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Genome-wide identification and comparative analysis of EPSPS (aroA) genes in different plant species


Shikimate pathway produces aromatic amino acids such as phenylalanine, tyrosine and tryptophan, which is essential for plant life. EPSPS (5-enolpyruvylshikimate-3-phosphate synthase, EC (aroA) that observed in plants and bacteria plays an important role in shikimate pathway as being the sixth crucial enzyme. In this study, genome-wide comparative analyses of EPSPS genes were performed in different plant species, including Glycine max, Medicago truncatula, Brachypodium disctahyon, Zea mays, Chlamydomonas reinhardtii (green alga), and Physcomitrella patens (moss). One EPSPS gene was identified in M. truncatula, B. distachyon, Z. mays, and C. reinhardtii while two EPSPS genes identified in G. max and P. patens. Based on nucleotide diversity analyses of EPSPS genes, nucleotide diversity was found to be π: 0.29 and θ: 0.34, respectively. Gene structure analysis revealed that 6 of 8 EPSPS genes (75 %) contained seven introns while the length of open reading frame (ORF) ranged from 1173 to 1611 bp. Two highly conserved motifs (LPGSKSLSNRILLLAAL and LFLGNAGTAMRPL) were detected in all plant species. All putative EPSPSs were predicted to be localized in chloroplast while 7 of 8 EPSPSs (87.5 %) contained N-glycosylation sites except for Chlamydomonas. Phylogenetic analyses showed that monocots, dicots, and moss were clustered in group A whereas green alga (C. reinhardtii) was located alone in group B. 3D structure analyses indicated that EPSP synthases contained N-terminal and C-terminal domains while electrostatic potential of EPSPSs were found to exhibit various patterns. In addition, ligand molecule glyphosate was docked with EPSPS of B. disctahyon and G. max. The result showed that there were strong favorable bonds between the proteins and the ligand with favorable conformation.

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5-enolpyruvylshikimate-3-phosphate synthase


3-deoxy-d-arabino-heptulosonate-7-phosphate synthase


3-dehydroquinate synthase


Shikimate kinase


Chorismate synthase


  1. Baerson SR, Rodriguez DJ, Tran M, Feng Y, Biest NA et al (2002) Glyphosate resistant goose grass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Plant Physiol 129:1265–1275

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  2. Bailey TL, Williams N, Misleh C, Li WW (2006) MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res 34:369–373

    Article  Google Scholar 

  3. Barry G, Padgette SR (1992) Glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthases. World Patent WO 92/04449

  4. Baylis AD (2000) Why glyphosate is a global herbicide: strengths, weaknesses and prospects. Pest Manag Sci 56:299–308

    CAS  Article  Google Scholar 

  5. Bjorklund AK, Ekman D, Light S, FreySkott J, Elofsson A (2005) Domain rearrangements in protein evolution. J Mol Biol 353:911–923

    PubMed  Article  Google Scholar 

  6. Cao G, Liu Y, Zhang S, Yang X, Chen R et al (2012) A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants. PLoS ONE 7(6):e38718. doi:10.1371/journal.pone.0038718

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  7. Chen Y, Zhang X, Wu W, Chen Z, Gu H, Qu LJ (2006) Overexpression of the wounding-responsive gene AtMYB15 activates the shikimate pathway in Arabidopsis. J Integr Plant Biol 48:1084–1095

    CAS  Article  Google Scholar 

  8. Comai L, Facciotti D, Hiatt WR, Thompson G, Rose RE et al (2012) Expression in plants of a mutant aroA gene from Salmonella typhimurium confers tolerance to glyphosate. Nature 317:741–744

    Article  Google Scholar 

  9. della-Cioppa G, Bauer SC, Klein BK, Shah DM, Fraley RT, Kishore G (1986) Translocation of the precursor of 5-enolpyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants in vitro. Proc Nati Acad Sci USA 83:6873–6877

  10. DePristo MA, Weinreich DM, Hartl DL (2005) Missense meanderings in sequence space: a biophysical view of protein evolution. Nat Rev Genet 6:678–687

    PubMed  CAS  Article  Google Scholar 

  11. Funke T, Han H, Healy-Fried ML, Fischer M, Schonbrunn E (2006) Molecular basis for the herbicide resistance of Roundup Ready crops. Proc Natl Acad Sci U S A 103:13010–13015

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  12. Garg B, Vaid N, Tuteja N (2014) In-silico analysis and expression profiling implicate diverse role of EPSPSfamily genes in regulating developmental and metabolic processes. BMC Res Notes 7:58

    PubMed  PubMed Central  Article  Google Scholar 

  13. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, Totowa, pp 571–607

    Chapter  Google Scholar 

  14. Geourjon C, Deleage G (1995) SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11:681–684

    PubMed  CAS  Google Scholar 

  15. Gong Y, Liao Z, Chen M, Guo B, Jin H, Sun X, Tang K (2006) Characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from Camptotheca acuminate. Biol Plant 50:542–550

    CAS  Article  Google Scholar 

  16. Guex N, Peitsch MC, Schwede T (2009) Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis 30(Suppl 1):S162–S173

    PubMed  Article  Google Scholar 

  17. Guo AY, Zhu QH, Chen X, Luo JC (2007) GSDS: a gene structure display server. Yi Chuan 29:1023–1026

    PubMed  CAS  Article  Google Scholar 

  18. Hu LF, Liu SQ (2011) Genome-wide identification and phylogenetic analysis of the ERF gene family in cucumbers. Genet Mol Biol 34:624–633

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  19. Kaur H, Heinzel N, Schottner M, Baldwin IT, Galis I (2010) R2R3-NaMYB8 regulates the accumulation of phenylpropanoid-polyamine conjugates, which are essential for local and systemic defense against insect herbivores in Nicotiana attenuata. Plant Physiol 152:1731–1747

