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Directed evolution of new catalytic activity using the α/β-barrel scaffold

Nature volume 403, pages 617–622 (2000)Cite this article

A Retraction to this article was published on 23 May 2002

Abstract

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the α/β-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the α/β-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.

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Figure 1: The reaction catalysed by phosphoribosylanthranilate isomerase (PRAI) and indoleglycerol-phosphate synthase (IGPS).
Figure 2: Superimposed Cα traces of IGPS (residues 1–255, green line) and PRAI (residues 255–452, blue line).
Figure 3: Ribbon representation of the experimental strategy for evolving a new function in the IGPS scaffold.
Figure 4: Complementation-based in vivo selection of active PRAI-containing clones using E. coli JA300 (a PRAI-deficient strain that does not grow in the absence of tryptophan).
Figure 5: Sequence alignment of in vitro evolved PRAI (ivePRAI), wild-type PRAI and wild-type IGPS.

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References

  1. Patthy, L. Protein Evolution (Blackwell Science, Oxford, 1999).

    Google Scholar 

  2. Henikoff, S. et al. Gene families: the taxonomy of protein paralogs and chimeras. Science 278, 609–614 (1997).

    Article  ADS  CAS  Google Scholar 

  3. Chothia, C. One thousand families for the molecular biologist. Nature 357, 543–544.

    Article  ADS  CAS  Google Scholar 

  4. Arnold, F. H. & Volkov, A. A. Directed evolution of biocatalysts. Curr. Opin. Chem. Biol. 3, 54– 59 (1999).

    Article  CAS  Google Scholar 

  5. Bränden, C. & Tooze, J. Introduction to Protein Structure 2nd edn (Garland, New York, 1999).

    Google Scholar 

  6. Gerlt, J. A. & Babbitt, P. C. Mechanistically diverse enzyme superfamilies: the importance of chemistry in the evolution of catalysis. Curr. Opin. Chem. Biol. 2, 607– 612 (1998).

    Article  CAS  Google Scholar 

  7. Holm, L. & Sander, C. An evolutionary treasure: unification of a broad set of amidohydrolases related to urease. Proteins 28, 72–82 (1997).

    Article  CAS  Google Scholar 

  8. Hasson, M. S. et al. Evolution of an enzyme active site: the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and nolase. Proc. Natl Acad. Sci. 95, 10396 –10401 (1998).

    Article  ADS  CAS  Google Scholar 

  9. Bränden, C. I. The TIM barrel—the most frequently occurring folding motif in proteins. Curr. Opin. Struct. Biol. 1, 978– 983 (1991).

    Article  Google Scholar 

  10. Murzin, A. G., Lesk, A. M. & Chothia, C. Principles determining the structure of beta-sheet barrels in proteins. II. The observed structures. J. Mol. Biol. 236, 1382–1400 (1994).

    Article  CAS  Google Scholar 

  11. Murzin, A. G., Lesk, A. M. & Chothia, C. Principles determining the structure of beta-sheet barrels in proteins. I. A theoretical analysis. J. Mol. Biol. 236, 1369–1381 (1994).

    Article  CAS  Google Scholar 

  12. Kirschner, K., Szadkowski, H., Jardetzky, T. S. & Hager, V. Phosphoribosylanthranilate isomerase-indoleglycerol-phosphate synthase from Escherichia coli. Methods Enzymol. 142, 386–397 (1987).

    Article  CAS  Google Scholar 

  13. Darimont, B., Stehlin, C., Szadkowski, H. & Kirschener, K. Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli. Protein Sci. 7, 1221–1232 (1998).

    Article  CAS  Google Scholar 

  14. Eberhard, M., Tsai-Pflugfelder, M., Bolewska, K., Hommel, U. & Kirschener, K. Indoleglycerol phosphate syntase-phophoribosyl anthranilate isomerase: comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains. Biochemistry 34, 5419–5428 (1995).

