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.

Computational redesign of enzymes for regio- and enantioselective hydroamination

Abstract

Introduction of innovative biocatalytic processes offers great promise for applications in green chemistry. However, owing to limited catalytic performance, the enzymes harvested from nature's biodiversity often need to be improved for their desired functions by time-consuming iterative rounds of laboratory evolution. Here we describe the use of structure-based computational enzyme design to convert Bacillus sp. YM55-1 aspartase, an enzyme with a very narrow substrate scope, to a set of complementary hydroamination biocatalysts. The redesigned enzymes catalyze asymmetric addition of ammonia to substituted acrylates, affording enantiopure aliphatic, polar and aromatic β-amino acids that are valuable building blocks for the synthesis of pharmaceuticals and bioactive compounds. Without a requirement for further optimization by laboratory evolution, the redesigned enzymes exhibit substrate tolerance up to a concentration of 300 g/L, conversion up to 99%, β-regioselectivity >99% and product enantiomeric excess >99%. The results highlight the use of computational design to rapidly adapt an enzyme to industrially viable reactions.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Computational active site redesign of AspB.
Fig. 2: Computational redesign of AspB for (R)-β-aminobutanoic acid synthesis.
Fig. 3: Computational redesign of AspB for (R)-β-aminopentanoic acid synthesis.
Fig. 4: Computational redesign of AspB for (S)-β-asparagine synthesis.
Fig. 5: Computational redesign of AspB for (S)-β-phenylalanine synthesis.

References

  1. Bornscheuer, U. T. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Reetz, M. T. Biocatalysis in organic chemistry and biotechnology: past, present, and future. J. Am. Chem. Soc. 135, 12480–12496 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Nestl, B. M., Hammer, S. C., Nebel, B. A. & Hauer, B. New generation of biocatalysts for organic synthesis. Angew. Chem. Int. Ed. Engl. 53, 3070–3095 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Savile, C. K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Pavlidis, I. V. et al. Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines. Nat. Chem. 8, 1076–1082 (2016).

    Article  CAS  PubMed  Google Scholar 

  6. Abrahamson, M. J., Vázquez-Figueroa, E., Woodall, N. B., Moore, J. C. & Bommarius, A. S. Development of an amine dehydrogenase for synthesis of chiral amines. Angew. Chem. Int. Ed. Engl. 51, 3969–3972 (2012).

    Article  CAS  PubMed  Google Scholar 

  7. Mutti, F. G., Knaus, T., Scrutton, N. S., Breuer, M. & Turner, N. J. Conversion of alcohols to enantiopure amines through dual-enzyme hydrogen-borrowing cascades. Science 349, 1525–1529 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kille, S., Zilly, F. E., Acevedo, J. P. & Reetz, M. T. Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution. Nat. Chem. 3, 738–743 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Kan, S. B., Lewis, R. D., Chen, K. & Arnold, F. H. Directed evolution of cytochrome c for carbon-silicon bond formation: bringing silicon to life. Science 354, 1048–1051 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 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 

  11. Jochens, H. & Bornscheuer, U. T. Natural diversity to guide focused directed evolution. ChemBioChem 11, 1861–1866 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Bendl, J. et al. HotSpot Wizard 2.0: automated design of site-specific mutations and smart libraries in protein engineering. Nucleic Acids Res. 44(W1), W479–W487 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Nobili, A. et al. Simultaneous use of in silico design and a correlated mutation network as a tool to efficiently guide enzyme engineering. ChemBioChem 16, 805–810 (2015).

    Article  CAS  PubMed  Google Scholar 

  14. Lutz, S. Beyond directed evolution—semi-rational protein engineering and design. Curr. Opin. Biotechnol. 21, 734–743 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sebestova, E., Bendl, J., Brezovsky, J. & Damborsky, J. Computational tools for design smart libraries. in Directed Evolution Library Creation (Springer, New York, 2014).

  16. Ebert, M. C. & Pelletier, J. N. Computational tools for enzyme improvement: why everyone can — and should — use them. Curr. Opin. Chem. Biol. 37, 89–96 (2017).

    Article  CAS  PubMed  Google Scholar 

  17. Santiago, G. et al. Computer-aided laccase engineering: towards biological oxidation of arylamines. ACS Catal. 6, 5415–5423 (2016).

