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Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids

Nature Chemistry volume 4, pages 478–484 (2012)Cite this article

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Abstract

The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyses the conversion of 3-methylaspartate to ammonia and 2-methylfumarate) to accept a variety of substituted amines and fumarates and catalyse the asymmetric synthesis of aspartic acid derivatives. We obtained two single-active-site mutants, one exhibiting a wide nucleophile scope including structurally diverse linear and cyclic alkylamines and one with broad electrophile scope including fumarate derivatives with alkyl, aryl, alkoxy, aryloxy, alkylthio and arylthio substituents at the C2 position. Both mutants have an enlarged active site that accommodates the new substrates while retaining the high stereo- and regioselectivity of the wild-type enzyme. As an example, we demonstrate a highly enantio- and diastereoselective synthesis of threo-3-benzyloxyaspartate (an important inhibitor of neuronal excitatory glutamate transporters in the brain).

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Figure 1: Crystal structure of wild-type MAL in complex with the natural substrate threo-(2S,3S)-3-methylaspartate.
Figure 2: Enzyme-catalysed transformations under optimized reaction conditions.
Figure 3: Structural comparison of wild-type MAL and the Gln73Ala and Leu384Ala mutants.
Figure 4: Molecular docking of substrates in the active sites of wild-type MAL and the two MAL mutants.

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References

  1. Sonke, T., Kaptein, B. & Schoemaker, H. E. Use of enzymes in the synthesis of amino acids, in Amino Acids, Peptides and Proteins in Organic Chemistry Vol. 1 (ed. Hughes, A. B.) 77–117 (Wiley-VCH, 2009).

    Google Scholar 

  2. Wohlgemuth, R. Biocatalysis—key to sustainable industrial chemistry. Curr. Opin. Biotechnol. 21, 713–724 (2010).

    Article  CAS  Google Scholar 

  3. Panke, S. & Wubbolts, M. Advances in biocatalytic synthesis of pharmaceutical intermediates. Curr. Opin. Chem. Biol. 9, 188–194 (2005).

    Article  CAS  Google Scholar 

  4. Panke, S., Held, M. & Wubbolts, M. Trends and innovations in industrial biocatalysis for the production of fine chemicals. Curr. Opin. Biotechnol. 15, 272–279 (2004).

    Article  CAS  Google Scholar 

  5. 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  Google Scholar 

  6. Barker, H. A., Smyth, R. D., Wilson, R. M. & Weissbach, H. The purification and properties of β-methylaspartase. J. Biol. Chem. 234, 320–328 (1959).

    CAS  PubMed  Google Scholar 

  7. Goda, S. K., Minton, N. P., Botting, N. P. & Gani, D. Cloning, sequencing, and expression in Escherichia coli of the Clostridium tetanomorphum gene encoding β-methylaspartase and characterization of the recombinant protein. Biochemistry 31, 10747–10756 (1992).

    Article  CAS  Google Scholar 

  8. Raj, H. et al. Alteration of the diastereoselectivity of 3-methylaspartate ammonia lyase by using structure-based mutagenesis. ChemBioChem 10, 2236–2245 (2009).

    Article  CAS  Google Scholar 

  9. Bright, H. J. Divalent metal activation of β-methylaspartase. The importance of ionic radius. Biochemistry 6, 1191–1203 (1967).

    Article  CAS  Google Scholar 

  10. Bright, H. J. On the mechanism of divalent metal activation of β-methylaspartase. J. Biol. Chem. 240, 1198–1210 (1965).

    CAS  PubMed  Google Scholar 

  11. Kato, Y. & Asano, Y. 3-Methylaspartate ammonia-lyase as a marker enzyme of the mesaconate pathway for (S)-glutamate fermentation in Enterobacteriaceae. Arch. Microbiol. 168, 457–463 (1997).

    Article  CAS  Google Scholar 

  12. Asuncion, M., Blankenfeldt, W., Barlow, J. N., Gani, D. & Naismith, J. H. The structure of 3-methylaspartase from Clostridium tetanomorphum functions via the common enolase chemical step. J. Biol. Chem. 277, 8306–8311 (2002).

    Article  CAS  Google Scholar 

  13. Levy, C. W. et al. Insights into enzyme evolution revealed by the structure of methylaspartate ammonia lyase. Structure 10, 105–113 (2002).

    Article  CAS  Google Scholar 

  14. Bright, H. J., Ingraham, L. L. & Lundin, R. E. The mechanism of the methylaspartate ammonia–lyase reaction: deuterium exchange. Biochim. Biophys. Acta 81, 576–584 (1964).

    CAS  PubMed  Google Scholar 

  15. Bright, H. The mechanism of the β-methylaspartase reaction. J. Biol. Chem. 239, 2307–2315 (1964).

    CAS  PubMed  Google Scholar 

  16. Babbitt, P. C. et al. The enolase superfamily: a general strategy for enzyme-catalyzed abstraction of the α-protons of carboxylic acids. Biochemistry 35, 16489–16501 (1996).

    Article  CAS  Google Scholar 

  17. Akhtar, M., Botting, N. P., Cohen, M. A. & Gani, D. Enantiospecific synthesis of 3-substituted aspartic acids via enzymic amination of substituted fumaric acids. Tetrahedron 43, 5899–5908 (1987).

    Article  CAS  Google Scholar 

  18. Botting, N. P., Akhtar, M., Cohen, M. A. & Gani, D. Substrate specificity of the 3-methylaspartate ammonia-lyase reaction: observation of differential relative reaction rates for substrate-product pairs. Biochemistry 27, 2953–2955 (1988).

