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The use of ene adducts to study and engineer enoyl-thioester reductases

Nature Chemical Biology volume 11, pages 398–400 (2015)Cite this article

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Abstract

An improved understanding of enzymes' catalytic proficiency and stereoselectivity would further enable applications in chemistry, biocatalysis and industrial biotechnology. We use a chemical probe to dissect individual catalytic steps of enoyl-thioester reductases (Etrs), validating an active site tyrosine as the cryptic proton donor and explaining how it had eluded definitive identification. This information enabled the rational redesign of Etr, yielding mutants that create products with inverted stereochemistry at wild type–like turnover frequency.

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Figure 1: Proposed reaction mechanisms of enoyl-thioester reductases.
Figure 2: Identification and characterization of the proton donation–deficient Etr1pY79F mutant.
Figure 3: Screening of Etr1p mutants for si-face proton donors using the C2-ene adduct as a molecular probe.

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References

  1. Kiss, G., Celebi-Olcum, N., Moretti, R., Baker, D. & Houk, K.N. Angew. Chem. Int. Edn Engl. 52, 5700–5725 (2013).

    Article  CAS  Google Scholar 

  2. Baker, D. Protein Sci. 19, 1817–1819 (2010).

    Article  CAS  Google Scholar 

  3. Jiang, L. et al. Science 319, 1387–1391 (2008).

    Article  CAS  Google Scholar 

  4. Siegel, J.B. et al. Science 329, 309–313 (2010).

    Article  CAS  Google Scholar 

  5. Hedlund, J., Jornvall, H. & Persson, B. BMC Bioinformatics 11, 534 (2010).

    Article  Google Scholar 

  6. Chen, Z.J. et al. J. Mol. Biol. 379, 830–844 (2008).

    Article  CAS  Google Scholar 

  7. Zheng, J., Gay, D.C., Demeler, B., White, M.A. & Keatinge-Clay, A.T. Nat. Chem. Biol. 8, 615–621 (2012).

    Article  CAS  Google Scholar 

  8. Airenne, T.T. et al. J. Mol. Biol. 327, 47–59 (2003).

    Article  CAS  Google Scholar 

  9. Kwan, D.H. & Leadlay, P.F. ACS Chem. Biol. 5, 829–838 (2010).

    Article  CAS  Google Scholar 

  10. Mesa, J. et al. Chem. Biol. Interact. 10.1016/j.cbi.2015.01.021 (22 January 2015).

  11. Wu, Y.H. et al. Structure 16, 1714–1723 (2008).

    Article  CAS  Google Scholar 

  12. Libby, R.D. & Mehl, R.A. Bioorg. Chem. 40, 57–66 (2012).

    Article  CAS  Google Scholar 

  13. Rosenthal, R.G. et al. Nat. Chem. Biol. 10, 50–55 (2014).

    Article  CAS  Google Scholar 

  14. Hamilton, G.A. in Progress in Bioorganic Chemistry (eds. Kaiser, E.T. and Kezdy, T.J.) 83–157 (Wiley Interscience, New York, 1971).

  15. Bryan, P., Pantoliano, M.W., Quill, S.G., Hsiao, H.Y. & Poulos, T. Proc. Natl. Acad. Sci. USA 83, 3743–3745 (1986).

    Article  CAS  Google Scholar 

  16. Fersht, A.R. et al. Nature 314, 235–238 (1985).

    Article  CAS  Google Scholar 

  17. Kwan, D.H. et al. Chem. Biol. 15, 1231–1240 (2008).

    Article  CAS  Google Scholar 

  18. Toogood, H.S. & Scrutton, N.S. Curr. Opin. Chem. Biol. 19, 107–115 (2014).

    Article  CAS  Google Scholar 

  19. Zheng, J., Piasecki, S.K. & Keatinge-Clay, A.T. ACS Chem. Biol. 8, 1964–1971 (2013).

    Article  CAS  Google Scholar 

  20. Rafi, S. et al. J. Biol. Chem. 281, 39285–39293 (2006).

    Article  CAS  Google Scholar 

  21. Maier, T., Leibundgut, M. & Ban, N. Science 321, 1315–1322 (2008).

    Article  CAS  Google Scholar 

  22. Erb, T.J. et al. Proc. Natl. Acad. Sci. USA 104, 10631–10636 (2007).

    Article  CAS  Google Scholar 

  23. Erb, T.J., Brecht, V., Fuchs, G., Muller, M. & Alber, B.E. Proc. Natl. Acad. Sci. USA 106, 8871–8876 (2009).

    Article  CAS  Google Scholar 

  24. Tabor, S. & Richardson, C.C. Proc. Natl. Acad. Sci. USA 82, 1074–1078 (1985).

    Article  CAS  Google Scholar 

  25. Studier, F.W. Protein Expr. Purif. 41, 207–234 (2005).

    Article  CAS  Google Scholar 

  26. Dawson, R.M.C. Data for Biochemical Research (Clarendon Press, Oxford, 1986).

  27. Torkko, J.M. et al. J. Biol. Chem. 278, 41213–41220 (2003).

    Article  CAS  Google Scholar 

  28. Kabsch, W. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  29. Mccoy, A.J. et al. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  30. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  31. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  32. Painter, J. & Merritt, E.A. Acta Crystallogr. D Biol. Crystallogr. 62, 439–450 (2006).

    Article  Google Scholar 

  33. Peyraud, R. et al. Proc. Natl. Acad. Sci. USA 106, 4846–4851 (2009).

    Article  CAS  Google Scholar 

  34. Hoops, S. et al. Bioinformatics 22, 3067–3074 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the Swiss National Science Foundation (SNF-Ambizione program PZ00P3_136828/1; granted to T.J.E.).

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Author notes

  1. Raoul G Rosenthal and Bastian Vögeli: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Microbiology, ETH (Swiss Federal Institute of Technology) Zurich, Zurich, Switzerland

    Raoul G Rosenthal, Patrick Kiefer, Julia A Vorholt & Tobias J Erb

  2. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

    Bastian Vögeli & Tobias J Erb

  3. Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland

    Nick Quade

  4. Paul Scherrer Institute, Villigen, Switzerland

    Guido Capitani

  5. Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland

    Marc-Olivier Ebert

Authors
  1. Raoul G Rosenthal
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Contributions

R.G.R., B.V. and T.J.E. conceived and designed all experiments, with the exception of the NMR experiments, which were designed together with M.-O.E., and the MS analyses, which were designed together with P.K. and J.A.V. NMR experiments were performed by R.G.R., B.V. and M.-O.E. MS experiments were performed by B.V., R.G.R. and P.K. B.V. prepared enzyme crystals of Etr1p and mutants, N.Q. and G.C. collected the diffraction data and interpreted the results. Enzyme kinetic assays and stopped-flow measurements, as well as purification of the intermediate, were performed by R.G.R. and B.V. R.G.R., B.V. and T.J.E. wrote the paper.

Corresponding author

Correspondence to Tobias J Erb.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Tables 1–5 and Supplementary Figures 1–8 (PDF 1928 kb)

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Rosenthal, R., Vögeli, B., Quade, N. et al. The use of ene adducts to study and engineer enoyl-thioester reductases. Nat Chem Biol 11, 398–400 (2015). https://doi.org/10.1038/nchembio.1794

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