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.

  • Article
  • Published:

Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase

Nature Chemistry volume 9, pages 50–56 (2017)Cite this article

Subjects

Abstract

Designing catalysts that achieve the rates and selectivities of natural enzymes is a long-standing goal in protein chemistry. Here, we show that an ultrahigh-throughput droplet-based microfluidic screening platform can be used to improve a previously optimized artificial aldolase by an additional factor of 30 to give a >109 rate enhancement that rivals the efficiency of class I aldolases. The resulting enzyme catalyses a reversible aldol reaction with high stereoselectivity and tolerates a broad range of substrates. Biochemical and structural studies show that catalysis depends on a Lys-Tyr-Asn-Tyr tetrad that emerged adjacent to a computationally designed hydrophobic pocket during directed evolution. This constellation of residues is poised to activate the substrate by Schiff base formation, promote mechanistically important proton transfers and stabilize multiple transition states along a complex reaction coordinate. The emergence of such a sophisticated catalytic centre shows that there is nothing magical about the catalytic activities or mechanisms of naturally occurring enzymes, or the evolutionary process that gave rise to them.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Substrates and inhibitor for the computationally designed aldolase.
Figure 2: Microfluidic platform for the directed evolution of aldolases.
Figure 3: Evolution and structural analysis of the optimized RA95.5-8F aldolase.
Figure 4: Hypothetical mechanism for aldol cleavage by the evolved enzyme.

Similar content being viewed by others

References

  1. Gefflaut, T., Blonski, C., Perie, J. & Willson, M. Class I aldolases: substrate specificity, mechanism, inhibitors and structural aspects. Prog. Biophys. Mol. Biol. 63, 301–340 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Dean, S. M., Greenberg, W. A. & Wong, C.-H. Recent advances in aldolase-catalyzed asymmetric synthesis. Adv. Synth. Catal. 349, 1308–1320 (2007).

    Article  CAS  Google Scholar 

  3. Clapés, P., Fessner, W.-D., Sprenger, G. A. & Samland, A. K. Recent progress in stereoselective synthesis with aldolases. Curr. Opin. Chem. Biol. 14, 154–167 (2010).

    Article  PubMed  CAS  Google Scholar 

  4. Windle, C. L., Müller, M., Nelson, A. & Berry, A. Engineering aldolases as biocatalysts. Curr. Opin. Chem. Biol. 19, 25–33 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Mukherjee, S., Yang, J. W., Hoffmann, S. & List, B. Asymmetric enamine catalysis. Chem. Rev. 107, 5471–5569 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Müller, M. M., Windsor, M. A., Pomerantz, W. C., Gellman, S. H. & Hilvert, D. A rationally designed aldolase foldamer. Angew. Chem. Int. Ed. 48, 922–925 (2009).

    Article  CAS  Google Scholar 

  7. Tanaka, F., Fuller, R. & Barbas III, C. F. Development of small designer aldolase enzymes: catalytic activity, folding, and substrate specificity. Biochemistry 44, 7583–7592 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Wagner, J., Lerner, R. A. & Barbas III, C. F. Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes. Science 270, 1797–1800 (1995).

    Article  CAS  PubMed  Google Scholar 

  9. Barbas III, C. F. et al. Immune versus natural selection: antibody aldolases with enzymic rates but broader scope. Science 278, 2085–2092 (1997).

    Article  Google Scholar 

  10. Jiang, L. et al. De novo computational design of retro-aldol enzymes. Science 319, 1387–1391 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Althoff, E. A. et al. Robust design and optimization of retroaldol enzymes. Protein Sci. 21, 717–726 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Giger, L. et al. Evolution of a designed retro-aldolase leads to complete active site remodeling. Nat. Chem. Biol. 9, 494–498 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Guo, M. T., Rotem, A., Heyman, J. A. & Weitz, D. A. Droplet microfluidics for high-throughput biological assays. Lab Chip 12, 2146–2155 (2012).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  15. Agresti, J. J. et al. Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proc. Natl Acad. Sci. USA 107, 4004–4009 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Baret, J.-C. et al. Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. Lab Chip 9, 1850–1858 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Kintses, B. et al. Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution. Chem. Biol. 19, 1001–1009 (2012).

    Article  CAS  PubMed  Google Scholar 

  18. Frenz, L., Blank, K., Brouzes, E. & Griffiths, A. D. Reliable microfluidic on-chip incubation of droplets in delay-lines. Lab Chip 9, 1344–1348 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Blomberg, R. et al. Precision is essential for efficient catalysis in an evolved Kemp eliminase. Nature 503, 418–421 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Albery, W. J. & Knowles, J. R. Evolution of enzyme function and the development of catalytic efficiency. Biochemistry 15, 5631–5640 (1976).