    Article  CAS  Google Scholar 

  15. Hommel, U., Eberhard, M. & Kirschener, K. Phosphoribosyl anthranilate isomerase catalyzes a reversible Amadori reaction. Biochemistry 34, 5429– 5439 (1995).

    Article  CAS  Google Scholar 

  16. Stemmer, W. P. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl Acad. Sci. USA 91, 12747–10751 (1994).

    Article  Google Scholar 

  17. Stemmer, W. P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 ( 1994).

    Article  ADS  CAS  Google Scholar 

  18. Zhao, H. & Arnold, F. H. Optimization of DNA shuffling for high fidelity recombination. Nucleic Acids Res. 25, 1307–1308 (1997).

    Article  CAS  Google Scholar 

  19. Shao, Z., Zhao, H., Giver, L. & Arnold, F. H. Random-priming in vitro recombination: an effective tool for directed evolution. Nucleic Acids Res. 26, 681–683 (1998).

    Article  CAS  Google Scholar 

  20. Giver, L. & Arnold, F. H. Combinatorial protein design by in vitro recombination. Curr. Opin. Chem. Biol. 2, 335–338 (1998).

    Article  CAS  Google Scholar 

  21. Zhao, H., Giver, L., Shao, Z., Affholter, J. A. & Arnold, F. H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nature Biotechnol. 16, 258–261 (1998).

    Article  CAS  Google Scholar 

  22. Crameri, A., Whitehorn, E. A., Tate, E. & Stemmer, W. P. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnol. 14, 315–319 (1996).

    Article  CAS  Google Scholar 

  23. Crameri, A., Cwirla, S. & Stemmer, W. P. Construction and evolution of antibody-phage libraries by DNA shuffling. Nature Med. 2, 100– 102 (1996).

    Article  CAS  Google Scholar 

  24. Crameri, A., Raillard, S. A., Bermudez, E. & Stemmer, W. P. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288– 291 (1998).

    Article  ADS  CAS  Google Scholar 

  25. Tawfik, D. S. & Griffiths, A. D. Man-made cell-like compartments for molecular evolution. Nature Biotechnol. 16, 652–656 (1998).

    Article  CAS  Google Scholar 

  26. Kauffman, S. A. (ed.) The Origins of Order (Oxford Univ. Press, New York, 1993).

    Google Scholar 

  27. Wilmanns, M., Priestle, J. P., Niermann, T. & Jansonius, J. N. Three-dimensional structure of the bifunctional enzyme phophoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 Å resolution. J. Mol. Biol. 223, 477–507 (1992).

    Article  CAS  Google Scholar 

  28. Wilmanns, M., Hyde, C. C., Davies, D. R., Kirschener, K. & Jansonius, J. N. Structural conservation in parallel beta/alpha-barrel enzymes that catalyze three sequential reactions in the pathway of tryptophan biosynthesis. Biochemistry 30, 9161– 9169 (1991).

    Article  CAS  Google Scholar 

  29. Knöchel, T. R. et al. The crystal structure of indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus in three different crystal forms: effects of ionic strength. J. Mol. Biol. 262, 502–515 ( 1996).

    Article  Google Scholar 

  30. Luger, K., Hommel, U., Herold, M., Hofsteenge, J. & Kirschner, K. Correct folding of circularly permuted variants of a beta alpha barrel enzyme in vivo. Science 243 , 206–210 (1989).

    Article  ADS  CAS  Google Scholar 

  31. Stehlin, C., Dahm, A. & Kirschner, K. Deletion mutagenesis as a test of evolutionary relatedness of indoleglycerol phosphate synthase with other TIM barrel enzymes. FEBS Lett. 403, 268–272 (1997).

    Article  CAS  Google Scholar 

  32. Neidhart, D. J., Kenyon, G. L., Gerlt, J. A. & Petsko, G. A. Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous. Nature 347, 692–694 (1990).

    Article  ADS  CAS  Google Scholar 

  33. Altamirano, M. M., Golbik, R., Zahn, R., Buckle, A. M. & Fersht, A. R. Refolding chromatography with immobilized mini-chaperones. Proc. Natl Acad. Sci. USA 94, 3576– 3578 (1997).

