Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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Site-directed Mutagenesis of Cysteine Residues in Phi-class Glutathione S-transferase F3 from Oryza sativa

  • Jo, Hyun-Joo (Biomolecular Chemistry Laboratory, Department of Chemistry, College of Natural Sciences, Chung-Ang University) ;
  • Lee, Ju-Won (Biomolecular Chemistry Laboratory, Department of Chemistry, College of Natural Sciences, Chung-Ang University) ;
  • Noh, Jin-Seok (Biomolecular Chemistry Laboratory, Department of Chemistry, College of Natural Sciences, Chung-Ang University) ;
  • Kong, Kwang-Hoon (Biomolecular Chemistry Laboratory, Department of Chemistry, College of Natural Sciences, Chung-Ang University)
  • Received : 2012.09.19
  • Accepted : 2012.10.04
  • Published : 2012.12.20
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Abstract

To elucidate the roles of cysteine residues in rice Phi-class GST F3, in this study, all three cysteine residues were replaced with alanine by site-directed mutagenesis in order to obtain mutants C22A, C73A and C77A. Three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Cys73 and Cys77 residues in OsGSTF3 with alanine did not affect the glutathione conjugation activities, showing non-essentiality of these residues. On the other hand, the substitution of Cys22 residue with alanine resulted in approximately a 60% loss of specific activity toward ethacrynic acid. Moreover, the ${K_m}^{CDNB}$ value of the mutant C22A was approximately 2.2 fold larger than that of the wild type. From these results, the evolutionally conserved cysteine 22 residue seems to participate rather in the structural stability of the active site in OsGSTF3 by stabilizing the electrophilic substrates-binding site's conformation than in the substrate binding directly.