    Article  CAS  Google Scholar 

  19. Gulzar, M. S., Akhtar, M. & Gani, D. Preparation of N-substituted aspartic acids via enantiospecific conjugate addition of N-nucleophiles to fumaric acids using methylaspartase: synthetic utility and mechanistic implications. J. Chem. Soc. Perkin Trans. 1, 649–656 (1997).

    Article  Google Scholar 

  20. Lutz, S. & Bornscheuer, U. T. Protein Engineering Handbook. Vols 1–2 (Wiley-VCH, 2009).

    Google Scholar 

  21. Reetz, M. T., Bocola, M., Carballeira, J. D., Zha, D. & Vogel, A. Expanding the range of substrate acceptance of enzymes: combinatorial active-site saturation test. Angew. Chem. Int. Ed. 44, 4192–4196 (2005).

    Article  CAS  Google Scholar 

  22. Morley, K. L. & Kazlauskas, R. J. Improving enzyme properties: when are closer mutations better? Trends Biotechnol. 23, 231–237 (2005).

    Article  CAS  Google Scholar 

  23. Arnold, F. H. & Georgiou, G. (eds) Methods in Molecular Biology (Directed Enzyme Evolution) Vol. 230 (Humana Press, 2003).

    Google Scholar 

  24. Turner, N. J. Directed evolution drives the next generation of biocatalysts. Nature Chem. Biol. 5, 567–573 (2009).

    Article  CAS  Google Scholar 

  25. Shimamoto, K. et al. Characterization of novel L-threo-β-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters. Mol. Pharmacol. 65, 1008–1015 (2004).

    Article  CAS  Google Scholar 

  26. Shimamoto, K. Glutamate transporter blockers for elucidation of the function of excitatory neurotransmission systems. Chem. Rec. 8, 182–199 (2008).

    Article  CAS  Google Scholar 

  27. Esslinger, C. S. et al. The substituted aspartate analogue L-β-threo-benzyl-aspartate preferentially inhibits the neuronal excitatory amino acid transporter EAAT3. Neuropharmacology 49, 850–861 (2005).

    Article  CAS  Google Scholar 

  28. Bridges, R. J. & Esslinger, C. S. The excitatory amino acid transporters: pharmacological insights on substrate and inhibitor specificity of the EAAT subtypes. Pharmacol. Ther. 107, 271–285 (2005).

    Article  CAS  Google Scholar 

  29. Mavencamp, T. L., Rhoderick, J. F., Bridges, R. J. & Esslinger, C. S. Synthesis and preliminary pharmacological evaluation of novel derivatives of L-β-threo-benzylaspartate as inhibitors of the neuronal glutamate transporter EAAT3. Bioorg. Med. Chem. 16, 7740–7748 (2008).

    Article  CAS  Google Scholar 

  30. Shimamoto, K. et al. Syntheses of optically pure β-hydroxyaspartate derivatives as glutamate transporter blockers. Bioorg. Med. Chem. Lett. 10, 2407–2410 (2000).

    Article  CAS  Google Scholar 

  31. Spengler, J., Pelay, M., Tulla-Puche, J. & Albericio, F. Synthesis of orthogonally protected L-threo-β-ethoxyasparagine. Amino Acids 39, 161–165 (2010).

    Article  CAS  Google Scholar 

  32. LaBean, T. H. & Kauffman, S. A. Design of synthetic gene libraries encoding random sequence proteins with desired ensemble characteristics. Protein Sci. 2, 1249–1254 (1993).

    Article  CAS  Google Scholar 

  33. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank B. Wu, B. Kaptein and O. May for helpful discussions. This research was financially supported by VENI grant 700.54.401 and ECHO grant 700.59.042 (both to G.J.P.) from the Division of Chemical Sciences of the Netherlands Organisation of Scientific Research (NWO-CW), and by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations (www.b-basic.nl) through B-Basic, a public-private NWO–ACTS programme (ACTS, Advanced Chemical Technologies for Sustainability). Financial support from the Royal Netherlands Academy of Arts and Sciences (KNAW to W.S. and B.L.F.) is also gratefully acknowledged.

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Authors and Affiliations

  1. Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen, 9713, AV, The Netherlands

    Hans Raj, Jandré de Villiers, Vinod Puthan Veetil, Carlos R. Reis, Marianne de Villiers, Wim J. Quax & Gerrit J. Poelarends

  2. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, Groningen, 9747, AG, The Netherlands

    Wiktor Szymański & Dick B. Janssen

  3. Center for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, Groningen, 9747, AG, The Netherlands

    Wiktor Szymański & Ben L. Feringa

  4. Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, Groningen, 9747, AG, The Netherlands

    Henriëtte J. Rozeboom & Andy-Mark W. H. Thunnissen

  5. Department of Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen, 9713, AV, The Netherlands

    Frank J. Dekker

  6. DSM Pharmaceutical Products, PO Box 18, Geleen, 6160 MD, The Netherlands

    Stefaan de Wildeman

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  1. Hans Raj
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Contributions

H.R. performed mutagenesis, library screening and activity measurements. W.S. and J.d.V. synthesized starting substrates and reference compounds. H.R., W.S., M.d.V. and F.J.D. performed preparative biocatalysis. H.J.R. performed X-ray crystallography experiments. V.P.V. performed chiral HPLC. C.R.R. performed the molecular docking experiments. S.d.W., W.J.Q., A.M.W.H.T., B.L.F., D.B.J. and G.J.P. supervised scientific work. All authors contributed to writing the paper.

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Correspondence to Gerrit J. Poelarends.

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The authors declare no competing financial interests.

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Raj, H., Szymański, W., de Villiers, J. et al. Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids. Nature Chem 4, 478–484 (2012). https://doi.org/10.1038/nchem.1338

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