Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

The 'evolvability' of promiscuous protein functions

Abstract

How proteins with new functions (e.g., drug or antibiotic resistance or degradation of man-made chemicals) evolve in a matter of months or years is still unclear. This ability is dependent on the induction of new phenotypic traits by a small number of mutations (plasticity). But mutations often have deleterious effects on functions that are essential for survival. How are these seemingly conflicting demands met at the single-protein level? Results from directed laboratory evolution experiments indicate that the evolution of a new function is driven by mutations that have little effect on the native function but large effects on the promiscuous functions that serve as starting point. Thus, an evolving protein can initially acquire increased fitness for a new function without losing its original function. Gene duplication and the divergence of a completely new protein may then follow.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Changes in activities of the newly evolved PON1 variants.
Figure 2: Changes in activities of the newly evolved PTE variants.
Figure 3: Changes in activities of the newly evolved CAII variants.
Figure 4: The location of the selectivity-changing mutations observed in the directly evolved PON1 variants.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Jensen, R.A. Enzyme recruitment in evolution of new function. Annu. Rev. Microbiol. 30, 409–425 (1976).

    Article  CAS  Google Scholar 

  2. Copley, S.D. Enzymes with extra talents: moonlighting functions and catalytic promiscuity. Curr. Opin. Chem. Biol. 7, 265–272 (2003).

    Article  CAS  Google Scholar 

  3. James, L.C. & Tawfik, D.S. Conformational diversity and protein evolution - a 60-year-old hypothesis revisited. Trends Biochem. Sci. 28, 361–368 (2003).

    Article  CAS  Google Scholar 

  4. Arnold, F.H., Wintrode, P.L., Miyazaki, K. & Gershenson, A. How enzymes adapt: lessons from directed evolution. Trends Biochem. Sci. 26, 100–106 (2001).

    Article  CAS  Google Scholar 

  5. Krebs, J.F., Ippolito, J.A., Christianson, D.W. & Fierke, C.A. Structural and functional importance of a conserved hydrogen bond network in human carbonic anhydrase II. J. Biol. Chem. 268, 27458–27466 (1993).

    CAS  PubMed  Google Scholar 

  6. Raushel, F.M. & Holden, H.M. Phosphotriesterase: an enzyme in search of its natural substrate. Adv. Enzymol. Relat. Areas Mol. Biol. 74, 51–93 (2000).

    CAS  PubMed  Google Scholar 

  7. Draganov, D.I. & La Du, B.N. Pharmacogenetics of paraoxonases: a brief review. Naunyn Schmiedebergs Arch. Pharmacol. 369, 78–88 (2004).

    Article  CAS  Google Scholar 

  8. Stemmer, W.P.C. DNA Shuffling by Random Fragmentation and Reassembly - in-Vitro Recombination for Molecular Evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751 (1994).

    Article  CAS  Google Scholar 

  9. Aharoni, A. et al. Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization. Proc. Natl. Acad. Sci. USA 101, 482–487 (2004).

    Article  CAS  Google Scholar 

  10. Bone, R., Silen, J.L. & Agard, D.A. Structural plasticity broadens the specificity of an engineered protease. Nature 339, 191–195 (1989).

    Article  CAS  Google Scholar 

  11. Perona, J.J. & Craik, C.S. Evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold. J. Biol. Chem. 272, 29987–29990 (1997).

    Article  CAS  Google Scholar 

  12. James, L.C. & Tawfik, D.S. The specificity of cross-reactivity: promiscuous antibody binding involves specific hydrogen bonds rather than nonspecific hydrophobic stickiness. Protein Sci. 12, 2183–2193 (2003).

    Article  CAS  Google Scholar 

  13. Bornscheuer, U.T. & Kazlauskas, R.J. Catalytic plasticity in biocatalysis: using old enzymes to form new bonds and follow new pathways. Angew. Chem. Int. Ed. Engl. 43, 2–10 (2004).

    Google Scholar 

  14. O'Brien, P.J. & Herschlag, D. Catalytic promiscuity and the evolution of new enzymatic activities. Chem. Biol. 6, R91–R105 (1999).

    Article  CAS  Google Scholar 

  15. Yang, K. & Metcalf, W.W. A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase. Proc. Natl. Acad. Sci. USA 101, 7919–7924 (2004).

    Article  CAS  Google Scholar 

  16. James, L., Roversi, P. & Tawfik, D. Antibody multi-specificity mediated by conformational diversity. Science 299, 1362–1367 (2003).