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References

  1. Frova, C. Physiol. Plant. 2003, 119, 469. https://doi.org/10.1046/j.1399-3054.2003.00183.x
  2. Shhehan, D.; Meade, G.; Foley, V. M.; Dowd, C. A. Biochem. J. 2001, 360, 1. https://doi.org/10.1042/0264-6021:3600001
  3. Dixon, D. P.; Lapthorne, A.; Edwards, R. Genome Biol. 2002, 3, 3004.1.
  4. Edwards, R.; Dixon, D. P.; Walbot, V. Trends Plant Sci. 2000, 5,193.
  5. Roxas, V. P.; Smith, R. K., Jr; Allen, E. R.; Allen, R. D. Nature Biotechnol. 1997, 15, 988. https://doi.org/10.1038/nbt1097-988
  6. Zettl, R.; Schell, J.; Palme, K. Proc. Natl. Acad. Sci. USA 1994, 91, 689. https://doi.org/10.1073/pnas.91.2.689
  7. Marrs, K. A.; Alfenito, M. R.; Lloyd, A. M.; Walbot, V. Nature 1995, 375, 397. https://doi.org/10.1038/375397a0
  8. Mueller, L. A.; Goodman, C. D.; Silady, R. A.; Walbot, V. Plant Physiol. 2000, 123, 1561. https://doi.org/10.1104/pp.123.4.1561
  9. Mannervik, B.; Danielson, U. H. CRC Crit. Rev. Biochem. 1988, 23, 283. https://doi.org/10.3109/10409238809088226
  10. Fahey, R. C.; Sundquist, A. R. Adv. Enzymol. Relat. Areas Mol. Biol. 1991, 64, 1.
  11. Dixon, D. P.; Cole, D. J.; Edwards, R. Plant Mol. Biol. 1998, 36, 75. https://doi.org/10.1023/A:1005958711207
  12. Dixon, D. P.; Davies, B. G.; Edwards, R. J. Biol. Chem. 2002, 277, 30859. https://doi.org/10.1074/jbc.M202919200
  13. Droog, F. J. Palt Growth Regul. 1997, 16, 95. https://doi.org/10.1007/PL00006984
  14. McGonigle, B.; Keeler, S. J.; Lau, S.-M. C.; Koeppe, M. K.; O'Keefe, D. P. Plant Physiol. 2000, 124, 1105. https://doi.org/10.1104/pp.124.3.1105
  15. Hong, S.-H.; Park, H.-J.; Kong, K.-H. Comp. Biochem. Physiol. 1999, 122, 21. https://doi.org/10.1016/S0305-0491(98)10135-9
  16. Cho, H.-Y.; Kong, K.-H. Pestic. Biochem. Phys. 2005, 83, 29. https://doi.org/10.1016/j.pestbp.2005.03.005
  17. Yang, X.; Sun, W.; Liu, J.-P.; Liu, Y.-J.; Zeng, Q.-Y. Plant Phys. Biochem. 2009, 47, 1061. https://doi.org/10.1016/j.plaphy.2009.07.003
  18. Cho, H.-Y.; Lee, H. J.; Kong, K.-H. J. Biochem. Mol. Biol. 2007, 40, 511. https://doi.org/10.5483/BMBRep.2007.40.4.511
  19. Cho, H.-Y.; Yoo, S.-Y.; Kong, K.-H. Pestic. Biochem. Phys. 2006, 86, 110. https://doi.org/10.1016/j.pestbp.2006.02.003
  20. Lee, J.-J.; Jo, H.-J.; Kong, K.-H. Bull. Korean Chem. Soc. 2011, 32, 3756. https://doi.org/10.5012/bkcs.2011.32.10.3756
  21. Jo, H.-J.; Lee, J.-J.; Kong, K.-H. Pestic. Biochem. Phys. 2011, 101, 265. https://doi.org/10.1016/j.pestbp.2011.10.005
  22. Yoon, S.-Y.; Kong, J.-N.; Jo, D.-H.; Kong, K.-H. Food Chem. 2011, 129, 1327. https://doi.org/10.1016/j.foodchem.2011.06.054
  23. Habig, W. H.; Jakoby, W. B. Methods Enzymol. 1981, 77, 398. https://doi.org/10.1016/S0076-6879(81)77053-8
  24. Flohe, L.; Gunzler, W. A. Methods. Enzymol. 1984, 105, 114. https://doi.org/10.1016/S0076-6879(84)05015-1
  25. Thom, R.; Cummins, I.; Dixon, D. P.; Edwards, R.; Cole, D. J. Biochemistry 2002, 41, 7008. https://doi.org/10.1021/bi015964x
  26. Desideri, A.; Caccuri, A. M.; Poligio, F.; Bastoni, R.; Federici, G. J. Biol. Chem. 1991, 266, 2063.
  27. Barycki, J. J.; Colman, R. F. Arch. Biochem. Biophys. 1997, 345, 16. https://doi.org/10.1006/abbi.1997.0244
  28. Nishida, M.; Harada, S.; Noguchi, S.; Satow, Y.; Inoue, H.; Takahashi, K. J. Mol. Biol. 1998, 281, 135. https://doi.org/10.1006/jmbi.1998.1927
  29. Vega, M. C.; Walsh, S. B.; Mantles, T. J.; Coll, M. J. Biol. Chem. 1998, 273, 2844. https://doi.org/10.1074/jbc.273.5.2844
  30. Reinemer, P.; Prade, L.; Hof, P.; Neuefeind, T.; Huber, R.; Palme, R.; Zettl, K.; Schell, J.; Koelln, I.; Bartunik, H. D.; Bieseler, B. J. Mol. Biol. 1996, 255, 289. https://doi.org/10.1006/jmbi.1996.0024
  31. Neuefeind, T.; Huber, R.; Dasenbrock, H.; Prade, L.; Bieseler, B. J. Mol. Biol. 1997, 274, 446. https://doi.org/10.1006/jmbi.1997.1402
  32. Neuefeind, T.; Reinemer, P.; Bieseler, B. Biol. Chem. 1997, 378, 199.
  33. Wongtrakul, J.; Sramala, I.; Ketterman, A. J. Protein Pept. Lett. 2003, 10, 375. https://doi.org/10.2174/0929866033478870

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