    Article  CAS  PubMed  Google Scholar 

  21. Esposito, G. et al. Structural and functional analysis of aldolase B mutants related to hereditary fructose intolerance. FEBS Lett. 531, 152–156 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Heine, A. et al. Observation of covalent intermediates in an enzyme mechanism at atomic resolution. Science 294, 369–374 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Henn-Sax, M., Höcker, B., Wilmanns, M. & Sterner, R. Divergent evolution of (βα)8-barrel enzymes. Biol. Chem. 382, 1315–1320 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Nagano, N., Orengo, C. A. & Thornton, J. M. One fold with many functions: the evolutionary relationships between TIM barrel families based on their sequences, structures and functions. J. Mol. Biol. 321, 741–765 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Galkin, A. et al. Characterization, kinetics, and crystal structures of fructose-1,6-bisphosphate aldolase from the human parasite, Giardia lamblia. J. Biol. Chem. 282, 4859–4867 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Lorentzen, E., Siebers, B., Hensel, R. & Pohl, E. Mechanism of the Schiff base forming fructose-1,6-bisphosphate aldolase: structural analysis of reaction intermediates. Biochemistry 44, 4222–4229 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Lassila, J. K., Baker, D. & Herschlag, D. Origins of catalysis by computationally designed retroaldolase enzymes. Proc. Natl Acad. Sci. USA 107, 4937–4942 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Meyer, H.-P. et al. The use of enzymes in organic synthesis and the life sciences: perspectives from the Swiss Industrial Biocatalysis Consortium (SIBC). Catal. Sci. Technol. 3, 29–40 (2013).

    Article  CAS  Google Scholar 

  29. Rajagopalan, S. et al. Design of activated serine-containing catalytic triads with atomic-level accuracy. Nat. Chem. Biol. 10, 386–391 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Glasner, M. E., Gerlt, J. A. & Babbitt, P. C. Mechanisms of protein evolution and their application to protein engineering. Adv. Enzymol. Areas Mol. Biol. 75, 193–239 (2007).

    CAS  Google Scholar 

  31. Garrabou, X., Beck, T. & Hilvert, D. A promiscuous de novo retro-aldolase catalyzes asymmetric Michael additions via Schiff base intermediates. Angew. Chem. Int. Ed. 54, 5609–5612 (2015).

    Article  CAS  Google Scholar 

  32. Garrabou, X., Wicky, B. I. M. & Hilvert, D. Fast Knoevenagel condensations catalyzed by an artificial Schiff-base-forming enzyme. J. Am. Chem. Soc. 138, 6972–6974 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Hilvert, D. Design of protein catalysts. Annu. Rev. Biochem. 82, 447–470 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. Taylor, S. V., Kast, P. & Hilvert, D. Investigating and engineering enzymes by genetic selection. Angew. Chem. Int. Ed. 40, 3310–3335 (2001).

    Article  Google Scholar 

  35. Yang, G. & Withers, S. G. Ultrahigh-throughput FACS-based screening for directed enzyme evolution. ChemBioChem 10, 2704–2715 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Zinchenko, A. et al. One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution. Anal. Chem. 86, 2526–2533 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chen, B. et al. High-throughput analysis and protein engineering using microcapillary arrays. Nat. Chem. Biol. 12, 76–81 (2016).

    Article  CAS  PubMed  Google Scholar 

  38. Neylon, C. Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. Nucleic Acids Res. 32, 1448–1459 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68 (1989).

    Article  CAS  PubMed  Google Scholar 

  40. Zhao, H. & Zha, W. In vitro ‘sexual’ evolution through the PCR-based staggered extension process (StEP). Nat. Protocols 1, 1865–1871 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  42. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Emsley, P. & Cowtan, K. COOT: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  PubMed  CAS  Google Scholar 

  46. Schuttelkopf, A. W. & van Aalten, D. M. F. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D 60, 1355–1363 (2004).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Stutz-Ducommun and B. Blattmann from the Protein Crystallization Core Facility at the University of Zurich and the staff at the Swiss Light Source (Paul Scherrer Institute) for outstanding technical support, as well as E. Mayot for surfactant synthesis. We are grateful to the mass spectrometry service of the Laboratory of Organic Chemistry at ETHZ and the Protein Analysis Group at the Functional Genomics Center of the University of Zurich. We thank A. Green, R. Zschoche, M. Reichen, R. Calbrix and all the members of the Hilvert and Griffiths labs for fruitful discussions. This work was generously supported by the Swiss National Science Foundation (D.H.).

Author information

Author notes

  1. Richard Obexer and Alexei Godina: These authors contributed equally to the work

Authors and Affiliations

  1. Laboratory of Organic Chemistry, ETH Zürich, Zürich, 8093, Switzerland

    Richard Obexer, Alexei Godina, Xavier Garrabou & Donald Hilvert

  2. Laboratory of Biochemistry, École Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), CNRS UMR 8231, 10, rue Vauquelin, Paris Cedex 05, 75231, France

    Alexei Godina & Andrew D. Griffiths

  3. Department of Biochemistry, University of Zürich, Zürich, 8057, Switzerland

    Peer R. E. Mittl

  4. Department of Biochemistry, University of Washington, Seattle, 98195, Washington, USA.

    David Baker

Authors
  1. Richard Obexer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexei Godina
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xavier Garrabou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peer R. E. Mittl
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Andrew D. Griffiths
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Donald Hilvert
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.H., A.D.G., D.B., P.R.E.M., X.G., A.G. and R.O. designed the experiments. R.O. and A.G. developed the assay and evolved retro-aldolases. R.O. and X.G. biochemically characterized the variants. X.G. performed biocatalytic aldol reactions and the synthesis of standards. P.R.E.M. and R.O. crystallized the proteins and solved their structures. The manuscript and figures were prepared by R.O., X.G., A.D.G. and D.H.

Corresponding authors

Correspondence to Andrew D. Griffiths or Donald Hilvert.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 10130 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Obexer, R., Godina, A., Garrabou, X. et al. Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase. Nature Chem 9, 50–56 (2017). https://doi.org/10.1038/nchem.2596

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.2596

This article is cited by

Search

Advanced 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