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, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Houghton, J. E., Bencini, D. A., O'Donovan, G. A. & Wild, J. R. Proc. natn. Acad. Sci. U.S.A. 81, 4864–4868 (1984).
Van Vliet, F. et al. Nucleic Acids Res. 12, 6277–6299 (1984).
Gerhart, J. C. & Schachman, H. K. Biochemistry 4, 1054–1062 (1965).
Legrain, C. et al. Eur. J. Biochem. 80, 401–409 (1984).
Foltermann, K. F., Beck, D. A. & Wild, J. R. J. Bact. 167, 285–295 (1986).
Bencini, D. A. et al. Nucleic Acids Res. 11, 8509–8518 (1983).
Krause, K. L., Volz, K. W. & Lipscomb, W. N. Proc. natn. Acad. Sci. U.S.A. 82, 643–1647 (1985).
Honzatko, R. B. et al. J. molec. Biol. 160, 219–263 (1982).
Kantrowitz, E. R. & Lipscomb, W. N. Science 241, 669–674 (1988).
Hoover, T. A. et al. Proc. natn. Acad. Sci. U.S.A. 80, 2462–2466 (1983).
Roof, W. D., Foltermann, K. F. & Wild, J. R. Molec. Gen. Genet. 187, 391–400 (1982).
Hirota, Y. Proc. natn. Acad. Sci. U.S.A. 46, 57–64 (1960).
Glansdorff, N., Sand, G. & Verhoef, C. Mutat. Res. 4, 743–751 (1967).
Cleary, M. L., Garvin, R. T. & James, E. Molec. Gen. Genet. 157, 155–165 (1977).
Lissens, W., Cunin, R., Kelker, N., Glansdorff, N. & Pierard, A. J. Bact. 141, 55–66 (1980).
Foltermann, K. F., Shanley, M. S. & Wild, J. R. J. Bact. 157, 891–898 (1984).
Baur, H., Stalon, V., Falmagne, P., Luethi, E. & Haas, D. Eur. J. Biochem. 166, 111–117 (1987).
Nyunoya, H., Broglie, K. E. & Lusty, C. J. Proc. natn. Acad. Sci. U.S.A. 82, 2244–2246 (1985).
Gilbert, W. Nature 271, 501 (1978).
Richards, J. H., Nature 323, 187 (1986).
Pauza, C. D., Karels, M. J., Navre, M. & Schachman, H. K. Proc. natn. Acad. Sci. U.S.A. 79, 4020–4024 (1983).
Zoller, M. J. & Smith, M. Meth. Enzym. 100, 468–500 (1983).
Vieira, J. & Messing, J. Gene 19, 259–268 (1982).
Vieira, J. & Messing, J. Gene 19, 269–275 (1982).
Sanger, F., Coulson, A. R., Barell, B. G., Smith, A. J. H. & Roe, B. A. J. molec. Biol. 143, 161–178 (1980).
Author information
Authors and Affiliations
Rights 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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/338172a0
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
