Characterization of a glyphosate-insensitive 5-enolpyruvylshikimic acid-3-phosphate synthase

A glyphosate (N-[phosphonomethyl]glycine)-insensitive 5-enolpyruvylshikimic acid-3-phosphate (EPSP) synthase has been purified from a strain of Klebsiella pneumoniae which is resistant to this herbicide [(1984) Arch. Microbiol. 137, 121-123] and its properties compared with those of the glyphosate-sensitive EPSP synthase of the parent strain. The apparent K_m values of the insensitive enzyme for phosphoenolpyruvate (PEP) and shikimate 3-phosphate (S-3-P) were increased 15.6- and 4.3-fold, respectively, as compared to those of the sensitive enzyme, and significant differences were found for the optimal pH and temperature, as well as the isoelectric points of the two enzymes. While PEP protected both enzymes against inactivation by N-ethylmaleimide, 3-bromopyruvate, and phenylglyoxal, glyphosate protected only the sensitive enzyme.     

Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5 flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.     

Insensitivity of 5-enolpyruvylshikimic acid-3-phosphate synthase to glyphosate confers resistance to this herbicide in a strain of Aerobacter aerogenes

The broadspectrum herbicide glyphosate (N-[phosphonomethyl]glycine), an inhibitor of the shikimate pathway enzyme 5-enolpyruvyl-shikimic acid-3-phosphate (EPSP)-synthase, inhibits the growth of Aerobacter aerogenes and causes the excretion of shikimic acid-3-phosphate. A strain of A. aerogenes, resistant to inhibition of growth by glyphosate, was isolated and found to have a glyphosate-insensitive EPSP-synthase and to no longer excrete shikimic acid-3-phosphate in the presence of glyphosate. Partial identity of EPSP-synthases from the glyphosate-sensitive and-resistant A. aerogenes strains was demonstrated by immunological procedures.Abbreviation EPSP-synthase 5-enolpyruvylshikimic acid-3-phosphate synthase (EC 2.5.1.19; 3-phosphoshikimate 1-carboxyvinyltransferase)     

Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells

Summary CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - EPSPS 5-enolpyruvylshikimate-3-phosphate synthase - I₅₀ 50% inhibitory concentration - Kb Kilobase (pairs) - PEP phosphoenolpyruvate - PMSF phenylmethylsulfonyl fluoride - PVPP polyvinylpolypyrrolidone - S-3-P shikimate-3-phosphate     

5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate

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.     

A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate

A novel class II 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) was identified from Pseudomonas stutzeri A1501 by complementation of an Escherichia coli auxotrophic aroA mutant. The single amino acid substitution of serine (Ser) for asparagine (Asn)-130 of the A1501 EPSPS enhanced resistance to 200 mM glyphosate. The mutated EPSPS had a 2.5-fold increase for IC(50) [glyphosate] value, a 2-fold increase for K (i) [glyphosate] value, but a K (m) [PEP] value similar to that of wild type. The effect of the single residue mutation on glyphosate resistance was also analyzed using a computer-based three-dimensional model.     

Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells

Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.     

Ultrastructural localisation by protein A-gold immunocytochemistry of 5-enolpyruvylshikimic acid 3-phosphate synthase in a plant cell culture which overproduces the enzyme

Recently we have shown that cultured cells of the higher plant Corydalis sempervirens Pers., adapted to growth in the presence of high concentrations of the herbicide glyphosate, a potent specific inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase (EC 2.5.1.19, 3-phosphoshikimate 1-carboxyvinyltransferase) oversynthesize the EPSP synthase protein (Smart et al., 1985, J. Biol. Chem. 260, 16338–16346). We now report that the EPSP synthase protein can be detected in cells of the adapted as well as of the non-adapted strain by the use of protein A-colloidal gold immunocytochemistry. The overproduced EPSP synthase in the glyphosate-adapted cells is located exclusively in the plastid and we find no evidence for the existence of extra-plastidic EPSP synthase in either strain.Abbreviations EPSP 5-enolpyruvylshikimic acid 3-phosphate     

Purification and properties of 5-enolpyruvylshikimate 3-phosphate synthase from seedlings of Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) from shoot tissue of pea seedlings was purified to apparent homogeneity by sequential ammonium-sulphate precipitation, ion-exchange and hydrophobic-interaction chromatography and substrate elution from cellulose phosphate. Gel electrophoresis and gel-permeation chromatography showed that the purified enzyme was monomeric with molecular weight 50,000. The herbicide glyphosate was a potent inhibitor of the forward enzyme-catalyzed reaction.Abbreviations DEAE diethylaminoethyl - EPSP 5-enolpyruvylshikimate 3-phosphate     

The purification of 5-enolpyruvylshikimate 3-phosphate synthase from an overproducing strain of Escherichia coli

The Escherichia coli aroA gene which codes for the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase) has been cloned from the lambda-transducing bacteriophage lambda pserC. The gene has been located on a 4.7 kilobase pair PstI DNA fragment which has been inserted into the multiple copy plasmid pAT153. E. coli cells transformed with this recombinant plasmid overproduce EPSP synthase 100-fold. A simple method for the purification of homogeneous enzyme in milligram quantities has been devised. The resulting enzyme is indistinguishable from enzyme isolated from untransformed E. coli.     