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  20. Kelley LA, Sternberg MJE (2009) Protein structure prediction on the web: a case study using the Phyre server. Nat Protoc 4:363–371

    PubMed  CAS  Article  Google Scholar 

  21. Kukić P, Nielsen JE (2010) Electrostatics in proteins and protein–ligand complexes. Future Med Chem 2:647–666

    PubMed  Article  Google Scholar 

  22. Lesk AM, Chothia C (1980) How different amino acid sequences determine similar protein structures: the structure and evolutionary dynamics of the globins. J Mol Biol 136:225–270

    PubMed  CAS  Article  Google Scholar 

  23. Liang A, Sha J, Lu W, Chen M, Li L et al (2008) A single residue mutation of 5-enolpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate. Biotechnol Lett 30:1397–1401

    PubMed  CAS  Article  Google Scholar 

  24. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452

    PubMed  CAS  Article  Google Scholar 

  25. Lovell SC, Davis IW, Bryan AW, Bakker, PIW, Word JM, Prisant MG, Richardson JS,  Richardson DC (2003). Structure validation by Cα geometry: ϕ,ψ and Cβ deviation.  Proteins: Structure, Function and Genetics 50: 437–450

  26. Maeda H, Dudareva N (2012) The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu Rev Plant Biol 63:73–105

    PubMed  CAS  Article  Google Scholar 

  27. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  28. Nei M (1987) Molecular evolutionary genetics. Columbia University, New York

    Google Scholar 

  29. Rogozin IB, Sverdlov AV, Babenko VN, Koonin EV (2005) Analysis of evolution of exon–intron structure of eukaryotic genes. Brief Bioinform 6:118–134

    PubMed  CAS  Article  Google Scholar 

  30. Rost B (1997) Protein structures sustain evolutionary drift. Fold Des 2:S19–S24

    PubMed  CAS  Article  Google Scholar 

  31. Schönbrunn E, Eschenburg S, Shuttleworth WA, Schloss JV, Amrhein N, Evans JNS, Kabsch W (2001) Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail. PNAS 98:1376–1380

    PubMed  PubMed Central  Article  Google Scholar 

  32. Schwarz F, Aebi M (2011) Mechanisms and principles of N-linked protein glycosylation. Curr Opin Struct Biol 21:576–582

    PubMed  CAS  Article  Google Scholar 

  33. Shuttleworth WA, Pohl ME, Helms GL, Jakeman DL, Evans JNS (1999) Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase. Biochemistry 38:296–302

    PubMed  CAS  Article  Google Scholar 

  34. Sonnhammer EL, Eddy SR, Durbin R (1997) Pfam: a comprehensive database of protein domain families based on seed alignments. Proteins 28:405–420

    PubMed  CAS  Article  Google Scholar 

  35. Spratt BG, Greenwood BM (2000) Prevention of pneumococcal disease by vaccination: does serotype replacement matter? Lancet 356:1210–1211

    PubMed  CAS  Article  Google Scholar 

  36. Stallings WC, Abdel-Meguid SS, Lim LW, Shie HS, Dayringer HE, Leimgruber NK, Stegemann RA, Anderson KS, Sikorski JA, Padgette SR, Kishore GM (1991) Proc Natl Acad Sci U S A 88:5046–5050

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  37. Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585–595

    PubMed  CAS  PubMed Central  Google Scholar 

  38. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  39. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  40. Tzin V, Galili G (2010) New insights into the shikimate and aromatic amino acids biosynthesis pathways in plants. Mol Plant 3:956–972

    PubMed  CAS  Article  Google Scholar 

  41. Voet A, Berenger F, Zhang KYJ (2013) Electrostatic similarities between protein and small molecule ligands facilitate the design of protein-protein interaction inhibitors. PLoS ONE 8(10):e75762. doi:10.1371/journal.pone.0075762

    PubMed  CAS  PubMed Central  Article  Google Scholar 

  42. Wang Y, Deng D, Bian Y, Lv Y, Xie Q (2010) Genome-wide analysis of primary auxin-responsive Aux/IAA gene family in maize (Zea mays. L.). Mol Biol Rep 37:3991–4001

    PubMed  CAS  Article  Google Scholar 

  43. Watterson GA (1975) On the number of segregating sites in genetical models without recombination. Theor Popul Biol 7:188–193

    Article  Google Scholar 

  44. Weaver LM, Herrmann KM (1997) Dynamics of the shikimate pathway in plants. Trends Plant Sci 2:346–351

    Article  Google Scholar 

  45. Ye GN, Hajdukiewicz TJ, Broyles D, Rodriguez D, Xu CW, Nehra N, Staub JM (2001) Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 25:261–270

    PubMed  CAS  Article  Google Scholar 

  46. Yu CS, Chen YC, Lu CH, Hwang JK (2006) Prediction of protein subcellular localization. Proteins 64:643–651

    PubMed  CAS  Article  Google Scholar 

  47. Zhou M, Xu H, Wei X, Ye Z, Wei L et al (2006) Identification of a glyphosate resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy. Plant Physiol 140:184–195

    PubMed  CAS  PubMed Central  Article  Google Scholar 

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Correspondence to Ertugrul Filiz.

Additional information

Ertugrul Filiz holds a PhD in Duzce University.

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Filiz, E., Koc, I. Genome-wide identification and comparative analysis of EPSPS (aroA) genes in different plant species. J. Plant Biochem. Biotechnol. 25, 21–29 (2016).

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  • Shikimate pathway
  • Aromatic amino acids
  • In silico analysis
  • 3D structure
  • Nucleotide diversity
  • Molecular docking