Skip to main content
SpringerLink
Log in

Reconstruction of an enzyme by domain substitution effectively switches substrate specificity

  • Letter
  • Published:

From Nature

View current issue Submit your manuscript

Abstract

THE polar domains of the two transcarbamoylases, aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase, (OTCase) from Escherichia coli bind the common substrate carbamoyl phosphate and share extensive amino-acid sequence homology1,2. The equatorial domains of the two enzymes differ in their substrate specificity (ATCase binds aspartate, OTCase binds ornithine) and have decreased sequence identity. While addressing the conservation of specific protein interactions during the evolution of these enzymes, we were able to switch one of their amino-acid-specific equatorial domains to produce a viable chimaeric enzyme. This was achieved by the in vitro fusion of DNA encoding the polar domain of OTCase to DNA encoding the equatorial domain of ATCase. The resulting gene fusion successfully transformed an argl-pyrB deletion strain of E. coli to pyrimidine prototrophy, giving rise to Pyr+ transformants that expressed ATCase but not OTCase activity. The formation of this active chimaeric enzyme shows that by exchanging protein domains between two functionally divergent enzymes we have achieved a switching in substrate specificity.

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

Similar content being viewed by others

References

  1. Houghton, J. E., Bencini, D. A., O'Donovan, G. A. & Wild, J. R. Proc. natn. Acad. Sci. U.S.A. 81, 4864–4868 (1984).

    Article  ADS  CAS  Google Scholar 

  2. Van Vliet, F. et al. Nucleic Acids Res. 12, 6277–6299 (1984).

    Article  CAS  Google Scholar 

  3. Gerhart, J. C. & Schachman, H. K. Biochemistry 4, 1054–1062 (1965).

    Article  CAS  Google Scholar 

  4. Legrain, C. et al. Eur. J. Biochem. 80, 401–409 (1984).

    Article  Google Scholar 

  5. Foltermann, K. F., Beck, D. A. & Wild, J. R. J. Bact. 167, 285–295 (1986).

    Article  CAS  Google Scholar 

  6. Bencini, D. A. et al. Nucleic Acids Res. 11, 8509–8518 (1983).

    Article  CAS  Google Scholar 

  7. Krause, K. L., Volz, K. W. & Lipscomb, W. N. Proc. natn. Acad. Sci. U.S.A. 82, 643–1647 (1985).

    Article  Google Scholar 

  8. Honzatko, R. B. et al. J. molec. Biol. 160, 219–263 (1982).

    Article  CAS  Google Scholar 

  9. Kantrowitz, E. R. & Lipscomb, W. N. Science 241, 669–674 (1988).

    Article  ADS  CAS  Google Scholar 

  10. Hoover, T. A. et al. Proc. natn. Acad. Sci. U.S.A. 80, 2462–2466 (1983).

    Article  ADS  CAS  Google Scholar 

  11. Roof, W. D., Foltermann, K. F. & Wild, J. R. Molec. Gen. Genet. 187, 391–400 (1982).

    Article  CAS  Google Scholar 

  12. Hirota, Y. Proc. natn. Acad. Sci. U.S.A. 46, 57–64 (1960).

    Article  ADS  CAS  Google Scholar 

  13. Glansdorff, N., Sand, G. & Verhoef, C. Mutat. Res. 4, 743–751 (1967).

    Article  CAS  Google Scholar 

  14. Cleary, M. L., Garvin, R. T. & James, E. Molec. Gen. Genet. 157, 155–165 (1977).

    Article  CAS  Google Scholar 

  15. Lissens, W., Cunin, R., Kelker, N., Glansdorff, N. & Pierard, A. J. Bact. 141, 55–66 (1980).

    Google Scholar 

  16. Foltermann, K. F., Shanley, M. S. & Wild, J. R. J. Bact. 157, 891–898 (1984).

    CAS  PubMed  Google Scholar 

  17. Baur, H., Stalon, V., Falmagne, P., Luethi, E. & Haas, D. Eur. J. Biochem. 166, 111–117 (1987).

    Article  CAS  Google Scholar 

  18. Nyunoya, H., Broglie, K. E. & Lusty, C. J. Proc. natn. Acad. Sci. U.S.A. 82, 2244–2246 (1985).

    Article  ADS  CAS  Google Scholar 

  19. Gilbert, W. Nature 271, 501 (1978).

    Article  ADS  CAS  Google Scholar 

  20. Richards, J. H., Nature 323, 187 (1986).

    Article  ADS  Google Scholar 

  21. Pauza, C. D., Karels, M. J., Navre, M. & Schachman, H. K. Proc. natn. Acad. Sci. U.S.A. 79, 4020–4024 (1983).

    Article  ADS  Google Scholar 

  22. Zoller, M. J. & Smith, M. Meth. Enzym. 100, 468–500 (1983).

    Article  CAS  Google Scholar 

  23. Vieira, J. & Messing, J. Gene 19, 259–268 (1982).

    Article  CAS  Google Scholar 

  24. Vieira, J. & Messing, J. Gene 19, 269–275 (1982).

    Article  Google Scholar 

  25. Sanger, F., Coulson, A. R., Barell, B. G., Smith, A. J. H. & Roe, B. A. J. molec. Biol. 143, 161–178 (1980).

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. John E. Houghton

    Present address: Department of Biology, Yale University, New Haven, Connecticut, 06511, USA

  2. Gerard A. O'Donovan

    Present address: Department of Biological Sciences, University of North Texas, Denton, Texas, 76203, USA

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas, 77843, USA

    John E. Houghton, Gerard A. O'Donovan & James R. Wild

Authors
  1. John E. Houghton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gerard A. O'Donovan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. James R. Wild
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Houghton, J., O'Donovan, G. & Wild, J. Reconstruction of an enzyme by domain substitution effectively switches substrate specificity. Nature 338, 172–174 (1989). https://doi.org/10.1038/338172a0

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/338172a0

  • Springer Nature Limited

Search

Navigation