    Article  CAS  Google Scholar 

  17. James, L.C. & Tawfik, D.S. Catalytic and binding poly-reactivities shared by two unrelated proteins: The potential role of promiscuity in enzyme evolution. Protein Sci. 10, 2600–2607 (2001).

    Article  CAS  Google Scholar 

  18. Miller, B.G. & Raines, R.T. Identifying latent enzyme activities: substrate ambiguity within modern bacterial sugar kinases. Biochemistry 43, 6387–6392 (2004).

    Article  CAS  Google Scholar 

  19. Taverna, D.M. & Goldstein, R.A. Why are proteins so robust to site mutations? J. Mol. Biol. 315, 479–484 (2002).

    Article  CAS  Google Scholar 

  20. Pata, J.D., Stirtan, W.G., Goldstein, S.W. & Steitz, T.A. Structure of HIV-1 reverse transcriptase bound to an inhibitor active against mutant reverse transcriptases resistant to other nonnucleoside inhibitors. Proc. Natl. Acad. Sci. USA 101, 10548–10553 (2004).

    Article  CAS  Google Scholar 

  21. Chong, Y.H. & Chu, C.K. Understanding the molecular mechanism of drug resistance of anti-HIV nucleosides by molecular modeling. Front. Biosci. 9, 164–186 (2004).

    Article  CAS  Google Scholar 

  22. Wouters, M.A., Liu, K., Riek, P. & Husain, A. A despecialization step underlying evolution of a family of serine proteases. Mol. Cell. 12, 343–354 (2003).

    Article  CAS  Google Scholar 

  23. Matsumura, I. & Ellington, A.D. In vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates. J. Mol. Biol. 305, 331–339 (2001).

    Article  CAS  Google Scholar 

  24. Lynch, M. & Katju, V. The altered evolutionary trajectories of gene duplicates. Trends Genet. 20, 544–549 (2004).

    Article  CAS  Google Scholar 

  25. Kirschner, M. & Gerhart, J. Evolvability. Proc. Natl. Acad. Sci. USA 95, 8420–8427 (1998).

    Article  CAS  Google Scholar 

  26. Earl, D.J. & Deem, M.W. Evolvability is a selectable trait. Proc. Natl. Acad. Sci. USA 101, 11531–11536 (2004).

    Article  CAS  Google Scholar 

  27. Radman, M., Matic, I. & Taddei, F. Evolution of evolvability. Ann. NY Acad. Sci. 870, 146–155 (1999).

    Article  CAS  Google Scholar 

  28. Giraud, A. et al. Costs and benefits of high mutation rates: Adaptive evolution of bacteria in the mouse gut. Science 291, 2606–2608 (2001).

    Article  CAS  Google Scholar 

  29. True, H.L. & Lindquist, S.L. A yeast prion provides a mechanism for genetic variation and phenotypic diversity. Nature 407, 477–483 (2000).

    Article  CAS  Google Scholar 

  30. Harel, M. et al. Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes. Nat. Struct. Mol. Biol. 11, 412–419 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank F. Kondrashov for his constructive and insightful criticism. We acknowledge financial support from the Benoziyo Institute of Molecular Medicine, the Minerva foundation and the Israel Science Foundation. S.G. is funded by the EU's ENDIRPRO program. D.S.T. is the incumbent of the Elaine Blond Career Development Chair.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dan S Tawfik.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

PON1, PTE and CAII substrates used in the different directed evolution experiments. (PDF 43 kb)

Supplementary Table 1

PON1 variants with improved γ-butyrolactonase activity. (PDF 35 kb)

Supplementary Table 2

PON1 variants with improved long-chain acyl esterase activity. (PDF 17 kb)

Supplementary Table 3

PON1 variants with improved acetoxy coumarin esterase activity. (PDF 67 kb)

Supplementary Table 4

PON1 variants evolved for an OP substrate. (PDF 103 kb)

Supplementary Table 5

PON1 variants analyzed for phenyl acetate, paraoxon, dihydrocoumain, δ-valerolactone and γ-heptaolid. (PDF 32 kb)

Supplementary Table 6

Analysis of the bacterial PTE variants evolved for 2-Naphtyl acetate hydrolysis. (PDF 58 kb)

Supplementary Table 7

Analysis of the newly evolved Carbonic anhydrase II (CAII) variants. (PDF 107 kb)

Supplementary Table 8

Examples for variants exhibiting large improvements in promiscuous activities and small changes in native activity. (PDF 90 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Aharoni, A., Gaidukov, L., Khersonsky, O. et al. The 'evolvability' of promiscuous protein functions. Nat Genet 37, 73–76 (2005). https://doi.org/10.1038/ng1482

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng1482

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing