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Improving the Thermostability of a Methyl Parathion Hydrolase by Adding the Ionic Bond on Protein Surface

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Abstract

The thermostability of the methyl parathion hydrolase (MPH_OCH) from Ochrobactrum sp. M231 was improved using site-directed mutagenesis. Two prolines (Pro76 and Pro78) located on the protein surface were selected for mutations after inspection of the sequence alignment of MPH_OCH and OPHC2, a thermostable organophosphorus hydrolase from Pseudomonas pseudoalcaligenes C2-1. The temperature of the double-point mutant (P76D/P78K) at which the mutant lost 50% of its activity (T50) was approximately 68 °C, which is higher than that of WT enzyme (64 °C), P76D (67 °C), and P78K (59 °C). Structural analysis of P76D/P78K indicated that the substituted residues (Asp76 and Lys78) could generate an ionic bond and increase the structural electrostatic energy, which could then increase the stability of the protein. These results also suggest that the thermal stability of proteins could be improved by adding the ionic bond on protein surface.

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References

  1. Brooks, B. R., Brooks, C. L., 3rd, Mackerell, A. D., Jr., Nilsson, L., Petrella, R. J., Roux, B., et al. (2009). Charmm: the biomolecular simulation program. Journal of Computational Chemistry, 30(10), 1545–1614.

    Article  CAS  Google Scholar 

  2. Chen, J., Brooks, C. L., 3rd, & Khandogin, J. (2008). Recent advances in implicit solvent-based methods for biomolecular simulations. Current Opinion in Structural Biology, 18(2), 140–148.

    Article  CAS  Google Scholar 

  3. Chu, X., Wu, N., Deng, M., Tian, J., Yao, B., & Fan, Y. (2006). Expression of organophosphorus hydrolase ophc2 in Pichia pastoris: purification and characterization. Protein Expression and Purification, 49(1), 9–14.

    Article  CAS  Google Scholar 

  4. Chu, X., Tian, J., Wu, N., & Fan, Y. (2010). An intramolecular disulfide bond is required for the thermostability of methyl parathion hydrolase, ophc2. Applied Microbiology and Biotechnology, 88(1), 125–131.

    Article  CAS  Google Scholar 

  5. Cui, Z., Li, S., & Fu, G. (2001). Isolation of methyl parathion-degrading strain m6 and cloning of the methyl parathion hydrolase gene. Applied and Environmental Microbiology, 67(10), 4922–4925.

    Article  CAS  Google Scholar 

  6. Dong, Y., Bartlam, M., Sun, L., Zhou, Y., Zhang, Z., Zhang, C., et al. (2005). Crystal structure of methyl parathion hydrolase from Pseudomonas sp. Wbc-3. Journal of Molecular Biology, 353(3), 655–663.

    Article  CAS  Google Scholar 

  7. Gribenko, A. V., Patel, M. M., Liu, J., McCallum, S. A., Wang, C., & Makhatadze, G. I. (2009). Rational stabilization of enzymes by computational redesign of surface charge–charge interactions. Proc Natl Acad Sci, 106(8), 2601–2606.

    Article  CAS  Google Scholar 

  8. Li, W., Zhou, X., & Lu, P. (2005). Structural features of thermozymes. Biotechnology Advances, 23(4), 271–281.

    Article  CAS  Google Scholar 

  9. Liu, H., Zhang, J. J., Wang, S. J., Zhang, X. E., & Zhou, N. Y. (2005). Plasmid-borne catabolism of methyl parathion and p-nitrophenol in Pseudomonas sp. strain wbc-3. Biochemical and Biophysical Research Communications, 334(4), 1107–1114.

    Article  CAS  Google Scholar 

  10. Pack, S. P., & Yoo, Y. J. (2004). Protein thermostability: structure-based difference of amino acid between thermophilic and mesophilic proteins. Journal of Biotechnology, 111(3), 269–277.

    Article  CAS  Google Scholar 

  11. Palackal, N., Brennan, Y., Callen, W. N., Dupree, P., Frey, G., Goubet, F., et al. (2004). An evolutionary route to xylanase process fitness. Protein Science, 13(2), 494–503.

    Article  CAS  Google Scholar 

  12. Perl, D., Mueller, U., Heinemann, U., & Schmid, F. X. (2000). Two exposed amino acid residues confer thermostability on a cold shock protein. Nature Structural Biology, 7(5), 380–383.

