Journal of Microbiology

  • 제45권2호
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  • Pages.153-157
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  • 2007
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  • 1225-8873(pISSN)
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  • 1976-3794(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
권호 표지

Cloning, Expression, and Functional Characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate Synthase Gene in Escherichia coli

  • Yi, Yi (Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University) ;
  • Qiao, Dairong (Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University) ;
  • Bai, Linhan (Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University) ;
  • Xu, Hui (Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University) ;
  • Li, Ya (Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University) ;
  • Wang, Xiaolin (Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University) ;
  • Cao, Yi (Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University)
  • 발행 : 2007.04.30
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초록

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, 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 secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.

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참고문헌

  1. Alibhai, M.F. and W.C. Stallings. 2001. Closing down on glyphosate inhibition-with a new structure for drug discovery. Proc. Natl. Acad. Sci. USA 98, 2944-2946
  2. Chen, L., S. Pradhan, and T.C. Evans, Jr. 2001. Herbicide resistance from a divided EPSPS protein: the split Synechocystis DnaE intein as an in vivo affinity domain. Gene 263, 39-48 https://doi.org/10.1016/S0378-1119(00)00568-0
  3. Du, W., N.G. Wallis, M.J. Mazzulla, A.F. Chalker, L. Zhang, W.S. Liu, H. Kallender, and D.J. Payne. 2000. Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations. Eur. J. Biochem. 267, 222-227 https://doi.org/10.1046/j.1432-1327.2000.00994.x
  4. Emanuelsson, O., H. Nielsen, and G. von Heijne. 1999. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci. 8, 978-984 https://doi.org/10.1110/ps.8.5.978
  5. Forlani, G., B. Parisi, and E. Nielsen. 1994. 5-enol-Pyruvyl-Shikimate- 3-Phosphate Synthase from Zea mays Cultured Cells (Purification and Properties). Plant Physiol. 105, 1107-1114 https://doi.org/10.1104/pp.105.4.1107
  6. Gasser, C.S., J.A. Winter, C.M. Hironaka, and D.M. Shah. 1988. Structure, expression, and evolution of the 5-enolpyruvylshikimate- 3-phosphate synthase genes of petunia and tomato. J. Biol. Chem. 263, 4280-4287
  7. He, M., Y.F. Nie, and P. Xu. 2003. A T42M substitution in bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) generates enzymes with increased resistance to glyphosate. Biosci. Biotechnol. Biochem. 67, 1405-1409 https://doi.org/10.1271/bbb.67.1405
  8. Huynh, Q.K. 1990. Mechanism of inactivation of Escherichia coli 5- enolpyruvoylshikimate-3-phosphate synthase by o-phthalaldehyde. J. Biol. Chem. 265, 6700-6704
  9. Huynh, Q.K., S.C. Bauer, G.S. Bild, G.M. Kishore, and J.R. Borgmeyer. 1988. Site-directed mutagenesis of Petunia hybrida 5-enolpyruvylshikimate-3-phosphate synthase: Lys-23 is essential for substrate binding. J. Biol. Chem. 263, 11636-11639
  10. McDowell, L.M., B. Poliks, D.R. Studelska, R.D. O'Connor, D.D. Beusen, and J. Schaefer. 2004. Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase. J. Biomol. NMR. 28, 11-29 https://doi.org/10.1023/B:JNMR.0000012864.70184.48
  11. Oliveira, J.S., C.A. Pinto, L.A. Basso, and D.S. Santos. 2001. Cloning and overexpression in soluble form of functional shikimate kinase and 5-enolpyruvylshikimate 3-phosphate synthase enzymes from Mycobacterium tuberculosis. Protein Expr. Purif. 22, 430-435 https://doi.org/10.1006/prep.2001.1457
  12. Padgette, S.R., D.B. Re, C.S. Gasser, D.A. Eichholtz, R.B. Frazier, C.M. Hironaka, E.B. Levine, D.M. Shah, R.T. Fraley, and G.M. Kishore. 1991. Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site. J. Biol. Chem. 266, 22364-22369
  13. Pick, U., L. Karni, and M. Avron. 1986. Determination of ion content and ion fluxes in the halotolerant alga Dunaliella salina. Plant Physiol. 81, 92-96 https://doi.org/10.1104/pp.81.1.92
  14. Priestman, M.A., T. Funke, I.M. Singh, S.S. Crupper, and E. Schonbrunn. 2005. 5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate. FEBS Lett. 579, 728-732 https://doi.org/10.1016/j.febslet.2004.12.057
  15. Schonbrunn, E., S. Eschenburg, W.A. Shuttleworth, J.V. Schloss, N. Amrhein, J.N. Evans, and W. Kabsch. 2001. Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail Proc. Natl.Acad. Sci. USA 98, 1376-1380
  16. Shuttleworth, W.A., M.E. Pohl, G.L. Helms, D.L. Jakeman, and J.N. Evans. 1999. Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase. Biochemistry 38, 296-302 https://doi.org/10.1021/bi9815142
  17. Stalker, D.M., W.R. Hiatt, and L. Comai. 1985. A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate. J. Biol. Chem. 260, 4724-4728
  18. Stallings, W.C., S.S. Abdel-Meguid, L.W. Lim, H.S. Shieh, H.E. Dayringer, N.K. Leimgruber, R.A. Stegeman, K.S. Anderson, J.A. Sikorski, S.R. Padgette, and G.M. Kishore. 1991. Structure and topological symmetry of the glyphosate target 5-enolpyruvylshikimate- 3-phosphate synthase: a distinctive protein fold. Proc. Natl. Acad. Sci. USA 88, 5046-5050=14&py
  19. Stauffer, M.E., J.K. Young, G.L. Helms, and J.N. Evans. 2001. Sequential assignments of the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase. FEBS Lett. 499, 182-186 https://doi.org/10.1016/S0014-5793(01)02555-8
  20. Sun, Y.C., Y. Li, H. Zhang, H.Q. Yan, D.N. Dowling, and Y.P. Wang. 2006. Reconstitution of the enzyme AroA and its glyphosate tolerance by fragment complementation. FEBS Lett. 580, 1521-1527 https://doi.org/10.1016/j.febslet.2006.01.075