    Article  CAS  Google Scholar 

  18. Moroz, Y. S. et al. New tricks for old proteins: single mutations in a nonenzymatic protein give rise to various enzymatic activities. J. Am. Chem. Soc. 137, 14905–14911 (2015).

    Article  CAS  PubMed  Google Scholar 

  19. Romero-Rivera, A., Garcia-Borràs, M. & Osuna, S. Computational tools for the evaluation of laboratory-engineered biocatalysts. Chem. Commun. (Camb.) 53, 284–297 (2016).

    Article  CAS  Google Scholar 

  20. Huang, P. S., Boyken, S. E. & Baker, D. The coming of age of de novo protein design. Nature 537, 320–327 (2016).

    Article  CAS  PubMed  Google Scholar 

  21. Constable, D. J. C. et al. Key green chemistry research areas – a perspective from pharmaceutical manufacturers. Green Chem. 9, 411–420 (2007).

    Article  CAS  Google Scholar 

  22. Kudo, F., Miyanaga, A. & Eguchi, T. Biosynthesis of natural products containing β-amino acids. Nat. Prod. Rep. 31, 1056–1073 (2014).

    Article  CAS  PubMed  Google Scholar 

  23. Ashfaq, M. et al. Enantioselective synthesis of β-amino acids: a review. Med. Chem. 5, 295–309 (2015).

    Article  CAS  Google Scholar 

  24. Liljeblad, A. & Kanerva, L. T. Biocatalysis as a profound tool in the preparation of highly enantiopure β-amino acids. Tetrahedron 62, 5831–5854 (2006).

    Article  CAS  Google Scholar 

  25. Rehdorf, J., Mihovilovic, M. D. & Bornscheuer, U. T. Exploiting the regioselectivity of Baeyer-Villiger monooxygenases for the formation of β-amino acids and β-amino alcohols. Angew. Chem. Int. Ed. Engl. 49, 4506–4508 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Zhang, D. et al. Development of β-amino acid dehydrogenase for the synthesis of β-amino acids via reductive amination of β-keto acids. ACS Catal. 5, 2220–2224 (2015).

    Article  CAS  Google Scholar 

  27. Turner, N. J. Ammonia lyases and aminomutases as biocatalysts for the synthesis of α-amino and β-amino acids. Curr. Opin. Chem. Biol. 15, 234–240 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Weise, N. J., Parmeggiani, F., Ahmed, S. T. & Turner, N. J. The bacterial ammonia lyase EncP: a tunable biocatalyst for the synthesis of unnatural amino acids. J. Am. Chem. Soc. 137, 12977–12983 (2015).

    Article  CAS  PubMed  Google Scholar 

  29. Kawata, Y. et al. Cloning and over-expression of thermostable Bacillus sp. YM55-1 aspartase and site-directed mutagenesis for probing a catalytic residue. Eur. J. Biochem. 267, 1847–1857 (2000).

    Article  CAS  PubMed  Google Scholar 

  30. Parmeggiani, F., Weise, N. J., Ahmed, S. T. & Turner, N. J. Synthetic and therapeutic applications of ammonia-lyases and aminomutases. Chem. Rev. 118, 73–118 (2018).

    Article  CAS  PubMed  Google Scholar 

  31. Viola, R. E. L-Aspartase: new tricks from an old enzyme. Adv. Enzymol. 74, 295–341 (2000).

    CAS  PubMed  Google Scholar 

  32. Renata, H., Wang, Z. J. & Arnold, F. H. Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution. Angew. Chem. Int. Ed. Engl. 54, 3351–3367 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Levin, K. B. et al. Following evolutionary paths to protein-protein interactions with high affinity and selectivity. Nat. Struct. Mol. Biol. 16, 1049–1055 (2009).

    Article  CAS  PubMed  Google Scholar 

  34. Asano, Y., Kira, I. & Yokozeki, K. Alteration of substrate specificity of aspartase by directed evolution. Biomol. Eng. 22, 95–101 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Vogel, A., Schmiedel, R., Hofmann, U., Gruber, K. & Zangger, K. Converting aspartase into a β-amino acid lyase by cluster screening. ChemCatChem 6, 965–968 (2014).

    Article  CAS  Google Scholar 

  36. Wu, B. et al. Versatile peptide C-terminal functionalization via a computationally engineered peptide amidase. ACS Catal. 6, 5405–5414 (2016).

    Article  CAS  Google Scholar 

  37. Wijma, H. J. et al. Enantioselective enzymes by computational design and in silico screening. Angew. Chem. Int. Ed. Engl. 54, 3726–3730 (2015).

