ABSTRACT
Applying the genomic library construction process and colony screening, a novel aroA gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.
The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) is the sixth enzyme in the shikimate pathway, which is essential for the synthesis of aromatic amino acids and many aromatic metabolites in plants, fungi, and microorganisms (2, 11, 16), including apicomplexan parasites (22). It converts shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. Interest in the characterization of EPSPS has increased significantly since the enzyme was identified as the primary target of the broad-spectrum, nonselective herbicide glyphosate [N-(phosphonomethyl)glycine] (25). Glyphosate is a competitive inhibitor with respect to PEP and binds adjacent to S3P in the active site of EPSPS, thereby mimicking an intermediate state of the ternary enzyme-substrate complex (23).
Two classes of EPSPS, class I and II enzymes, sharing less than 30% amino acid similarity have been reported (9). Class I includes those found in plants and bacteria such as Escherichia coli and Salmonellaenterica serovar Typhimurium, whose catalytic activity is inhibited at low micromolar concentrations of glyphosate (8). Class II EPSPS, found in Pseudomonas sp. strain PG2982, Agrobacterium tumefaciens strain CP4, Streptococcus pneumoniae, and Staphylococcus aureus, was distinguished by its ability to sustain efficient catalysis in the presence of high glyphosate concentrations (6, 9).
Although a large number of AroA enzymes (EPSPS) have been cloned, identified, and tested as glyphosate resistant, only AroA variants derived from the A. tumefaciens strain CP4 have been successfully used commercially (9). To find a new enzyme similar to that of the AroAA.tumefaciensCP4, in this study a highly glyphosate-tolerant strain from the rhizosphere of rice in a field where glyphosate is frequently used has been selected and identified on M9 minimal medium containing 200 mM glyphosate, and its 16S rRNA gene sequence confirmed that this strain was strongly related to Ochrobactrum anthropi (99.9%). Additionally, the aroAO. anthropi gene was isolated and kinetic characteristics of the Ochrobactrum anthropi strain EPSP synthase were determined in this study.
Isolation of gene aroAO. anthropi.
To isolate the gene aroAO. anthropi, we used the genomic library construction process and colony screening (26). The protocol of genomic library construction and colony screening was performed as described by Sun et al. (26) with modifications as follows. DNA fragments of 2 to 4 kb from Ochrobactrum anthropi were purified and inserted into pUC18 digested by BamHI. The resulting ligation mixture was transformed into ElectroMAX DH10B T1 phage-resistant E. coli by electroporation, and more than 106 clones grown on LB agar plates were extracted using a plasmid maxikit. Then the genomic library plasmid was transformed into mutant E. coli strain ER2799 (with the EPSP synthase gene deleted from its genome), and one clone containing the gene aroAO. anthropi was identified by the ability to restore growth of the mutant ER2799 cells on M9 minimal medium containing 60 mM glyphosate.
Sequence analysis.
The clone contained an insert of about 2.0 kb. This fragment was sequenced and analyzed subsequently. The fragment had a complete open reading frame of 1,353 bp with a G+C content of 61.9% and encoded a protein of 451 amino acid residues, which was named AroAO. anthropi. It started with an AUG start codon, and the deduced molecular mass was 47.59 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (see Fig. S1 in the supplemental material). Sequence analysis indicated that AroAO. anthropi shared more than 82.89% amino acid identity with AroAA. tumefaciensCP4 but less than 30% amino acid identity with AroAE. coli. Phylogenetic tree analysis indicated that AroAO. anthropi was a class II EPSPS (Fig. 1).
