Skip to main content
SpringerLink
Log in

Improving the Activity of Cytochrome P450 BM-3 Catalyzing Indole Hydroxylation by Directed Evolution

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Cytochrome P450 BM-3 (A74G/F87V/L188Q) could catalyze indole to produce indigo. To further improve this capability, random mutagenesis was performed on the heme domain of P450 BM-3 (A74G/F87V/L188Q) with error-prone PCR. A single mutant V445A was selected out from the error-prone library and exhibited the highest specific activity toward indole among the mutants obtained. The kinetic parameters of V445A were also highly improved. Compared with the parent enzyme, the turnover rate (k cat) of V445A was increased by 7.5 times, while its K m value decreased by 9.2 %. Consequently, the catalytic efficiency (k cat/K m) of V445A was raised to 8.2 times than that of the parent enzyme. Moreover, alanine was confirmed as the best amino acid substitution by saturated mutagenesis in Val445 position. Three-dimensional structure analysis was also used to rationalize the effect on the enzyme properties of the mutation. This study showed that random mutagenesis was efficient to identify mutants with potential values in industry and increased our insight into P450 BM-3.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Božič, M., Díaz-González, M., Tzanov, T., Guebitz, G. M., & Kokol, V. (2009). Enzyme and Microbial Technology, 45, 317–323.

    Article  Google Scholar 

  2. Papanastasiou, M., Allen, N. S., McMahon, A., Naegel, L. C., Edge, M., & Protopappas, S. (2012). Dyes and Pigments, 92, 1192–1198.

    Article  CAS  Google Scholar 

  3. Blackburn, R. S., Bechtold, T., & John, P. (2009). Coloration Technology, 125, 193–207.

    Article  CAS  Google Scholar 

  4. Meksi, N., Ben, T. M., Kechida, M., & Mhenni, M. F. (2012). Journal of Cleaner Production, 24, 149–158.

    Article  CAS  Google Scholar 

  5. Whitehouse, C. J., Bell, S. G., & Wong, L. L. (2012). Chemical Society Reviews, 41, 1218–1260.

    Article  CAS  Google Scholar 

  6. Zhang, Z. G., Liu, Y., Guengerich, F. P., Matse, J. H., Chen, J., & Wu, Z. L. (2009). Journal of Biotechnology, 139, 12–18.

    Article  CAS  Google Scholar 

  7. Huang, W., Johnston, W. A., Hayes, M. A., De Voss, J. J., & Gillam, E. M. (2007). Archives of Biochemistry and Biophysics, 467, 193–205.

    Article  CAS  Google Scholar 

  8. Gillam, E. M., Notley, L. M., Cai, H. L., De Voss, J. J., & Guengerich, F. P. (2000). Biochemistry, 39, 13817–13824.

    Article  CAS  Google Scholar 

  9. van Vugt-Lussenburg, B. M. A., Stjernschantz, E., Lastdrager, J., Oostenbrink, C., Vermeulen, N. P. E., & Commandeur, J. N. M. (2007). Journal of Medicinal Chemistry, 50, 455–461.

    Article  Google Scholar 

  10. Li, Q. S., Schwaneberg, U., Fischer, P., & Schmid, R. D. (2000). Chemical-European Journal, 6, 1531–1536.

    Article  CAS  Google Scholar 

  11. Narhi, L. O., & Fulco, A. J. (1986). Journal of Biological Chemistry, 261, 7160–7169.

    CAS  Google Scholar 

  12. Warman, A. J., Robinson, J. W., Luciakova, D., Lawrence, A. D., Marshall, K. R., Warren, M. J., et al. (2012). FEBS Journal, 279, 1675–1693.

    Article  CAS  Google Scholar 

  13. Munro, A. W., Leys, D. G., McLean, K. J., Marshall, K. R., Ost, T. W. B., Daff, S., et al. (2002). Trends in Biochemical Sciences, 27, 250–257.

    Article  CAS  Google Scholar 

  14. Li, H. M., Mei, L. H., Urlacher, V. B., & Schmid, R. D. (2008). Applied Biochemistry and Biotechnology, 144, 27–36.

    Article  CAS  Google Scholar 

  15. Maurer, S. C., Schulze, H., Schmid, R. D., & Urlacher, V. B. (2003). Advanced Synthesis and Catalysis, 345, 802–810.

    Article  CAS  Google Scholar 

  16. Omura, T., & Sato, R. (1964). Journal of Biological Chemistry, 239, 2370–2378.

    CAS  Google Scholar 

  17. Urlacher, V. B., Makhsumkhanov, A., & Schmid, R. D. (2006). Applied Microbiology and Biotechnology, 70, 53–59.

    Article  CAS  Google Scholar 

  18. Choi, H. S., Kim, J. K., Cho, E. H., Kim, Y. C., Kim, J. I., & Kim, S. W. (2003). Biochemical and Biophysical Research Communications, 306, 930–936.

    Article  CAS  Google Scholar 

  19. Sevrioukova, I. F., Li, H., Zhang, H., Peterson, J. A., & Poulos, T. L. (1999). Proceedings of the National Academy of Sciences, 96, 1863–1868.

    Article  CAS  Google Scholar 

  20. Sven, H., Michael, S., Bert, S., & Svante, W. (1987). Journal of Medicinal Chemistry, 30, 1126–1135.

    Article  Google Scholar 

  21. Oliver, C. F., Modi, S., Primrose, W. U., Lian, L. Y., & Roberts, G. C. (1997). Biochemistry Journal, 327, 537–544.

    CAS  Google Scholar 

  22. Hutzler, J. M., Wienkers, L. C., Wahlstrom, J. L., Carlson, T. J., & Tracy, T. S. (2003). Archives Biochemistry Biophysics, 410, 16–24.

    Article  Google Scholar 

  23. Hu, S., Huang, J., Mei, L. H., Yu, Q., Yao, S. J., & Jin, Z. H. (2010). Journal of Molecular Catalysis B: Enzymatic, 67, 29–35.

    Article  CAS  Google Scholar 

  24. Liao, Y., Zeng, M., Wu, Z. F., Chen, H., Wang, H. N., Wu, Q., et al. (2011). Applied Biochemistry Biotechnology, 166, 549–562.

    Article  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (30970638, 21176220, and 31240054) and Natural Science Foundation of Zhejiang Province (LZ13B060002).

Author information

Authors and Affiliations

  1. Institute of Bioengineering, Zhejiang University, Hangzhou, 310027, Zhejiang, People’s Republic of China

    Zhang Pengpai & Mei Lehe

  2. College of Biological and Chemical Engineering, Ningbo Institute of Technology, Ningbo, 315100, China

    Hu Sheng, Mei Lehe, Lei Yinlin, Jin Zhihua & Hu Guixiang

Authors
  1. Zhang Pengpai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hu Sheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mei Lehe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lei Yinlin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jin Zhihua
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hu Guixiang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mei Lehe.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pengpai, Z., Sheng, H., Lehe, M. et al. Improving the Activity of Cytochrome P450 BM-3 Catalyzing Indole Hydroxylation by Directed Evolution. Appl Biochem Biotechnol 171, 93–103 (2013). https://doi.org/10.1007/s12010-013-0353-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-013-0353-5

Keywords

Search

Navigation