    Article  ADS  CAS  Google Scholar 

  34. Clarke, L. Isolation of the centromere-linked CDC10 gene by complementation in yeast. Proc. Natl Acad. Sci. USA 77, 2173– 2177 (1980).

    Article  ADS  CAS  Google Scholar 

  35. Yanofsky, C., Horn, V., Bonner, M. & Stasiowki, S. Polarity and enzyme functions in mutants of the first three genes of the tryptophan operon of Escherichia coli. Genetics 69, 409–433 (1971).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Yanofsky, C. Tryptophan biosynthesis in Escherichia coli. Genetic determination of the proteins involved. J. Am. Med. Assoc. 218, 1026–1035 (1971).

    Article  CAS  Google Scholar 

  37. Sterner, R. et al. Phosphoribosyl anthranilate isomerase from Thermotaga maritima is an extremely stable and active homodimer. Protein Sci. 5, 2000–2008 ( 1996).

    Article  CAS  Google Scholar 

  38. Winter, G. & Milstein, C. Man-made antibodies. Nature 349, 293–299 ( 1991).

    Article  ADS  CAS  Google Scholar 

  39. Bisswanger, H., Kirschner, K., Cohn, W., Hager, V. & Hansson, E. N-(5-phosphoribosyl)anthranilate isomerase-indoleglycerol-phosphate synthase. 1. A substrate analogue binds to two different binding sites on the bifunctional enzyme from Escherichia coli. Biochemistry 18, 5946–5953 ( 1979).

    Article  CAS  Google Scholar 

  40. Sternberg, N. Display of peptides and proteins on the surface of bacteriophage λ. Proc. Natl. Acad. Sci. USA 92, 1609– 1613 (1995).

    Article  ADS  CAS  Google Scholar 

  41. Yanofsky, C. & Horn, V. Role of regulatory features of the Trp operon of Escherichia coli in mediating a response to a nutritional shift. J. Bacteriol. 176, 6245– 6254 (1994).

    Article  CAS  Google Scholar 

  42. Stephen, A., Gish, W., Miller, W., Myers, E. & Lipman, D. Basic local alignment search tool. J. Mol. Biol. 215 , 403–410 (1990).

    Article  Google Scholar 

Download references

Acknowledgements

M.M.A. was a Marie Curie-EU and an EMBO fellow; J.M.B. was a research fellow at Fitzwilliam College and thanks the Royal Society for a University Research Fellowship; C.A. was a Fundación UNAM fellow. We thank P. Barker for valuable advice and discussion, and the Visual Aids Department, especially A. Lenton, for invaluable help.

Author information

Author notes

  1. Jonathan M. Blackburn

    Present address: Department of Biochemistry, Cambridge University, 80 Tennis Court Road, Cambridge, CB2 1GA, UK

  2. Cristina Aguayo

    Present address: Facultad de Medicina, UNAM, P.O. Box 70-159, México City, 04510, D.F., México

  3. Alan R. Fersht: Correspondence and requests for material should be addressed to A.R.F.

Authors and Affiliations

  1. Cambridge Centre for Protein Engineering, and Cambridge University Chemical Laboratory, MRC Centre, Hills Road, Cambridge, CB2 2QH, UK

    Myriam M. Altamirano, Jonathan M. Blackburn, Cristina Aguayo & Alan R. Fersht

  2. Departamento de Bioquímica, Facultad de Medicina, UNAM, P. O. Box 70-159, México City, 04510 D. F., México

    Myriam M. Altamirano

  3. Departamento de Bioquímica, Instituto de Fisiología Celular, UNAM, P. O. Box 70-242, México City, 04510, D.F. México

    Myriam M. Altamirano

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Altamirano, M., Blackburn, J., Aguayo, C. et al. Directed evolution of new catalytic activity using the α/β-barrel scaffold. Nature 403, 617–622 (2000). https://doi.org/10.1038/35001001

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