(A) Amino acid sequence alignment of AroAO. anthropi, AroAA. tumefaciensCP4, and AroAE. coli. Asterisks and circles indicate residues important for S3P binding and PEP binding, respectively, in AroAE.coli. Domains important for glyphosate tolerance and maintenance of productive PEP binding in class II AroA are boxed. Underlining indicates the regions containing many amino acid differences between AroAO. anthropi and AroAA. tumefaciens. (B) Phylogenetic tree analysis of AroAO. anthropi using MEGA. Class I AroA proteins shown are from E. coli (Swiss-Prot accession no. P07638), Aeromonas salmonicida (Swiss-Prot Q03321), Arabidopsis thaliana (Swiss-Prot P05466), Nicotiana tabacum (Swiss-Prot P23981), Petunia hybrida (Swiss-Prot P11043), Zea mays (GenBank accession no. CAA44974), and Bordetella pertussis (Swiss-Prot P12421); class II AroA proteins shown are from Pseudomonas sp. strain PG2982 (Swiss-Prot P56952), A. tumefaciens CP4 (Swiss-Prot Q9R4E4), Bacillus subtilis (Swiss-Prot P20691), Staphylococcus aureus (Swiss-Prot Q05615), Dichelobacter nodosus (Swiss-Prot Q46550), and Streptococcus pneumoniae (Swiss-Prot Q9S400).
In vitro glyphosate sensitivity assays.
In vitro glyphosate sensitivity assays of the mutant ER2799 harboring plasmid p251-aroAO. anthropi or p251-aroAE. coli were utilized to determine whether AroAO. anthropi is able to confer glyphosate resistance in intact cells (12). The plasmid p251-aroAO. anthropi was generated by inserting EPSPS gene aroAO. anthropi, which was amplified by PCR with primers P1Z and P1F (see primers in the supplemental material) into the BamHI-to-SacI sites in pYPX251 (GenBank accession no. AY178046). Similarly, the plasmid p251-aroAE. coli was generated using primers P2Z and P2F (see primers in the supplemental material). As shown in Fig. 2, the results show that the growth of cells harboring p251-aroAE. coli was strongly inhibited at 50 mM glyphosate. In contrast, cells harboring p251-aroAO. anthropi can grow at 150 mM glyphosate, and growth was strongly inhibited at 200 mM glyphosate. It is obvious that AroAO. anthropi is more tolerant of glyphosate than AroAE. coli.
Growth of ER2799 harboring p251-aroAO. anthropi and p251-aroAE. coli in liquid M9 minimal medium supplemented with glyphosate at concentrations of 0 (A), 50 (B), 100 (C), 150 (D), and 200 (E) mM.
Kinetic characteristics of AroAO. anthropi.
To further evaluate the kinetic characteristics of AroAO. anthropi, the protein was purified using a HisTrap HP kit by the method described previously (26). The protein AroAE.coli was also used for comparison. The kinetic characteristics of proteins were explored using the malachite green dye assay method as previously reported (5, 12, 17). Notably, the Ki was demonstrated by Dixon plot in this study. In the Dixon plot, the competition inhibition shows the linear intersection of different substrate concentrations that are correspondingly projected on the x axis at −Ki. The kinetic parameters of AroAO. anthropi and AroAE. coli are shown in Table 1 and in Fig. S2, S3, S4, and S5 in the supplemental material. Although the results of enzyme assays in different studies vary based on experimental conditions, it is useful to compare the kinetic parameters of different AroA enzymes. As shown in Table 1, AroAO. anthropi showed about 48-fold higher Ki and 10-fold greater 50% inhibitory concentration (IC50) than AroAE. coli. However, compared with AroAA. tumefaciensCP4 (9), it does so poorly.
Kinetic properties of AroAE. coli, AroAO. anthropi, and the A103C mutant of AroAO. anthropi
As a class II EPSPS, AroAO. anthropi has kinetic characteristics significantly different from those of AroAA. tumefaciensCP4. Therefore, we analyzed the possible reasons resulting in the kinetics difference between them. Based on what is known about the crystal structure of EPSPS, many active sites of EPSPS have been largely studied and identified (3, 14, 18, 19). Mutation of these amino acids can result in a significant change in glyphosate tolerance. In addition, glyphosate tolerance can also be induced by other specific mutations, including Thr42Met (13), Gly96Ala (20), Thr97Ile (24), Ala183Thr (15), and Pro101Leu, Pro106Leu, Pro101Thr, Pro101Ala, and Pro101Ser (1, 7, 27). As for the proteins AroAO. anthropi and AroAA. tumefaciensCP4, the important residues for S3P binding and PEP binding and the domains for glyphosate tolerance were found to be conservative (Fig. 1). So it is that some mutant residues exert an indirect effect on glyphosate/PEP binding and lead to the kinetics difference between AroAO. anthropi and AroAA. tumefaciensCP4.
