaines, 2014). In contrast, P106 substitutions confer weak glyphosate resistance but preserve adequate EPSPS functionality (Healy-Fried et al, 2007; for review, see Sammons and Gaines, 2014).…”

Resistência da planta daninha capim-branco (Chloris polydactyla) ao herbicida glyphosate

“…No primeiro caso, o herbicida atinge o sítio de ação, mas não consegue inibir a enzima do biótipo resistente, como acontece com os biótipos resistentes ao glyphosate que possuem mutação na posição 106 da EPSPs, onde pode haver uma substituição do aminoácido prolina por uma serina, threonina, alanina ou leucina, mudando a forma com que o glyphosate se "acopla" à EPSPs (PEREZ-JONES et al, 2007;POWLES, 2007;PRESTON et al, 2009;PRESTON, 2006;KAUNDUN et al, 2008;FUNKE et al, 2009). Há também casos em que o herbicida é capaz de realizar sua função, ou seja, inibir a ação da EPSPs, mas os biótipos resistentes possuem a habilidade de produzir mais EPSPs do que o glyphosate é capaz de inibir, conhecida como superexpressão gênica (GAINES et al, 2010).…”