Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Engineering and Characterization of the Isolated C-Terminal Domain of 5-Enolpyruvylshikimate-3-phosphate (EPSP) Synthase

  • Kim, Hak-Jun (Department of Applied Polar Science, Korea Polar Research Institute) ;
  • Kim, Hyun-Woo (Department of Marine Biology, Pukyong National University) ;
  • Kang, Sung-Ho (Department of Applied Polar Science, Korea Polar Research Institute)
  • Published : 2007.08.30
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Abstract

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP and inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in the biosynthesis of aromatic amino acids. To delineate the domain-specific function, we successfully isolated the discontinuous C-terminal domain (residues 1-21, linkers, 240-427) of EPSP synthase (427 residues) by site-directed mutagenesis. The engineered C-terminal domains containing no linker (CTD), or with gly-gly ($CTD^{GG}$) and gly-ser-ser-gly ($CTD^{GSSG}$) linkers were purified and characterized as having distinct native-like secondary and tertiary structures. However, isothermal titration calorimetry (ITC), $^{15}N-HSQC$,\;and\;^{31}P-NMR$ revealed that neither its substrate nor inhibitor binds the isolated domain. The isolated domain maintained structural integrity, but did not function as the half of the full-length protein.

Keywords

References

  1. Anderson, K. S., J. A. Sikorski, and K. A. Johnson. 1988. A tetrahedral intermediate in the EPSP synthase reaction observed by rapid quench kinetics. Biochemistry 27: 7395-7406 https://doi.org/10.1021/bi00419a034
  2. Du, W., N. G. Wallis, and D. J. Payne. 2000. The kinetic mechanism of 5-enolpyruvylshikimate-3-phosphate synthase from a Gram-positive pathogen Streptococcus pneumoniae. J. Enzyme Inhib. 15: 571-581 https://doi.org/10.3109/14756360009040711
  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. Eschenburg, S., M. L. Healy, M. A. Priestman, G. H. Lushington, and E. Schonbrunn. 2002. How the mutation glycine96 to alanine confers glyphosate insensitivity to 5- enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli. Planta 216: 129-135 https://doi.org/10.1007/s00425-002-0908-0
  5. Franz, J., M. K. Mao, and Sikorski, J. A. 1997. Glyphosate: A Unique Global Herbicide. Oxford Univ. Press, New York
  6. Garner, E., P. Romero, A. K. Dunker, C. Brown, and Z. Obradovic. 1999. Predicting binding regions within disordered proteins. Genome Inform. Ser. Workshop Genome Inform. 10: 41-50
  7. Gruys, K. J., M. C. Walker, and J. A. Sikorski. 1992. Substrate synergism and the steady-state kinetic reaction mechanism for EPSP synthase from Escherichia coli. Biochemistry 31: 5534-5544 https://doi.org/10.1021/bi00139a016
  8. Haslam, E. 1993. Shikimic Acid: Metabolism and Metabolites. John Wiley, Chichester
  9. Hwang, E. I., B. S. Yun, S. W. Choi, J. S. Kim, S. J. Lim, J. S. Moon, S. H. Lee, and S. U. Kim. 2005. Isolation of sangivamycin from Streptomyces sp. A6497 and its herbicidal activity. J. Microbiol. Biotechnol. 15: 434-437
  10. Kim, H. J., J. K. Young, G. L. Helms, and J. N. Evans. 2002. Letter to the editor: $^1H$, TEX>$^{13}C$, and $^{15}N$ backbone resonance assignments of the C-terminal domain of 5- enolpyruvylshikimate-3-phosphate synthase. J. Biomol. NMR 24: 269-270 https://doi.org/10.1023/A:1021604524843
  11. Kim, J.-H., H.-S. Song, D.-H. Kim, and H.-Y. Kim. 2006. Quantification of genetically modified Canola GT73 using TaqMan real-time PCR. J. Microbiol. Biotechnol. 16: 1778-1783
  12. Kim, Y.-H., S. J. Choi, H.-A. Lee, and T. W. Moon. 2006. Quantitation of CP4 5-enolpyruvylshikimate-3-phosphate synthase in soybean by two-dimensional gel electrophoresis. J. Microbiol. Biotechnol. 16: 25-31
  13. Kishore, G. M. and D. M. Shah. 1998. Amino acid biosynthesis inhibitors as herbicides. Annu. Rev. Biochem. 57: 627-663 https://doi.org/10.1146/annurev.bi.57.070188.003211
  14. Krekel, F., C. Oecking, N. Amrhein, and P. Macheroux. 1999. Substrate and inhibitor-induced conformational changes in the structurally related enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS). Biochemistry 38: 8864- 8878 https://doi.org/10.1021/bi990412o
  15. Lee, S.-H., S.-H. Kang, Y.-H. Park, D.-M. Min, and Y.-M. Kim. 2006. Quantitative analysis of two genetically modified maize lines by real-time PCR. J. Microbiol. Biotechnol. 16: 205-211
  16. Park, H. J., J. L. Hilsenbeck, H. J. Kim, W. A. Shuttleworth, Y. H. Park, J. N. S. Evans, and C. H. Kang. 2004. Structural studies of Strpetococcus pneumoniae EPSP synthase in unliganded state, tetrahedral intermediate-bound state and S3P-GLP-bound state. Mol. Microbiol. 51: 963-971 https://doi.org/10.1046/j.1365-2958.2003.03885.x
  17. Ream, J. E., H. K. Yuen, R. B. Frazier, and J. A. Sikorski. 1992. EPSP synthase binding studies using isothermal titration microcalorimetry and equilibrium dialysis and their implications for ligand recognition and kinetic mechanism. Biochemistry 31: 5528-5534 https://doi.org/10.1021/bi00139a015
  18. Roberts, F., C. W. Roberts, R. E. Lyons, M. J. Kiristis, E. J. Mui, J. Finnerty, J. J. Johnson, D. J. Ferguson, J. R. Coggins, T. Krell, G. H. Coombs, W. K. Milhous, D. E. Kyle, S. Tzipori, J. Barnwell, J. B. Dame, J. Carlton, and R. McLeod. 2002. The shikimate pathway and its branches in apicomplexan parasites. J. Infec. Dis. 185: 25-36 https://doi.org/10.1086/338004
  19. 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
  20. Stallings, W. C., S. S. Abdelmeguid, 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
  21. Stauffer, M. E., J. K. Young, and J. N. Evans. 2001. Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase. Biochemistry 40: 3951-3957 https://doi.org/10.1021/bi002912j
  22. 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. J. Biomol. NMR 20: 387-388 https://doi.org/10.1023/A:1011250423966
  23. Stauffer, M. E., J. K. Young, G. L. Helms, and J. N. Evans. 2001. Chemical shift mapping of shikimate-3-phosphate binding to 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
  24. Yon, J. M., D. Perahia, and C. Ghelis. 1998. Conformational dynamics and enzyme activity. Biochimie 80: 33-42 https://doi.org/10.1016/S0300-9084(98)80054-0