    Article  CAS  Google Scholar 

  13. Polizzi, K. M., Bommarius, A. S., Broering, J. M., & Chaparro-Riggers, J. F. (2007). Stability of biocatalysts. Current Opinion in Chemical Biology, 11(2), 220–225.

    Article  CAS  Google Scholar 

  14. Sambrook, J., & Russell, D. W. (2001). Molecular cloning: a laboratory manual (3rd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory.

    Google Scholar 

  15. Sterner, R., & Liebl, W. (2001). Thermophilic adaptation of proteins. Critical Reviews in Biochemistry and Molecular Biology, 36(1), 39–106.

    Article  CAS  Google Scholar 

  16. Tian, J., Wang, P., Gao, S., Chu, X., Wu, N., & Fan, Y. (2010). Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rationally engineering a glycine to proline mutation. FEBS Journal, 277, 4901–4908.

    Article  CAS  Google Scholar 

  17. Tian, J., Wu, N., Chu, X., & Fan, Y. (2010). Predicting changes in protein thermostability brought about by single- or multi-site mutations. BMC Bioinformatics, 11, 370.

    Article  Google Scholar 

  18. Van den Burg, B., Dijkstra, B. W., Vriend, G., Van der Vinne, B., Venema, G., & Eijsink, V. G. (1994). Protein stabilization by hydrophobic interactions at the surface. European Journal of Biochemistry/FEBS, 220(3), 981–985.

    Article  Google Scholar 

  19. Wei, M., Zhang, J. J., Liu, H., Wang, S. J., Fu, H., & Zhou, N. Y. (2009). A transposable class I composite transposon carrying mph (methyl parathion hydrolase) from Pseudomonas sp. strain wbc-3. FEMS Microbiology Letters, 292(1), 85–91.

    Article  CAS  Google Scholar 

  20. Xiao, W., Chu, X., Tian, J., Guo, J., & Wu, N. (2008). Cloning of a methyl parathion hydrolase gene from Ochrobactrum sp. (in Chinese). Journal of Agricultural Science and Technology, 10(S1), 99–102.

    Google Scholar 

  21. Yang, C., Liu, N., Guo, X., & Qiao, C. (2006). Cloning of mpd gene from a chlorpyrifos-degrading bacterium and use of this strain in bioremediation of contaminated soil. FEMS Microbiology Letters, 265(1), 118–125.

    Article  CAS  Google Scholar 

  22. Yang, C., Freudl, R., Qiao, C., & Mulchandani, A. (2010). Cotranslocation of methyl parathion hydrolase to the periplasm and of organophosphorus hydrolase to the cell surface of Escherichia coli by the tat pathway and ice nucleation protein display system. Applied and Environmental Microbiology, 76(2), 434–440.

    Article  CAS  Google Scholar 

  23. Yu, H., Yan, X., Shen, W., Hong, Q., Zhang, J., Shen, Y., et al. (2009). Expression of methyl parathion hydrolase in Pichia pastoris. Current Microbiology, 59(6), 573–578.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by grants from National High Technology Research and Development Program of China (863 Program, 2007AA100605).

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Authors and Affiliations

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, People’s Republic of China

    Yidan Su, Jian Tian, Ping Wang, Xiaoyu Chu, Ningfeng Wu & Yunliu Fan

  2. College of Life Science, Northwest Normal University, Lanzhou, 730070, People’s Republic of China

    Yidan Su & Guoan Liu

Authors
  1. Yidan Su
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  2. Jian Tian
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  3. Ping Wang
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  4. Xiaoyu Chu
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  5. Guoan Liu
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  6. Ningfeng Wu
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  7. Yunliu Fan
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Corresponding author

Correspondence to Ningfeng Wu.

Additional information

Yidan Su and Jian Tian contributed equally to this work.

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Table S1

Primers used in this study (DOC 30.5 kb)

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Su, Y., Tian, J., Wang, P. et al. Improving the Thermostability of a Methyl Parathion Hydrolase by Adding the Ionic Bond on Protein Surface. Appl Biochem Biotechnol 165, 989–997 (2011). https://doi.org/10.1007/s12010-011-9314-z

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  • DOI: https://doi.org/10.1007/s12010-011-9314-z

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