Purification and properties of a glyphosate-tolerant 5-enolpyruvylshikimate 3-phosphate synthase from the cyanobacterium Anabaena variabilis

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.9) from the glyphosate-tolerant cyanobacterium Anabaena variabilis (ATCC 29413) was purified to homogeneity. The enzyme had a similar relative molecular mass to other EPSP synthases and showed similar kinetic properties except for a greatly elevated K _i for the herbicide glyphosate (approximately ten times higher than that of enzymes from other sources). With whole cells, the monoisopropylamine salt of glyphosate was more toxic than the free acid but the effects of the free acid and monoisopropylamine salt on purified EPSP synthase were identical.Abbreviations EPSP 5-enolpyruvylshikimate 3-phosphate - M_r relative molecular mass - PEP phosphoenolpyruvate - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - S3P shikimate 3-phosphate The funding of this work by the Agricultural and Food Research Council and the University of Dundee Research Initiatives Programme is gratefully acknowledged.     

Differential sensitivity of bacterial 5-enolpyruvylshikimate-3-phosphate synthases to the herbicide glyphosate

Abstract The potent inhibition of the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase by the broad-spectrum herbicide glyphosate ( N -[phosphonomethyl]glycine) was confirmed for the enzymes extracted from various bacteria, a green alga and higher plants. However, 5 out of 6 species belonging to the genus Pseudomonas were found to have EPSP synthases with a 50- to 100-fold decreased sensitivity to the inhibitor. Correspondingly, growth of these 5 species was not inhibited by 5 mM glyphosate, and the organisms did not excrete shikimate-3-phosphate in the presence of the herbicide.     

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0 ×10⁴ clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indicajaponica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.     

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.     

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase-S3P-Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native (31)P in S3P and Glp, biosynthetically (13)C-labeled S3P, specifically (13)C and (15)N labeled Glp, and a variety of protein-(15)N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.     

Length of time in tissue culture can affect the selected glyphosate resistance mechanism

Usually, stepwise selection of plant suspension cultures with gradually increasing concentrations of the herbicide glyphosate results in the amplification of the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) gene that leads to resistance by increasing EPSPS mRNA and enzyme activity. We show that glyphosate selection with newly initiated suspension cultures can produce resistant lines with resistance mechanisms other than gene amplification and that usually as the cultures age gene amplification becomes the predominant mechanism. Gene amplification did not occur in 3 lines selected from 5-month-old Datura innoxia Mill. cultures but did occur in all 10 lines selected after 52 months. Selection with Nicotiana tabacum L. (tobacco) less than 5 months old produced 2 lines out of 24 with no EPSPS amplification while all 17 lines selected from older cultures contained amplified genes. Lines selected from the oldest culture (35 years) also exhibited amplification of several different genes, indicating the expression of different EPSPS genes or an enhanced gene amplification incidence. None of the 15 lines selected from 2 different 5-month-old Daucus carota L. (carrot) lines exhibited amplification while amplification led to the resistance of all 7 lines selected from one of the original carrot lines (DHL) after 3 years. However, the other line (Car4) was exceptional and produced only non-amplified lines (9 of 9) after 8 years in culture. These results show that plant tissue cultures change with time in culture and that several different new mechanisms can result in glyphosate resistance.Abbreviations AHAS acetohydroxyacid synthase - EPSPS 5-enolpyruvylshikimate-3-phosphate synthase     

Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of <Emphasis Type="Italic">E. coli</Emphasis> (k12) and transformation of rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) in order to make tolerance to glyphosate

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This is a key enzyme in the aromatic amino acid biosynthesis pathway of microorganisms and plants. The manipulation of bacterial EPSPS gene in order to reduce its affinity for glyphosate, followed by its transfer to plants is one of the most effective approaches for the production of glyphosate-tolerant plants. In this study, we chose to focus on amino acid residues glycine96 and alanine183 of the E. coli (k12) EPSPS enzyme. These two amino acids are important residues for glyphosate binding. We used site directed mutagenesis (SDM) to induce point mutations in the E. coli EPSPS gene, in order to convert glycine96 to alanine (Gly96Ala) and alanine183 to threonine (Ala183Thr). After confirming the mutation by sequencing, the altered EPSPS gene was transferred to rapeseed (Brassica napus L.) via Agrobacterium-mediated transformation. The transformed explants were screened in shoot induction medium containing 25 mg L⁻¹ kanamycin. Glyphosate tolerance was assayed in putative transgenic plants. Statistical analysis of data showed that there was a significant difference between the transgenic and control plants. It was observed that transgenic plants were resistant to glyphosate at a concentration of 10 mM whereas the non-transformed control plants were unable to survive 1 mM glyphosate. The presence and copy numbers of the transgene were confirmed with PCR and Southern blotting analysis, respectively.     

Involvement of Glyceraldehyde-3-phosphate Dehydrogenase in the X-Ray Resistance of HeLa Cells

We investigated changes in the sub-cellular distribution of glycelaldehyde-3-phosphate dehydrogenase (GAPDH) after X-ray irradiation in HeLa cells. Twenty-four h after irradiation at 5 Gy, nuclear GAPDH levels increased 2.6-fold, whereas total GAPDH levels increased only 1.2-fold. Knockdown of GAPDH using specific small interfering RNA (siRNA) led to sensitization to X-ray-induced cell death. These results suggest that GAPDH plays a role in the radioresponse.