    Article  CAS  PubMed  Google Scholar 

  38. Fibriansah, G., Veetil, V. P., Poelarends, G. J. & Thunnissen, A. M. Structural basis for the catalytic mechanism of aspartate ammonia lyase. Biochemistry 50, 6053–6062 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Zhang, J. & Liu, Y. A QM/MM study of the catalytic mechanism of aspartate ammonia lyase. J. Mol. Graph. Model. 51, 113–119 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Cohen, Y., Vaknin, M. & Mauch-Mani, B. BABA-induced resistance: milestones along a 55-year journey. Phytoparasitica 44, 513–538 (2016).

    Article  CAS  Google Scholar 

  41. Lima-Ramos, J., Neto, W. & Woodley, J. M. Engineering of biocatalysts and biocatalytic processes. Top. Catal. 57, 301–320 (2014).

    Article  CAS  Google Scholar 

  42. Huisman, G. W. & Collier, S. J. On the development of new biocatalytic processes for practical pharmaceutical synthesis. Curr. Opin. Chem. Biol. 17, 284–292 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Zhang, W. et al. Total synthesis and reassignment of stereochemistry of obyanamide. Tetrahedron 62, 9966–9972 (2006).

    Article  CAS  Google Scholar 

  44. Zhao, R. et al. Inhibition of the Bcl-xL deamination pathway in myeloproliferative disorders. N. Engl. J. Med. 359, 2778–2789 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Fortin, P. D., Walsh, C. T. & Magarvey, N. A. A transglutaminase homologue as a condensation catalyst in antibiotic assembly lines. Nature 448, 824–827 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Grayson, J. I., Roos, J. & Osswald, S. Development of a commercial process for (S)-β-phenylalanine. Org. Process Res. Dev. 15, 1201–1206 (2011).

    Article  CAS  Google Scholar 

  47. Owen, R. T. Dapoxetine: a novel treatment for premature ejaculation. Drugs Today (Barc.) 45, 669–678 (2009).

    Article  CAS  Google Scholar 

  48. Wu, B. et al. Mechanism-inspired engineering of phenylalanine aminomutase for enhanced β-regioselective asymmetric amination of cinnamates. Angew. Chem. Int. Ed. Engl. 51, 482–486 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Eisenthal, R., Danson, M. J. & Hough, D. W. Catalytic efficiency and k cat /K M: a useful comparator? Trends Biotechnol. 25, 247–249 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Fox, R. J. & Clay, M. D. Catalytic effectiveness, a measure of enzyme proficiency for industrial applications. Trends Biotechnol. 27, 137–140 (2009).

    Article  CAS  PubMed  Google Scholar 

  51. 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 

  52. Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Ericsson, U. B., Hallberg, B. M., Detitta, G. T., Dekker, N. & Nordlund, P. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal. Biochem. 357, 289–298 (2006).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank W. Szymanski for discussions. We thank for the 100 Talent Program grant (B.W.) and Biological Resources Service Network Initiative (ZSYS-012; B.W.) and a grant (SKT1604; C.Y.L.) from the Chinese Academy of Sciences, Natural Science Foundation of China grants (31601412 (B.W.), 21603013 (C.Y.L.)), and a BE-Basic grant (H.J.W. and D.B.J.) from the Dutch Ministry of Economic Affairs for the financial support.

Author information

Authors and Affiliations

Authors

Contributions

D.B.J. and B.W. initiated the project. B.W., H.J.W. and Y. Cui performed the computational work. L.S., R.L., M.O., Y. Tian, J.D., T.L., D.N., Y. Chen and J.F. performed biocatalytic experiments. J.H., H.C. and Y. Tao developed high-density fermentation methods. R.L. performed preparative-scale synthesis of the amino acids. D.B.J. and B.W. provided supervision and input on experimental design and wrote the manuscript, which was revised and approved by all authors. R.L., H.J.W. and L.S. contributed equally to this work.

Corresponding authors

Correspondence to Dick B. Janssen or Bian Wu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–17, Supplementary Tables 1–14

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, R., Wijma, H.J., Song, L. et al. Computational redesign of enzymes for regio- and enantioselective hydroamination. Nat Chem Biol 14, 664–670 (2018). https://doi.org/10.1038/s41589-018-0053-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0053-0

Further reading

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