Site-directed mutagenesis.
In order to prove that specific amino acid residues may affect the glyphosate affinities of AroAO. anthropi, three residues of AroAO. anthropi were replaced by corresponding residues of AroAA. tumefaciensCP4 by site-directed mutagenesis (21) (see primers in the supplemental material), including residue 103. Residue 103 in AroAA. tumefaciensCP4 is Cys, which is part of a hydrophobic pocket, and mutation in this position might impact the integrity of this pocket, leading to distinct kinetic properties. As predicted, the result shows that the A103C mutation increased the Ki of glyphosate and the IC50 significantly (Table 1). Also, the Km of PEP and the specific activity increased trivially, whereas those of the E259P and S336A mutant did not. This implied that glyphosate tolerance of AroAO. anthropi was related to residue 103.
3D structure of the A103C mutant.
Moreover, to better understand the effect of mutation on glyphosate affinity, the three-dimensional (3D) structure of the A103C mutant was derived using SWISS-MODEL (10). The structure shows that Ala-103 is buried in the third helix of the core of the N-terminal domain (Fig. 3). This helix is a universal mutation hot spot for glyphosate resistance, and changes in this helix may cause alterations in the active site. Furthermore, the Gly-100 residue constitutes part of the binding pocket for the phosphonate moiety of glyphosate (7) and Arg-104 located nearby. Therefore, as is the case for the G96A (7), P101S (1), and P106L (27) mutant enzymes, the A103C mutation affects the conformation of this helix and renders distinct kinetic properties.
Structural models of AroAA. tumefaciensCP4 and AroAO. anthropi. The labeled residues are shown as colored balls. (Left) Positions of amino acid residue A103 and strictly conserved residues R104 and G100 at the N-terminal domain in AroAO. anthropi. (Right) Positions of amino acid residue C103 and strictly conserved residues R104 and G100 at the N-terminal domain in AroAA. tumefaciensCP4.
Conclusion.
Despite the fact that many promising enzymes that appear to be advantageous for glyphosate-resistant crops were identified via single site-directed mutagenesis, such as the E. coli Gly96Ala EPSPS mutant, they failed to show sufficient glyphosate resistance for commercial utilization due to a decreased affinity for PEP (26). Remarkably, the multisite mutations with more favorable properties could be introduced into crops to express herbicide resistance. For instance, the Thr97Ala/Pro101Ser double mutant of the class I EPSP synthase from E. coli produces a catalytically efficient, glyphosate-resistant enzyme, and the Thr102Ile/Pro106Ser double mutant of Zea mays EPSPS has been utilized to produce the first commercial varieties of glyphosate-resistant maize (U.S. patent 6,040,497 [24]). Therefore, we believe that there exists a pressing need to identify multisite mutations with more favorable properties for engineering glyphosate resistance in crops. In this study, though the A103C mutation exerts an indirect effect on glyphosate binding and results in increased Ki (glyphosate) and IC50 (glyphosate) values, there is no explanation for the elevated Km (PEP). Therefore, in order to obtain multisite mutations with higher Ki (glyphosate) and normal Km (PEP), directed evolution or multiple-site mutations of the novel aroA gene from O. anthropi will need to be performed under the selective pressure caused by the presence of high glyphosate concentrations (4, 21). Only selective multisite mutations may be suitable for the generation of glyphosate-resistant crops.
Nucleotide sequence accession number.
The GenBank accession number for the sequences determined in this study is GU992200.
ACKNOWLEDGMENTS
We thank Thomas C. Evans, Jr. (New England Biolabs), for providing E. coli ER2799.
The research was supported by the Key Project Fund of the Shanghai Municipal Committee of Agriculture (grant no. 2008-7-5), the Shanghai Basic Research Project (grant no. 08JC1418000), and the 863 Programs (grant no. 2006AA10Z117, 2006AA06Z358, and 2008AA10Z401).
FOOTNOTES
- Received 29 March 2010.
- Accepted 19 June 2010.
- Copyright © 2010 American Society for Microbiology
